Methods in

Molecular Biology 2733

Daniel R. Pérez
Editor

Reverse Genetics
of RNA Viruses
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

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Reverse Genetics of RNA Viruses

Methods and Protocols

Second Edition

Edited by

Daniel R. Pérez
College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Editor
Daniel R. Pérez
College of Veterinary Medicine
University of Georgia
Athens, GA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
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Preface

Reverse Genetics of RNA Viruses: Methods and Protocols, Second Edition follows the footsteps
of the first edition with the incorporation of new chapters and modifications of some of the
old chapters to highlight alternative methods and approaches for the manipulation of RNA
viruses. This second edition complements and does not replace the first edition which
continues to be relevant to this date. The term “reverse genetics” is an approach to unravel
the function of a gene by establishing and analyzing the phenotypic effects of synthetically
engineered gene sequences. For RNA viruses, reverse genetics implies the de novo reconsti-
tution of the virus from a cDNA copy. Using molecular biology, cDNA copies of RNA
viruses are cloned into a variety of vectors, most typically and in order of preference,
plasmids, bacterial artificial chromosomes or bacmids, or recombinant viral vectors. Consid-
ered the holy grail to study RNA viruses, reverse genetics has truly revolutionized our
understanding of pathogenesis, host range, and transmission of a myriad of RNA viruses.
In recent years and despite the sounding alarms of those that warn against this type of
research, reverse genetics has been instrumental in identifying and characterizing virulent
markers in RNA viruses and in producing innocuous viral progenies and reporter systems
that can be safely handled in the lab as surrogates for development of vaccines and antivirals.
Reverse genetics and synthetic biology played a crucial role in the timely production of
protective vaccines against influenza during the 2009 H1N1 pandemic. And prior research
using reverse genetics systems of coronaviruses led to the realization and manipulation of
key elements of the Spike glycoprotein of the severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) to produce in record time the highly effective mRNAs vaccines that helped
curtail the effects of the COVID-19 pandemic.
Here we have compiled 15 chapters summarizing reverse genetics breakthroughs and
detailed protocols. As with the first edition, the book does not cover every reverse genetics
protocol for every RNA virus. And like the first edition, this book would not have been
possible without the outstanding and most generous contributions of our authors who are
leaders in their respective fields and that have shared their insights and step-by-step proto-
cols to help you, our colleagues, with your own research endeavors. Reverse genetics will
continue to play a fundamental role in studying RNA viruses, identifying markers of host
range, disease, and transmission, and in helping us with the further the development of in
silico computational biology tools and artificial intelligence algorithms that can better
predict the emergence of pathogens that threaten food security and/or carry greater
zoonotic and/or pandemic potential.
I hope you find this book helpful.

Athens, GA, USA Daniel R. Pérez

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Reverse Genetics Systems for Filoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Bianca S. Bodmer and Thomas Hoenen
2 Reverse Genetics Systems for the De Novo Rescue of Diverse Members
of Paramyxoviridae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Griffin Haas and Benhur Lee
3 Rescue of Recombinant Newcastle Disease Virus Expressing Heterologous
Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Arantza Cobela-Garcı́a, Ignacio Mena,
and Adolfo Garcı́a-Sastre
4 Use of Reverse Genetics for the Generation of Recombinant Influenza
Viruses Carrying Nanoluciferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
C. Joaquin Caceres, L. Claire Gay, Flavio Cargnin Faccin,
and Daniel R. Pérez
5 Reverse Genetics of Bat Influenza A Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Susanne Kessler, Adolfo Garcı́a-Sastre, Martin Schwemmle,
and Kevin Ciminski
6 Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA . . . . . . . 87
Daniela Toro-Ascuy, Matı́as Cárdenas, Yesseny Vásquez-Martı́nez,
and Marcelo Cortez-San Martı́n
7 Reverse Genetics System for Rift Valley Fever Virus . . . . . . . . . . . . . . . . . . . . . . . . . 101
Breanna Tercero and Shinji Makino
8 Plasmid-Based Lassa Virus Reverse Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Luis Martı́nez-Sobrido, Chengjin Ye, and Juan Carlos de la Torre
9 Bacterial Artificial Chromosome Reverse Genetics Approaches
for SARS-CoV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Kevin Chiem, Aitor Nogales, Fernando Almazán,
Chengjin Ye, and Luis Martı́nez-Sobrido
10 Construction of Infectious Clones for Human Enteroviruses . . . . . . . . . . . . . . . . . 155
Thinesshwary Yogarajah and Justin Jang Hann Chu
11 Reverse Genetics of Hepatitis C Virus Using an RNA Polymerase
I-Mediated Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Ryosuke Suzuki and Tetsuro Suzuki
12 Reverse Genetics of Zika Virus Using a Bacterial Artificial Chromosome. . . . . . . 185
Aitor Nogales, Luis Martı́nez-Sobrido, and Fernando Almazán

vii
viii Contents

13 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing

Group II Intron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Zhong-Yu Liu, Xiao-Feng Li, and Cheng-Feng Qin
14 Reverse Genetics of Dengue Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
José Valter Joaquim Silva Júnior, Andréa Nazaré Monteiro Rangel da Silva,
Jefferson José da Silva Santos, and Laura Helena Vega Gonzales Gil
15 Recovery of Recombinant Rotaviruses by Reverse Genetics . . . . . . . . . . . . . . . . . . 249
Chantal A. Agbemabiese, Asha A. Philip, and John T. Patton

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Contributors

CHANTAL A. AGBEMABIESE • Noguchi Memorial Institute for Medical Research, University

of Ghana, Accra, Ghana; Department of Biology, Indiana University, Bloomington,
IN, USA
FERNANDO ALMAZÁN • Department of Molecular and Cell Biology, Centro Nacional de
Biotecnologı́a (CNB), CSIC, Madrid, Spain
BIANCA S. BODMER • Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-
Institut, Greifswald, Insel Riems, Germany
C. JOAQUIN CACERES • Department of Population Health, College of Veterinary Medicine,
University of Georgia, Athens, GA, USA
MATÍAS CÁRDENAS • Department of Population Health College of Veterinary Medicine,
University of Georgia, Athens, GA, USA
KEVIN CHIEM • Texas Biomedical Research Institute, San Antonio, TX, USA
JUSTIN JANG HANN CHU • Department of Microbiology and Immunology, Yong Loo Lin
School of Medicine, National University Health System, Singapore, Singapore
KEVIN CIMINSKI • Institute of Virology, Medical Center – University of Freiburg, Freiburg,
Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany
ARANTZA COBELA-GARCÍA • Department of Microbiology, Icahn School of Medicine at Mount
Sinai, New York, NY, USA; Bernhard Nocht Institute for Tropical Medicine, Hamburg,
Germany; Faculty of Mathematics, Informatics and Natural Sciences, University
Hamburg, Hamburg, Germany
MARCELO CORTEZ-SAN MARTÍN • Laboratory of Molecular Virology and Pathogens Control,
Faculty of Chemistry and Biology, University of Santiago de Chile, Santiago, Chile
ANDRÉA NAZARÉ MONTEIRO RANGEL DA SILVA • Virology Laboratory, Institute of Biological
Sciences, Federal University of Pará, Belém, PA, Brazil
JEFFERSON JOSÉ DA SILVA SANTOS • Department of Microbiology, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
JUAN CARLOS DE LA TORRE • Department of Immunology and Microbial Science, The Scripps
Research Institute, La Jolla, CA, USA
FLAVIO CARGNIN FACCIN • Department of Population Health, College of Veterinary
Medicine, University of Georgia, Athens, GA, USA
ADOLFO GARCÍA-SASTRE • Department of Microbiology, Icahn School of Medicine at Mount
Sinai, New York, NY, USA; Global Health and Emerging Pathogens Institute, Icahn
School of Medicine at Mount Sinai, New York, NY, USA; Department of Medicine,
Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY,
USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY,
USA; Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of
Medicine at Mount Sinai, New York, NY, USA
L. CLAIRE GAY • Department of Population Health, College of Veterinary Medicine,
University of Georgia, Athens, GA, USA
LAURA HELENA VEGA GONZALES GIL • Laboratory of Virology and Experimental Therapy,
Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, PE, Brazil

ix
x Contributors

GRIFFIN HAAS • Icahn School of Medicine at Mount Sinai, New York, NY, USA
THOMAS HOENEN • Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-
Institut, Greifswald, Insel Riems, Germany
SUSANNE KESSLER • Institute of Virology, Medical Center – University of Freiburg, Freiburg,
Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany
BENHUR LEE • Icahn School of Medicine at Mount Sinai, New York, NY, USA
XIAO-FENG LI • Department of Virology, State Key Laboratory of Pathogen and Biosecurity,
Beijing Institute of Microbiology and Epidemiology, AMMS, Beijing, China
ZHONG-YU LIU • School of Medicine, Sun Yat-sen University, Shenzhen, China
SHINJI MAKINO • Departments of Microbiology and Immunology, Galveston, TX, USA;
Institute of Human Infection and Immunity, Galveston, TX, USA; Center for Biodefense
and Emerging Infectious Diseases, Galveston, TX, USA; UTMB Center for Tropical
Diseases, Galveston, TX, USA; The Sealy Institute for Vaccine Sciences, Galveston, TX, USA
LUIS MARTÍNEZ-SOBRIDO • Texas Biomedical Research Institute, San Antonio, TX, USA
IGNACIO MENA • Department of Microbiology, Icahn School of Medicine at Mount Sinai,
New York, NY, USA; Global Health and Emerging Pathogens Institute, Icahn School of
Medicine at Mount Sinai, New York, NY, USA; Department of Immunology and
Microbiology, The Scripps Research Institute, La Jolla, CA, USA
AITOR NOGALES • Centro de Investigacion en Sanidad Animal (CISA-INIA/CSIC),
Madrid, Spain
JOHN T. PATTON • Department of Biology, Indiana University, Bloomington, IN, USA
DANIEL R. PÉREZ • Department of Population Health, College of Veterinary Medicine,
University of Georgia, Athens, GA, USA
ASHA A. PHILIP • Department of Biology, Indiana University, Bloomington, IN, USA;
CSL Seqirus, Waltham, MA, USA
CHENG-FENG QIN • Department of Virology, State Key Laboratory of Pathogen and
Biosecurity, Beijing Institute of Microbiology and Epidemiology, AMMS, Beijing, China
MARTIN SCHWEMMLE • Institute of Virology, Medical Center – University of Freiburg,
Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany
JOSÉ VALTER JOAQUIM SILVA JÚNIOR • Virology Sector, Department of Preventive Veterinary
Medicine, Federal University of Santa Maria, Santa Maria, RS, Brazil; Virology Sector,
Laboratory of Immunopathology Keizo Asami, Federal University of Pernambuco, Recife,
PE, Brazil
RYOSUKE SUZUKI • Department of Virology II, National Institute of Infectious Diseases,
Tokyo, Japan
TETSURO SUZUKI • Department of Microbiology and Immunology, Hamamatsu University
School of Medicine, Hamamatsu, Japan
BREANNA TERCERO • Departments of Microbiology and Immunology, Galveston, TX, USA
DANIELA TORO-ASCUY • Laboratory of Virology, Department of Biology, Faculty of Sciences,
Universidad de Chile, Santiago, Chile
YESSENY VÁSQUEZ-MARTÍNEZ • Escuela de Medicina, Faculty of Health Sciences, University of
Santiago de Chile, Santiago, Chile
CHENGJIN YE • Texas Biomedical Research Institute, San Antonio, TX, USA
THINESSHWARY YOGARAJAH • Department of Microbiology and Immunology, Yong Loo Lin
School of Medicine, National University Health System, National University of Singapore,
Singapore, Singapore
 Chapter 1

Reverse Genetics Systems for Filoviruses

Bianca S. Bodmer and Thomas Hoenen

Abstract
Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For
studies of virus biology and the subsequent development of countermeasures, reverse genetic systems, and
especially those facilitating the generation of recombinant filoviruses, are indispensable. Here, we describe
the generation of recombinant filoviruses from cDNA.

Key words Ebola virus, Marburg virus, Filoviruses, Reverse genetics, Full-length clone system,
Infectious clone, Recombinant virus

1 Introduction

Filoviruses are classified in the order Mononegavirales and comprise

four genera that are associated with infections in mammals (i.e. the
genus Orthoebolavirus, Orthomarburgvirus, Cuevavirus, and Dia-
nlovirus). At least two of these (the ebolaviruses and the marburg-
viruses) include viruses that are responsible for human infections
where they cause severe hemorrhagic fevers with high case fatality
rates (CFR). Consequently, all filoviruses are currently classified as
biosafety level (BSL) 4 agents [1–3]. Of the six ebolaviruses that are
known as of now, four are linked to human disease (i.e., Ebola virus
(EBOV), Sudan virus, Bundibugyo virus, and Taı̈ Forest virus),
while Reston virus is apathogenic in humans. Of these, the most
virulent is EBOV, which was also responsible for the two most
extensive outbreaks of filovirus disease ever reported: one in
2013–2016, with over 28,600 cases and 11,300 deaths (CFR
40%) and the other in 2018–2020, with nearly 3470 cases and
2280 deaths (CFR 66%) [3–5]. Importantly, increasing virus dis-
covery efforts have led to the identification of several novel filo-
viruses of unknown pathogenic potential, e.g., Bombali virus
(genus: Orthoebolavirus) in West Africa, Lloviu virus (genus: Cue-
vavirus) in Europe, and Měnglà virus (genus: Dianlovirus) in

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

1
2 Bianca S. Bodmer and Thomas Hoenen

China—clearly showing that filoviruses have a much larger geo-

graphic distribution than previously believed. Further, this high-
lights the need for the development of broadly acting
countermeasures to combat both the possible emergence of new
filoviruses and future outbreaks of those that are already known [6–
8]. Indeed, the unprecedented extent of the EBOV outbreaks in
2013–2016 and 2018–2020 has spurred research on antivirals and
resulted in the approval of two EBOV-specific antibody-based
treatments by the Federal Drug Administration (FDA)
[9, 10]. Additionally, a vaccine based on an attenuated
replication-competent vesicular stomatitis virus (VSV) that encodes
the EBOV glycoprotein (GP) instead of the VSV glycoprotein has
been approved by the FDA and European Medicines Agency
(EMA) and was administered to over 300,000 people in ring
vaccinations during the 2018–2020 outbreak [5, 11, 12]. Further-
more, the EMA approved a heterologous prime boost regimen
based on two other vector vaccines (i.e., a GP-expressing adenovi-
rus followed by a GP-expressing modified vaccinia Ankara) for use
under exceptional circumstances in 2020 [13, 14]. Nevertheless,
the dimensions of these recent ebolavirus disease outbreaks (in the
case of the latter one, despite the use of ring vaccination) empha-
sizes the need for additional research efforts on filoviruses, and
particularly those focused on identifying new therapeutic
approaches. However, understanding the molecular biology of
filoviruses is a prerequisite for both the development and testing
of possible countermeasures. Importantly, reverse genetics can
assist in these endeavors, and can be of particular use for work on
novel filoviruses, since for many of these viruses, isolates are not
available, and only their sequences are known.
The single-stranded, negative-sense RNA genome of filoviruses
encodes for seven structural proteins. It is encapsidated by the
nucleoprotein (NP), which together with the viral polymerase L,
the viral protein 30 (VP30), a transcriptional activator for ebola-
viruses, and VP35, a polymerase co-factor and interferon (IFN)
antagonist, forms the ribonucleoprotein complex (RNP). These
proteins also represent the minimal components required for repli-
cation and transcription of the viral RNA [15]. Further, the avail-
able data suggest that the nucleocapsid-associated protein VP24
condenses the RNP into a form that is no longer able to serve as a
template for viral RNA synthesis, but is now able to be incorporated
into nascent virions [16, 17]. For ebolaviruses and cuevaviruses,
VP24 is also an IFN antagonist [18–20]. Budding and morpho-
genesis of the virus particles, which have a typical threadlike shape,
is driven by the matrix protein VP40, which also acts as an IFN
antagonist for marburgviruses [21, 22]. GP is embedded in the
host cell-derived virus membrane and mediates attachment and
fusion with target cells [23].
 Filovirus Reverse Genetics 3

Reverse genetics can be used to model specific aspects of the

filovirus life cycle, including replication and transcription, morpho-
genesis, budding, and entry outside of high-containment facilities
(i.e., under BSL1 or BSL2 conditions, depending on local regula-
tions) through the application of life cycle modeling systems
(reviewed in [24]). Further, reverse genetic-based full-length
clone systems allow the generation of recombinant viruses (includ-
ing those that contain mutations in the virus genome) that are
infectious and thus allow us to study the whole filovirus life cycle
but must be used under BSL4 conditions. Here, we focus on the
procedure for generating (or “rescuing”) full-length ebolaviruses
(Fig. 1). In such a full-length system, the genetic information for
the full-length ebolavirus genome is provided as a cDNA copy on a
plasmid. Initial transcription from this plasmid results in the pro-
duction of a cRNA antigenome. Traditionally, this transcription of
the viral genome is facilitated by T7 RNA polymerase, which is
co-expressed from another plasmid [25, 26]. The resulting cRNA is
then recognized by RNP proteins, co-expressed from additional
expression plasmids (usually PolII-driven, e.g., pCAGGS) and ille-
gitimately encapsidated and replicated into vRNA. This vRNA can
then be transcribed to produce all seven viral mRNAs, which in turn
are translated to produce each of the viral proteins. These then
support further viral RNA synthesis as well as all other steps in
the viral life cycle, up to and including the budding of infectious
ebolaviruses. By modifying the full-length genome plasmid, recom-
binant viruses can also be generated using this approach and used in
subsequent experiments to study any and all aspects of the molecu-
lar biology of ebolaviruses. For example, such viruses have already
been used to study the biology of inclusion bodies [27], virus-host
interactions [28, 29], and pathogenicity determinants [30–33]. At
the same time, the introduction of reporter genes (e.g., fluorescent
proteins or luciferases) has extended the applications of such viruses
to include high-throughput screening approaches (for instance, in
antiviral testing or the identification of interacting host factors), as
well as the analysis of cellular processes via imaging techniques
[34, 35] and even in vivo applications [36]. In the following sec-
tions, a detailed protocol is described for the rescue of recombinant
EBOV from cDNA; however, the rescue of other recombinant
filoviruses is similar so that this protocol can also be adapted
accordingly.

2 Materials

2.1 Rescue and 1. Cells for initial rescue and passaging, e.g., VeroE6, HEK293T,
Passaging of Viruses or Huh7 cells (see Note 1).
4 Bianca S. Bodmer and Thomas Hoenen

Fig. 1 Schematic depiction of processes taking place during a filovirus rescue. Cells are transfected with
plasmids encoding a cDNA copy of the full-length viral genome (rgZ) and each of the viral ribonucleoprotein
complex (RNP) components, i.e., L, VP35, VP30, and NP. A plasmid encoding the T7 RNA polymerase is also
transfected. Initial transcription of the viral genome-encoding plasmid by T7 RNA polymerase (A) results in a
“naked” full-length antigenome (cRNA), which is subsequently illegitimately encapsidated (B) by the viral RNP
proteins. At this point, it can then serve as a template for genome replication (C) to produce genomic RNA
(vRNA), which is encapsidated to form RNPs. These vRNA-containing RNPs can undergo not only replication
but also transcription of viral mRNAs encoding each of the viral proteins (D). This results in translation of the
viral proteins (E), which further encapsidate newly replicated genomes and antigenomes (F) to mediate further
viral RNA synthesis, but also mediate additional steps in the morphogenesis and budding of recombinant virus
particles (G)
 Filovirus Reverse Genetics 5

2. Dulbecco’s Modified Eagle’s Medium (DMEM) with 10%

(v/v) or 5% (v/v) fetal bovine serum (FBS, heat-inactivated
for 30 min at 56 °C), 1% L-glutamine (Q, 2 mM), and 1%
penicillin/streptomycin (PS, 100 U/mL).
3. OptiMEM.
4. 6-well plates and T150 tissue culture flasks.
5. Transit-LT1 transfection reagent.
6. Cell scrapers, 50 mL centrifuge tubes, and 1.5 mL cryovials.

2.2 Sequence 1. QIAamp viral RNA Mini Kit.

Confirmation of 2. 96–100% ethanol.
Rescued Viruses
3. Superscript IV Reverse Transcriptase kit (includes 5X SSIV
buffer, 0.1 M DTT, RNase-free water, RNaseOUT recombi-
nant RNase inhibitor, dNTPs (10 mM each), and RNase H).
4. iProof High-Fidelity DNA polymerase kit (includes 5× reaction
buffers, MgCl2 solution, DMSO, dNTPs, and iProof DNA
polymerase).
5. PCR cooling block and PCR cycler.
6. PCR purification kit.
7. Primers (see Note 2): #3185 cggacacacaaaaagaaagaag; #3186
caaatacttgactgcgccac; #3187 gagtgcggacagtttccttc; #3188
gtactcccgtgtgctgtgg; #3189 ggccaagcatggagagtatg; #3190
gttctgtgagggcctgggac; #3191 gggtggacaacagaagaacag; #3192
cctgttttcgttccttgactac; #3193 catggcaatcctgcaacatc; #3194
cagtagccaatgaagccaatg; #3195 cgaagccaaacccgaagac; #3196
caagctcggggaatgtcac; #3197 ctcctcaatgtgccctaattc; #3198 gat-
gaatgctgatgacacactg; #3199 ggacactccatcgaatccac; #3200
catggtgaggtctcctggag; #3201 gaccggtaagaaggtgacttc; #3202
gttgccccacaatatccttctag; #3203 gcgtaatcttcatctctcttag; #3204
gggtcctccgttgcattgac; #3205 gaaggtgtcgttgcatttctg; #3206
gtcgtggcagagggagtg; #3207 cttgacatctctgaggcaac; #3208
ccatcctgtccaccaattgtc; #3209 cgaaccacatgattggaccaag; #3210
ctcgataattctctctggatgatg; #3211 atatgagagaggacgcccac; #3212
cacacggtaactggagagc; #3213 cagttttgaagctgcactatg; #3214
cttatcagacctccgcattaatc; #3215 gaggtgtttggtattggctattg;
#3216 atgcaggggcaaagtcattag; #3217 ggtggaaggtttattgggctg;
#3218 cagtgaggatttatctgtggttaaac; #3219 gttatcttga-
catctctgctttc; #3220 gagagcatcttgcattgtgtac; #3221 caagg-
cactgtcaggcaatg; #3222 gggtgtgatttacagctaaatgc; #3223
cctcgaaccattgtgcttgg; #3224 gatattgtggtagtagatactcgag;
#3225 cctcacaaatttagtactaaacgtg; #3226 gtctgcgtcagtctctaag;
#3227 ctggacaagtatttcatgtgctc; #3228 gcgacccagggacatttaatc;
#3229 ctccgaatgattgagatggatg; #3230 ccgatagtccagcttattcg;
#3231 caaccaggtgggaaaccattc; #3232 cagccgtttaccttggaaaaatg;
#3233 ccgagaaaacgaattgatttatg; #3234
6 Bianca S. Bodmer and Thomas Hoenen

tgtcgtgaggatgtacatgatc; #3235 ggtcaaaacccaacacctgtg; #3236

ccgacttgaaactctctatttc; #3237 gagatccgtcattgataccacag; #3238
agttaaatgacttagccagtatgg; #3239 atgccacaccaaaaccatctc; #3240
cagggagagaggctaaatatag; #3241 cctgatacttgcaaaggttgg; #3242
tggacacacaaaaaagaagaaatag

3 Methods

3.1 Initial Rescue of 1. Seed p0 cells (see Note 1) in 2 mL DMEM10%FCS/PS/Q in

Recombinant Viruses 6-well plates for ~50% confluence on the next day. Incubate
the cells at 37 °C and 5% CO2 in a humidified incubator.
2. After 24 h, combine the helper plasmids in a 1.5 mL reaction-
tube: 125 ng pCAGGS-NP, 125 ng pCAGGS-VP35, 75 ng
pCAGGS-VP30, 1000 ng pCAGGS-L or pCAGGS-eGFP,
250 ng pCAGGS-T7 (amounts are per well to be transfected).
3. Add 100 μL Opti-MEM (per well to be transfected) to the
helper plasmid mixture, vortex, and spin down the sample. It is
advisable to transfect several wells in parallel since rescue effi-
cacy is usually below 100%, and to use one well as negative
control by replacing pCAGGS-L with an equal amount of
pCAGGS-eGFP.
4. Transfer cells, diluted helper plasmids, and full-length clone
plasmid into a BSL4 laboratory.
5. Add 250 ng full-length plasmid to the diluted helper plasmid
mixture.
6. Briefly vortex the vial containing Transit-LT1 transfection
reagent. Add 6 μL Transit-LT1 (per well to be transfected)
(see Note 3) to the plasmid mixture, vortex, and spin down.
Incubate for 15 min at room temperature to allow transfection
complexes to form.
7. After 15 min, add 100 μL of transfection complexes in a drop-
wise fashion to the cells, trying to distribute them over the
whole surface area of the well.
8. Rock the plates back and forth and from side to side. Do not
swirl the plates, to avoid transfection complexes being pushed
to the edge of the well. Return the cells to the incubator.
9. At 24 h post transfection, replace the medium with 4 mL
DMEM5%FCS/PS/Q (see Notes 4 and 5). Return cells to the
incubator.

3.2 Passage of 1. Six days post transfection, seed p1 cells (see Note 6) in 4 mL
Recombinant Viruses DMEM10%FCS/PS/Q in 6-well plates for ~90% confluence on
the next day. Incubate cells at 37 °C and 5% CO2 in a humidi-
fied incubator.
 Filovirus Reverse Genetics 7

2. On the next day (i.e., 7 days post transfection), take the p1 cells
into the BSL4 laboratory. Add 1 mL of supernatant from the
p0 cells to the medium of the p1 cells (see Note 7). Return the
cells to the incubator.
3. From now on, check for cytopathic effect (CPE) in p1 cells
daily (see Note 8). Alternatively, if reporter-expressing viruses
(e.g., viruses expressing GFP or luciferase) are being rescued,
reporter activity can be used as a read-out.
4. Once clear CPE is visible, passage 1 mL of p1 supernatant onto
a T150 flask with 90% confluent cells (p2 cells) in 60 mL
DMEM10%FCS/PS/Q (see Note 7). Check for CPE in p2 cells
daily.

3.3 Harvest of 1. Once p2 cells show clearly discernibly CPE, scrape the cells into
Recombinant Viruses the medium using a cell scraper. Then transfer ~30 mL of the
resulting cell suspension each into two 50 mL centrifuge tubes,
and spin down for 10 min at 1000× g at 4 °C.
2. Pour the clarified supernatant into fresh centrifuge tubes each
containing 3 mL FBS (heat-inactivated for 30 min at 56 °C;
i.e., 10% total sample volume), mix by inversion, and aliquot.
Store the aliquots in liquid nitrogen.

3.4 Sequence 1. To ensure that the rescued virus has the correct/expected
Confirmation of sequence (see Note 9), inactivate 140 μL virus stock by adding
Rescued Virus it to 560 μL AVL buffer containing carrier RNA (from the
QIAamp viral RNA Mini Kit). Vortex and incubate for 10 min.
2. Remove the inactivated virus from the biosafety cabinet and
transfer it into a clean tube containing 560 μL 96–100% etha-
nol (see Note 10). Mix by vortexing, incubate for a further
10 min, and then remove the sample from the BSL4 laboratory
following appropriate approved protocols.
3. Extract viral RNA using the QIAamp viral RNA Mini Kit,
following the manufacturer’s instructions.
4. In a PCR tube, combine 1 μL RNA, 1 μL RT primer #3185
(2 μM), 1 μL dNTPs, and 11 μL RNase-free water (see Note
11). Incubate for 5 min at 65 °C, and then place immediately
on ice for at least 1 min.
5. In the meantime, combine 4 μL 5× SSIV buffer, 1 μL 0.1 M
DTT, 1 μL RNaseOUT, and 1 μL SuperScript IV, vortex
briefly, and place on ice for at least 1 min.
6. Add 7 μL of this mixture to the RNA/primer/dNTP mix (see
Note 11), incubate for 10 min at 50 °C, and then heat-
inactivate for 10 min at 80 °C and cool to 4 °C.
7. Add 1 μL RNase H (see Note 11), incubate for 20 min at 37 °
C, and then store cDNA at 4 °C.
8 Bianca S. Bodmer and Thomas Hoenen

Table 1
Primer combinations for PCRs and subsequent sequencing

Forward PCR Reverse PCR Product

primer primer size Primers for sequencing
#3185 #3186 ~0.7 kB #3185, #3186
#3187 #3192 ~2.1 kB #3187, #3188, #3189, #3190, #3191,
#3192
#3193 #3198 ~2.1 kB #3193, #3194, #3195, #3196, #3197,
#3198
#3199 #3204 ~2.1 kB #3199, #3200, #3201, #3202, #3203,
#3204
#3205 #3210 ~2.1 kB #3205, #3206, #3207, #3208, #3209,
#3210
#3211 #3216 ~2.1 kB #3211, #3212, #3213, #3214, #3215,
#3216
#3217 #3222 ~2.1 kB #3217, #3218, #3219, #3220, #3221,
#3222
#3223 #3228 ~2.1 kB #3223, #3224, #3225, #3226, #3227,
#3228
#3229 #3234 ~2.1 kB #3229, #3230, #3231, #3232, #3233,
#3234
#3235 #3240 ~2.1 kB #3235, #3236, #3237, #3238, #3239,
#3240
#3241 #3242 ~0.7 kB #3241, #3242
#3185 #3190 ~2.1 kB #3185, #3186, #3187, #3188, #3189,
#3190
#3191 #3196 ~2.1 kB #3191, #3192, #3193, #3194, #3195,
#3196
#3197 #3202 ~2.1 kB #3197, #3198, #3199, #3200, #3201,
#3202
#3203 #3208 ~2.1 kB #3203, #3204, #3205, #3206, #3207,
#3208
#3209 #3214 ~2.1 kB #3209, #3210, #3211, #3212, #3213,
#3214
#3215 #3220 ~2.1 kB #3215, #3216, #3217, #3218, #3219,
#3220
#3221 #3226 ~2.1 kB #3221, #3222, #3223, #3224, #3225,
#3226
#3227 #3232 ~2.1 kB #3227, #3228, #3229, #3230, #3231,
#3232
#3233 #3238 ~2.1 kB #3233, #3234, #3235, #3236, #3237,
#3238

(continued)
 Filovirus Reverse Genetics 9

Table 1
(continued)

Forward PCR Reverse PCR Product

primer primer size Primers for sequencing
#3239 #3242 ~1.4 kB #3239, #3240, #3241, #3242
#3197 #3198 ~0.7 kB #3197, #3198
#3209 #3212 ~1.4 kB #3209, #3210, #3211, #3212
#3213 #3216 ~1.4 kB #3213, #3214, #3215, #3216

Table 2
PCR conditions

Step Duration Temperature Remarks

1 30 s 98 °C
2 15 s 98 °C
3 15 s 59–54.5 °C Reduce temperature by 0.5 °C per cycle
4 60 s 72 °C
5 Return to step 2 for a total of 10 cycles
6 15 s 98 °C
7 15 s 54 °C
8 60 s 72 °C
9 Return to step 6 for a total of 30 cycles
10 3 min 72 °C
11 Hold 4 °C

8. Label 24 PCR tubes (e.g., three eight-tube strips) and place in a

pre-chilled PCR cooling block.
9. Prepare the primers (Table 1) at a concentration of 5 μM each
in eight-tube strips (see Note 12).
10. Program the PCR cycler (Table 2) and preheat to 98 °C.
11. Prepare the PCR master mix by combining 773.75 μL water,
250 μL 5× GC buffer, 25 μL dNTPs (10 mM each), 37.5 μL
DMSO, 12.5 μL MgCl2, and 6.25 μL iProof DNA polymerase,
vortex, and place on ice (see Note 13).
12. Add 1105 μL PCR master mix to 20 μL cDNA, vortex, and
pipet 45 μL into each PCR tube.
10 Bianca S. Bodmer and Thomas Hoenen

13. Add 5 μL primers using a multichannel pipette, mix well (see

Note 11), and incubate the reactions in PCR cycler according
to the temperature profile shown in Table 2.
14. After the run, PCR-purify the products using a commercial
PCR purification kit following the manufacturer’s instructions,
e.g., the Macherey-Nagel PCR NucleoSpin Gel and PCR
Clean-up kit (see Notes 14 and 15).
15. Sanger-sequence the purified PCR products using the primers
listed in Table 1.

4 Notes

1. Various cell lines and cell line mixtures have been used for the
rescue of recombinant filoviruses, e.g., a HEK293T/Vero cell
mix, Vero cells, or Huh-7 cells. These cell lines vary in their
susceptibility to filovirus infection and their transfectability,
both of which impacts rescue efficiency. For example,
HEK293 and HEK293T cells are highly transfectable, but
only poorly susceptible to filovirus infection (although this
can be overcome by expressing the virus attachment factor
Tim-1 in these cells) [17, 37]. In contrast, Vero cells are highly
susceptible to filovirus infection, but somewhat harder to
transfect—although in our experience, this also varies between
different Vero clones. Further, it has been shown that the
choice of cell line can influence sequence fidelity in the recom-
binant viruses that are rescued, with rescue in Vero cells result-
ing in certain mutations (particularly A insertions in poly-A
stretches) in the genome with a higher frequency than when
using other cells [38]. These properties should be taken into
consideration when choosing a cell line for rescue. The authors
recommend Huh-7 cells for initial rescue, and either Huh-7 or
VeroE6 cells for further passaging.
2. All primer sequences provided here are for the species Zaire
ebolavirus and are based on the strain Mayinga sequence (Gen-
Bank accession number NC_002549) [39].
3. The amount of Transit-LT1 may need to be adjusted to obtain
optimal transfection efficacy in the cell line used. As a starting
point for most cell types, a ratio of 3 μL Transit-LT1 per 1 ug
DNA can be used. For Vero cells, this ratio should be increased
to 6:1.
4. At this point, the supernatant should be considered to contain
infectious ebolaviruses, and standard practices should be used
to avoid cross-contaminating wells (i.e., changing of pipettes/
pipette tips between different wells).
 Filovirus Reverse Genetics 11

5. It is important at this point to completely remove all superna-

tant prior to adding fresh medium, in order to remove traces of
the cDNA plasmid, which could otherwise cause problems in
downstream procedures (e.g., sequencing). This can be
achieved by first removing the supernatant with a serological
pipette, and then removing any remaining liquid with a 100 or
1000 μL pipette.
6. Again, the choice of cells can be varied. Vero cells are most
commonly used at this point; however, continuous passaging in
these cells can lead to nucleotide insertions in regions encoding
poly-A stretches, e.g., at the GP gene editing site [38, 40].
7. An alternative approach is to freeze 1 mL of p0 supernatant at
-80 °C at the same time the p0 supernatant is passaged to p1
cells in 6-well format. Once p1 cells show clear CPE, the
corresponding p0 supernatant can be used to inoculate the
T150 flask for stock growth. This reduces the passage number
for the virus stock, which in turn reduces the chance for the
appearance of unwanted mutations while still allowing for easy
screening of a larger number of wells for a successful virus
rescue.
8. The onset of cytopathic effect is dependent on the properties of
the rescued virus, as well as the rescue efficacy. As a rule of
thumb, clear CPE (compared to the -L control) should start to
be visible about a week after passaging. However, for some
ebolavirus species, this can take up to 3 weeks and may be less
pronounced than for EBOV.
9. Given the frequency with which rescued ebolaviruses show
unwanted mutations, it is imperative that they are completely
sequenced prior to use.
10. The addition of ethanol is important to ensure complete inac-
tivation before removal from the BSL4 laboratory, as incuba-
tion with AVL for 10 min (as recommended by the
manufacturer) is not always sufficient to completely inactivate
samples [41, 42].
11. To ensure proper mixing, samples should be mixed by flicking
the tubes repeatedly and then very briefly spinning them down
at low speed (i.e., in a benchtop microfuge).
12. If sequencing is done more often, primers can be frozen in
eight-tube strips. In this case, make sure the strips are spun
down thoroughly before opening, and label both the strip caps
and the tubes, to avoid cross-contamination. Note that strips
from some manufacturers can be difficult to open, resulting in
an increased risk of spilling the contents. To alleviate this issue,
the strips can be incubated empty at 99 °C in a PCR cycler for
5 min before they are first used.
12 Bianca S. Bodmer and Thomas Hoenen

13. The amounts given are for 24 + 1 reactions, which is sufficient

to sequence one virus completely. If more than one virus is
sequenced, the amount can be scaled-up proportionally. How-
ever, since the iProof DNA polymerase has 3′–5′ exonuclease
activity and does not have to be activated by heating (i.e.,
hot-start), it becomes very important that all reactions are
kept on ice or in a PCR cooling block, particularly when
processing a large number of samples.
14. If desired, PCR products can also be visualized using standard
gel electrophoresis prior to purification. In this case, loading
2 μL of PCR product should result in clearly visible bands after
ethidium-bromide staining. The expected product sizes are
indicated in Table 1.
15. Other PCR purification kits can also be used; however, the
Macherey-Nagel kit allows the use of diluted buffer NTI (1:4
in water) for the initial DNA binding step, which decreases the
amount of small, unspecific products (primer-dimers) in the
purified DNA.

Acknowledgments

This work was supported by the Friedrich-Loeffler-Institut

through intramural funding and the Deutsche Forschungsge-
meinschaft (DFG; grant number 452208680). The authors further
are grateful to Allison Groseth (Friedrich-Loeffler-Institut) for crit-
ical reading of this manuscript.

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 Chapter 2

Reverse Genetics Systems for the De Novo Rescue

of Diverse Members of Paramyxoviridae
Griffin Haas and Benhur Lee

Abstract
Paramyxoviruses place significant burdens on both human and wildlife health; while some paramyxoviruses
are established within human populations, others circulate within diverse animal reservoirs. Concerningly,
bat-borne paramyxoviruses have spilled over into humans with increasing frequency in recent years,
resulting in severe disease. The risk of future zoonotic outbreaks, as well as the persistence of paramyx-
oviruses that currently circulate within humans, highlights the need for efficient tools through which to
interrogate paramyxovirus biology. Reverse genetics systems provide scientists with the ability to rescue
paramyxoviruses de novo, offering versatile tools for implementation in both research and public health
settings. Reverse genetics systems have greatly improved over the past 30 years, with several key innovations
optimizing the success of paramyxovirus rescue. Here, we describe the significance of such advances and
provide a generally applicable guide for the development and use of reverse genetics systems for the rescue
of diverse members of Paramyxoviridae.

Key words Paramyxovirus, Paramyxoviridae, Reverse genetics, Virus rescue

1 Introduction

Paramyxoviruses, members of the viral family Paramyxoviridae of

the order Mononegavirales, are enveloped viruses with a negative-
sense, single-stranded RNA genome. Uniquely, paramyxoviruses
have genome sizes that are always equally divisible by 6 and encode
a minimum of six proteins (Fig. 1) [1, 2]. Paramyxoviridae is a
broad family and includes many species that place a significant
burden on human health. These include measles virus (MeV),
mumps virus (MuV), and the human parainfluenza viruses (HPIV
1–4) [3]. Numerous wildlife reservoirs, including birds, reptiles,
fish, rodents, and notably, bats harbor a myriad of paramyxovirus
species [4, 5]. With the advent of metagenomic screens, new para-
myxoviruses have been identified annually in increasingly diverse
hosts. Concerningly, bat-borne paramyxoviruses of the genera
Pararubulavirus and Henipavirus have demonstrated an aptitude

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

15
16 Griffin Haas and Benhur Lee

Fig. 1 General genome structure of paramyxoviruses. Paramyxoviruses encode a

bipartite promoter sequence in both the 3′ leader (3′ Ldr) and 5′ trailer (5′ Tr)
regions of the genome. Paramyxoviruses encode at least six genes, nucleocap-
sid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment
protein (RBP), and the large vRdRp protein (L). Some species may encode
additional proteins. The number of nucleotides in the genome is always equally
divisible by 6

for traversing the species barrier and have caused severe disease
when they have spilled over into human hosts; there is increasing
concern that such zoonotic paramyxoviruses could establish them-
selves in the human population, resulting in outbreaks [6–11]. Sys-
tems to advance our understanding of paramyxoviruses are
increasingly needed, and the use of such tools will undoubtedly
bolster our ability to prepare for and respond to future (zoonotic)
pandemics.
The ability to rescue paramyxovirus species de novo from
cDNA provides numerous applications for both research and public
health. Reverse genetics systems allow for the generation of pure
viral stocks without a need for excessive passaging, preventing
adaptation of virus to cell culture; this is desirable to produce
vaccine or research strains in which passaging may introduce unex-
pected mutations. Plasmid-encoded viral cDNA can be easily
manipulated, allowing for the integration of reporter genes, the
generation of desired mutations, and the ability to knock out entire
genes from viral genomes; such versatility greatly expands the
toolkit of virologists and likewise the scope of questions that may
be investigated. Excitingly, reverse genetics systems even allow for
the de novo rescue and characterization of sequenced viral species
that have been identified by metagenomics, but have yet to be
isolated, such as a mumps-like virus from Epomophorus bats (Bat-
MuV) and a morbillivirus from Myotis bats (Fig. 2) [12, 13].

1.1 History and Minireplicon systems laid the foundation for Paramyxoviridae
Optimization of reverse genetics. Between 1991 and 1993, minireplicon systems
Paramyxovirus were constructed for Sendai virus (SeV), respiratory syncytial virus
Reverse Genetics (RSV), and HPIV-3 [14–16]. These constructs were used as tem-
Systems plate for the in vitro transcription of synthetic, viral-like RNAs for
each species; transfection of the respective viral-like RNA species
into cells, complemented by superinfection with respective full-
length virus, resulted in “rescue” of the viral-like RNAs, character-
ized by replication of the viral-like RNA and concomitant reporter
gene expression by the viral RNA-dependent RNA polymerase
(vRdRp). Further, such viral-like RNAs were observed to be pack-
aged into propagable, viral-like particles (VLPs). These works
 Reverse Genetics Systems for Diverse Paramyxoviruses 17

Fig. 2 Phylogeny of fully sequenced members of Paramyxoviridae. The evolutionary history was inferred using
the neighbor-joining method for amino acid sequences of the large (L) vRdRp protein from each species. The
tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson
correction method and are in the units of the number of amino acid substitutions per site. Evolutionary
analyses were conducted in MEGA X. An asterisk (*) next to a species name denotes that reverse genetics
systems have been successfully established for the respective species

provided fundamental insights into paramyxovirus biology, includ-

ing as follows: (1) that the terminal ends of paramyxoviruses
encode the necessary cis-acting promoter elements required for
vRdRp recognition, and that such sequence elements are sufficient
to drive replication and gene expression; (2) that the terminal ends
of paramyxoviruses are sufficient for genome encapsidation by
nucleoprotein and for the packaging of RNA into virions; and
(3) that the expression of the viral replication complex—nucleopro-
tein (N), phosphoprotein (P), and the large vRdRp protein (L)—at
proper ratios in trans is sufficient to facilitate the rescue of viral-like
RNA species bearing the terminal ends of respective
paramyxoviruses.
18 Griffin Haas and Benhur Lee

In 1995, endeavors to encode full-length paramyxovirus gen-

omes into cDNA were successful, facilitating the first ever de novo
rescue of replication-competent MeV, SeV, and RSV [17–19],
although the latter is now classified as a member of the distinct
Pneumoviridae family. However, early reverse genetics systems were
inefficient, necessitating repeated rescue attempts in large quanti-
ties of transfected cells to successfully recover infectious virus.
Paramyxovirus reverse genetics systems have since been optimized
by taking the following factors into consideration:
1. Sufficiently high levels of the full-length antigenome must be
transcribed from the template cDNA plasmid.
2. The resultant, full-length vRNA species that are transcribed
from cDNA must have a genome size that is equally divisible
by 6, without the introduction of artificial, exogenous
sequence to the 3′ or 5′ extreme termini.
3. The viral replication complex proteins (N, P, and L) must be
expressed relative to one another at an optimal ratio.
Achieving sufficient transcription of the cDNA-encoded vRNA
has been fulfilled by cloning the viral sequence to be under the
control of a DNA-dependent RNA polymerase. The
DNA-dependent RNA polymerase from the T7 bacteriophage has
been widely applied in reverse genetics systems. T7 polymerase
possesses remarkably efficient processivity, making it ideal for the
transcription of long vRNAs that often exceed 15 kilobases in
length. Further, because the T7 polymerase specifically recognizes
a phage promoter element and not a mammalian promoter, all T7
polymerase expressed within a mammalian system is dedicated to
the reverse genetics system [20]. Despite the advantages of T7
polymerase, there are challenges that must be overcome for its
implementation; namely, the T7 polymerase must be sufficiently
expressed in mammalian cells during rescue. There have been sev-
eral strategies to achieve high expression of T7 within target cells,
such as by encoding T7 polymerase in recombinant helper virus
(e.g., vaccinia virus), or via the generation of cell lines (including
HEK-293T and BHK cells) that stably express T7 polymerase
[17, 21, 22]. We have demonstrated that in trans expression of
codon-optimized T7 polymerase significantly enhances T7 expres-
sion in mammalian cells and concomitantly improves paramyxovi-
rus rescue [23, 24]. Recently, reverse genetics systems have been
developed that utilize mammalian RNA polymerase II promoter
elements either alone or in combination with a T7 promoter ele-
ment [25–27]. Because RNA Pol II is constitutively expressed in
mammalian cells, its use alleviates the requirement to achieve high
expression of a polymerase species in target cells; however, this
comes at the cost of specificity, as there is competition between
the reverse genetics system and cellular DNA for RNA Pol II.
 Reverse Genetics Systems for Diverse Paramyxoviruses 19

Fig. 3 A Hh-Rbz allows for efficient transcription without compromising the rule
of six. (a) Minimal and optimal T7 promoter sequences. Templated G’s are
underlined. (b) A Hh-Rbz allows for implementation of the optimal T7 promoter
sequence, as it can be designed to cleave immediately upstream of the viral 3′
terminus (shown in blue)

Transcribing paramyxovirus vRNA from cDNA may conse-

quently introduce additional, exogenous sequences to the terminal
ends of a vRNA. Indeed, the optimal promoter element for T7
polymerase includes three templated “G” nucleotides, and removal
of one or more of these “G’s” significantly decreases activity by T7
polymerase (Fig. 3a) [28]. The addition of exogenous sequence to
the termini of paramyxovirus vRNAs is problematic, as the extreme
3′ and 5′ ends contain bipartite promoter elements that are pre-
cisely positioned relative to one another and are essential for recog-
nition by the vRdRp. During paramyxovirus replication, both the
negative-sense genomic vRNA and positive-sense antigenomic
vRNA are completely encapsidated in N. Each monomer of N
binds precisely to six consecutive nucleotides (a hexamer) of the
vRNA [2, 29, 30]. Full encapsidation of the vRNA by N drives a
helical, three-dimensional spatial arrangement of the viral ribonu-
cleoprotein complex (vRNP); the fully encapsidated, helical vRNP
positions two conserved, distal promoter elements in the 3 ends of
both the respective viral genome (encoded in the 3′ leader) and
viral antigenome (encoded in the 5′ trailer) such that two promoter
elements are arranged on the same face of the vRNP complex
[29, 31–33]. Proper three-dimensional arrangement of the bipar-
tite promoter is essential for recognition by the viral RdRp; having
evolved under this constraint, paramyxoviruses accommodate the
bipartite promoter structure by maintaining genome sizes that are
equally divisible by 6 (such that all nucleotides are encapsidated by
20 Griffin Haas and Benhur Lee

N) [2, 29, 30]. Exogenous sequence added to the 3′ or 5′ ends of

the vRNA disrupts proper phasing of these promoter elements,
preventing vRdRp recognition and optimal rescue.
To overcome the addition of exogenous sequence to para-
myxovirus termini, ribozyme elements—RNA structures with
autocatalytic energy to self-cleave—have been incorporated into
reverse genetics systems (Fig. 3b). A ribozyme derived from hepa-
titis delta virus (HDV-Rbz) had early implementation in reverse
genetics systems, ensuring that the 3′ end of the cDNA-encoded
viral sequence possessed no exogenous nucleotides; a
T7-terminator sequence is appended immediately downstream of
the HDV-Rbz to end T7 transcription. To satisfy the 5′ terminus,
suboptimal T7 promoter elements were often employed that lacked
one or more of the templated “G’s.” While this approach some-
times fulfilled the “rule of six,” it sacrificed efficient recognition of
the T7 promoter by T7 polymerase. In 2003, a reverse genetics
design to recover MeV encoded not only a 3′ HDV-Rbz but also an
autocatalytic hammerhead ribozyme (Hh-Rbz) immediately
upstream of the 5′ end of the viral cDNA; because the Hh-Rbz
self-cleaved immediately upstream of the first nucleotide of the viral
antigenomic 5′ end, an optimal T7 promoter element was able to
be encoded upstream of the Hh-Rbz [34]. This design facilitated
efficient transcription of the full-length vRNA by T7 polymerase
without compromising the rule of six. We have demonstrated that
such dual-ribozyme systems, under the control of an optimal T7
promoter, remarkably improve the rescue of paramyxoviruses [23].
Finally, successful rescue is highly dependent upon expression
of the viral replication complex (the N, P, and L proteins) at precise,
optimal ratios [35]. While the ratios of N, P, and L that can facilitate
rescue vary by viral species, we have observed that related members
of the same genus can generally be rescued using common ratios.
To achieve proper expression of the replication complex, the
respective viral N, P, and L proteins are typically encoded within
independent expression vectors and are co-transfected into cells at
the appropriate ratios at the time of rescue (Fig. 4). Table 1 pro-
vides a comprehensive summary of reported N:P:L ratios that have
been successfully employed for the de novo rescue of diverse para-
myxovirus species.

1.2 Design and Reporter Gene Integration

Manipulation of Diverse Paramyxoviridae reverse genetics systems have revealed
Recombinant that paramyxoviruses possess a remarkable flexibility to accommo-
Paramyxovirus date insertions of artificial sequence within diverse regions of the
Reverse Genetics viral genome; this has been demonstrated through the stable incor-
Systems poration of reporter genes in various regions of the viral genome
(Fig. 5). Indeed, several designs have been successfully employed to
generate reporter viruses. These include encoding reporter within
its own transcript by duplicating one of the noncoding regions
 Reverse Genetics Systems for Diverse Paramyxoviruses 21

Fig. 4 Typical plasmid ensemble for paramyxovirus reverse genetics. The

paramyxovirus antigenome and viral N, P, and L proteins are encoded within
expression vectors under the control of a T7 promoter element. The viral cDNA is
flanked on its 5′ end by a sequence-specific Hh-Rbz element that self-cleaves
immediately upstream of the 3′ Ldr; the viral cDNA is flanked on its 3′ end by an
HDV-Rbz that self-cleaves immediately downstream of the 5′ Tr sequence. A T7
terminator sequence is downstream of the HDV-Rbz

(NCR) between viral genes. This approach has been successful for
the insertion of reporter genes upstream of N, between N and P/V,
between P/V and M, and between RBP and L [26, 34, 46, 72,
73]. Alternatively, a P2A sequence may be used to encode reporter
genes within the same transcript as some viral genes, such as matrix
(M) or the receptor binding protein (RBP) [26, 24, 71]. Regardless
of approach, such gene insertions must not violate the rule of six
nor disrupt the phasing of the terminal bipartite promoter ele-
ments. If manipulation of a paramyxovirus genome violates the
rule of six, we recommend correcting this by adding additional
nucleotides immediately following the stop codon of a gene, as
this will have minimal, if any, effect on virus replication or kinetics.

Hh-Rbz Design
The Hh-Rbz encoded upstream of the 3′ leader sequence (3′Ldr)
must be designed specifically for each respective virus. This is
because a region of the Hh-Rbz is required to base-pair with the
extreme terminal nucleotide sequence of the 3′ Ldr (Fig. 3b). We
have compared numerous Hh-Rbz designs and have identified at
least two designs that are efficiently cleaved [24]. Base-pairing of
the Hh-Rbz with between five and eight nucleotides of the viral 3′
Ldr sequence is often sufficient for proper folding and cleavage;
however, due to sequence variation, designs that work for one
species may be suboptimal for another. Thus, we recommend
22 Griffin Haas and Benhur Lee

Table 1
Replication complex ratios successfully applied for the rescue of diverse paramyxovirus species

Accessory
plasmid
Family Genus Species N:P:L promoter Source(s)
Orthoparamyxovirinae Morbillivirus Measles virus 5.0:1.0: CAG [36]
(MeV) 5.0
3.0:3.0: T7 [26]
1.0
Myotis bat 3.0:3.0: T7 [13]
morbillivirus 1.0
(MBaMV)
Canine distemper 3.0:3.0: T7 [37, 38]
virus (CDV) 1.0
2.0:2.0: T7 [38]
1.0
Rinderpest virus 10.0: T7 [39]
(RPV) 10.0:
1.0
Peste-des-petits- 2.0:1.0: CAG [40]
ruminants 1.0
virus (PPRV) 1.5:1.0: CAG [41, 42]
1.0
[
20.0: T7 43]
20.0:
1.0
Jeilongvirus J-virus (JV) 2.0:1.0: T7 [44]
75.0
[
Narmovirus Tupaia virus 1.0:1.0: CAG 45]
(TupV) 1.0
Henipavirus Nipah virus 3.1:2.0: T7 [26, 46]
(NiV) 1.0
5.0:1.0: T7 [27]
2.0
3.1:2.0: CAG [47]
1.0
2.0:2.0: CAG [21]
1.0
Hendra virus 3.1:2.0: T7 [48]
(HeV) 1.0
5.0:1.0: T7 [27]
2.0
Cedar virus 3.1:2.0: CMV [49]
(CedV) 1.0
3.1:2.0: T7 Lee lab
1.0 unpublished
data
Respirovirus Human 8.0:8.0: T7 [50]
parainfluenza 1.0
virus type
1 (HPIV-1)

(continued)
 Reverse Genetics Systems for Diverse Paramyxoviruses 23

Table 1
(continued)

Accessory
plasmid
Family Genus Species N:P:L promoter Source(s)
Human 20.6: T7 [26]
parainfluenza 11.0:
virus type 1.0
3 (HPIV-3) 8.0:8.0: T7 [51]
1.0
Bovine 2.0:2.0: T7 [52]
parainfluenza 1.0
virus type
3 (BPIV3)
Sendai virus 20.6: T7 [26, 53]
(SeV) 11.0:
1.0
10.0: T7 [54]
10.0:
1.0
Avulavirinae Orthoavulavirus Newcastle disease 2.5:3.0: T7 [55]
virus (NDV) 1.0
(APMV-1) 2.0:1.0: T7 [26, 56–58]
1.0
2.0:1.0: CMV [20]
1.0
Paraavulavirus Avian 3.0:2.0: T7 [59]
paramyxovirus 1.0
type 3 (APMV-
3)
Metaavulavirus Avian 3.0:2.0: T7 [60]
paramyxovirus 1.0
type 2 (APMV-
2)
Avian 2.0:1.0: CAG [61]
paramyxovirus 1.0
type 6 (APMV-
6)
Avian 2.0:1.0: T7 [62]
paramyxovirus 1.0
type 7 (APMV-
7)
Avian 2.0:1.0: CAG [61]
paramyxovirus 1.0
type
10 (APMV-
10)

(continued)
24 Griffin Haas and Benhur Lee

Table 1
(continued)

Accessory
plasmid
Family Genus Species N:P:L promoter Source(s)
Rubulavirinae Orthorubulavirus Mumps virus 6.0:1.0: T7 [63–65]
(MuV) 4.0
6.0:1.0: T7 [66]
10.0
3.0:1.0: T7 [26]
2.0
Human 8.0:1.0: T7 [67]
parainfluenza 4.0
virus type 8.0:8.0: T7 [68]
2 (HPIV-2) 1.0
Parainfluenza 10.0: T7 [69]
virus type 1.0:
5 (PIV-5) 10.0
6.0:1.0: T7 [70]
7.5
Bat mumps virus 6.0:1.0: T7 [12]
(BatMuV) 4.6
Pararubulavirus Menangle virus 3.0:1.0: T7 Lee lab
(MenV) 2.0 unpublished
data
Sosuga virus 2.0:1.0: CAG [71]
(SosV) 4.0
Ratios of plasmids encoding N, P, and L were calculated from amounts of respective accessory plasmids employed for
rescue in the respective corresponding publication(s). Promoter type designates the promoter element driving transcrip-
tion of the accessory plasmids. T7 transcription by T7 polymerase, CMV transcription by RNA polymerase II, CAG
transcription by RNA polymerase II

pre-screening new Hh-Rbz designs for optimal folding; we tend to

use the RNAstructure software that is freely available via webserver
from the Matthews Lab at the University of Rochester (https://rna.
urmc.rochester.edu/) [74].

2 Materials

2.1 Transformation 1. Sequence-validated plasmid encoding the full-length para-

and Validation of Full- myxovirus cDNA (antigenome) flanked by a sequence-specific
Length Paramyxovirus 5′ Hh-Rbz and a 3′ HDV-Rbz, all under the control of a T7
cDNA and Accessory promoter element. A T7 terminator sequence should be down-
Rescue Plasmids stream of the HDV-Rbz.
2. Plasmids containing the respective viral N, P, and L genes
cloned into an expression vector (e.g., pTM1).
 Reverse Genetics Systems for Diverse Paramyxoviruses 25

Fig. 5 Summary of successful designs for the stable integration of a reporter

gene within paramyxovirus genomes. Reverse genetics systems have revealed
that paramyxoviruses can tolerate the insertion of artificial genetic material (e.g.,
GFP) by duplication of various noncoding regions (NCRs) or via the use of P2A
sequence

3. Plasmid encoding T7 polymerase that is codon-optimized for

mammalian expression (Addgene, plasmid #65974).
4. Chemically competent bacterial cells (Stellar Competent Cells,
Takara Bio; MAX Efficiency Stbl2 Competent Cells, Thermo
Fisher).
5. Water bath capable of maintaining a temperature of 42 °C.
6. Super optimal broth with catabolite repression (S.O.C.) for
bacterial competent cell recovery following plasmid
transformation.
7. LB agar plates supplemented with carbenicillin (100 μg/mL)
for growth and isolation of bacterial colonies following plasmid
transformation.
26 Griffin Haas and Benhur Lee

8. Lysogeny broth (LB) and Terrific broth (TB) supplemented

with carbenicillin (100 μg/mL) for growth of bacterial cells
following plasmid transformation.
9. Sterile glassware (e.g., Erlenmeyer flasks) for bacterial cultures.
10. Incubators for bacterial plates/cultures (capable of maintain-
ing a temperature of 30 °C or above).
11. Filter-sterilized 50% glycerol.
12. QIAprep Spin Miniprep Kit (Qiagen).
13. Gel electrophoresis-grade agarose.
14. SYBR Safe DNA gel stain or other DNA-visualizing dyes.
15. DNA standard.
16. Transilluminator or gel documentation system for visualization
and imaging of resolved DNA agarose gels.
17. DNA gel electrophoresis equipment.
18. Restriction enzyme(s) for diagnostic digest.
19. PureLink HiPure Plasmid Midiprep Kit (Invitrogen).

2.2 Cell Preparation 1. Sterile tissue culture materials (plasticware, serological pipettes,
etc.).
2. BSRT7/5 cells (4 × 105 cells per well of a six-well plate), or
HEK-293T cells (1 × 106 cells per well of a six-well plate).
3. Media for mammalian cell maintenance: 1× DMEM (Gibco)
supplemented with 10% fetal bovine serum (Gibco).

2.3 Preparation of 1. Opti-MEM (Gibco) or other reduced serum media.

Transfection 2. Lipofectamine LTX/PLUS transfection reagent (Thermo
Complexes and Virus Fisher) or other highly efficient transfection reagents (e.g.,
Rescue Mirus TransIT-LT1).

2.4 Virus Recovery 1. Screw-cap tubes with O-ring (Sarstedt).

and Amplification 2. Trypsin EDTA (2.5%) (Gibco).
3. Vero CCL81 cells or other permissive cell lines (one or more
80–90% confluent T150 or T175 flasks).
4. Chemical disinfectants approved for virus work per institu-
tional SOP (e.g., 70% ethanol, CaviCide, bleach, etc.) and
spray bottle or reservoir for disinfectants.
5. Personal protective equipment approved for virus work per
institutional SOP (e.g., impermeable gown, double gloves,
eye protection, mask, etc.).
 Reverse Genetics Systems for Diverse Paramyxoviruses 27

3 Methods

3.1 Transformation 1. Thaw chemically competent bacterial cells on ice for 10 min.
and Validation of Full- Transfer thawed competent cells to pre-labeled tubes (50 μL
Length Paramyxovirus aliquots, one tube per plasmid). Use Stellar competent cells for
cDNA and Accessory the smaller, accessory rescue plasmids (e.g., pTM1-N, pTM1-
Rescue Plasmids P, pTM1-L, and pCAGGs-T7CoOpt). Use Max-Efficiency
Stbl2 competent cells for propagation/amplification of the
larger, antigenomic viral cDNA plasmids.
2. On ice, add 5–10 ng of plasmid per respective tube of compe-
tent cells and gently flick to mix. Incubate tube on ice for
30 min.
3. Heat-shock competent cells at 42 °C for either 45 s (Stellar
competent cells) or 30 s (Max-Efficiency Stbl2 competent
cells). Immediately return heat-shocked tubes to ice. Incubate
on ice for 2 min.
4. Add S.O.C. to each tube (with no antibiotic) to a final volume
of 500 μL.
5. Incubate tubes at 30 °C with gentle shaking for 1 h.
6. Using a sterile cell spreader (or pipette tip), inoculate appropri-
ate antibiotic-containing LB agar plate with 50–100 μL of
transformants. Use sterile cell spreader or pipette tips to streak
out transformants to isolate single colonies. Incubate for
24–48 h between 30 and 33 °C (see Note 1).
7. Pick several colonies (using sterile pipette tips) to inoculate
5 mL of LB supplemented with appropriate antibiotic. Mark
the agar plate to indicate which colonies were picked. Incubate
outgrowth cultures with shaking at between 30 and 33 °C
overnight.
8. Save an aliquot of the outgrowth (250–500 μL) to make a
glycerol stock by adding equal volume of 50% glycerol. Glyc-
erol stocks must be frozen at -80 °C. Alternatively, wrap the
transformation plate in parafilm and store at 4 °C; validated,
marked colonies may be regrown and picked again.
9. Pellet and miniprep the bacterial outgrowths.
10. Use appropriate restriction enzyme(s) to perform a diagnostic
digest of the derived clones for each plasmid.
11. Resolve restriction digests by gel electrophoresis to validate
plasmid integrity. If the diagnostic digest is as expected,
sequence-verify each construct (see Note 2).
28 Griffin Haas and Benhur Lee

12. For validated clones, use a scraping from the glycerol stock
(or re-pick respective colonies from the LB agar plate) to
inoculate 50–100 mL of TB supplemented with the appropri-
ate antibiotic. Incubate in an appropriately sized Erlenmeyer
flask overnight with shaking, at between 30 and 33 °C.
13. When the outgrowth is turbid and sufficient for harvest, pellet
the bacteria via centrifugation at 4000× g for 10 min, and
process the pellet using a midiprep kit according to the manu-
facturer’s instructions.

3.2 Cell Preparation 1. BSRT7/5 cells and/or HEK-293T cells should be 80–90%
confluent on the day of transfection. Cells are maintained in
DMEM supplemented with 10% FBS.
2. Immediately prior to transfection, replace the media on all cells
with fresh, pre-warmed DMEM + 10% FBS.

3.3 Preparation of 1. Add 200 μL of Opti-MEM to each DNAse-/RNAse-free tube;

Transfection use one tube per rescue reaction (Fig. 6).
Complexes and Virus 2. Dilute viral cDNA plasmid and accessory rescue plasmids
Rescue (pTM1-N, pTM1-P, pTM1-L, and pCAGGs-T7CoOpt) at
the appropriate ratios in the Opti-MEM (see Table 1 and
Note 3).
3. Add the transfection reagent directly to the DNA:Opti-MEM
solution according to the manufacturer’s instructions. Pipette
the entire mixture up and down only twice, very slowly. Pipette
and handle the tubes gently to avoid disruption of transfection
complexes (see Note 4).
4. Incubate the transfection mix at room temperature for
20–30 min, or according to the manufacturer’s instructions
specific to the transfection reagent.
5. Add the transfection complexes to each well of the six-well
plate dropwise. Do not pipette the transfection mix up and
down, as this can disrupt the complexes.
6. Incubate the rescue plate at 37 °C (see Note 5).
7. Between 12 and 24 h post-transfection, replace the media on
each well with fresh, pre-warmed DMEM + 10%FBS.

3.4 Virus Recovery 1. Monitor the rescue plate daily for cytopathic effect. If using a
and Amplification reporter virus (e.g., GFP), check for reporter gene expression
and monitor for secondary infection events.
2. If the cells become over-confluent or if the media becomes
acidic, replace the media as needed to prolong cell viability.
3. Upon observation of cytopathic effect or secondary infection
events, prepare for generation of passage 1 (P1) stocks:
 Reverse Genetics Systems for Diverse Paramyxoviruses 29

Fig. 6 Overview of plasmid-mediated paramyxovirus rescue. (1) Plasmids encoding the viral antigenome/
cDNA, codon-optimized T7 polymerase, and the accessory viral N, P, and L proteins are transfected into cells.
(2) T7 polymerase is transcribed and translated by the cell. (3) T7 polymerase transcribes the accessory
viral N, P, and L proteins, as well as the viral antigenome. The Rbz elements encoded on the 5′ and 3′ ends of
the antigenomic vRNA are cleaved, resulting in viral antigenome without exogenous sequence on the termini.
(4) N encapsidates the viral antigenome, facilitating the helical arrangement of the vRNP and proper
positioning of the antigenomic bipartite promoter (highlighted in green). (5) The vRdRp associates with the
vRNP via P and recognizes the bipartite promoter element. (6) The vRdRp replicates the antigenome, making
viral genome. The vRNP properly forms the genomic bipartite promoter (highlighted in blue). (7) The genomic
bipartite promoter is recognized by the vRdRp, and viral genome provides a template for vRdRp-mediated
transcription of viral gene products. (8) The accumulation of viral gene products facilitates replication of the
viral genome. (9) The accumulation of viral proteins facilitates the assembly of virions and packaging of the
viral genome. Matrix protein facilitates the budding of virions

(a) Supernatant may be harvested from successful rescue

wells. Virus-containing rescue supernatant (P0) may be
frozen at -80 °C in the long term, or immediately used
for inoculation of Vero CCL81 or other permissive cells to
generate passage 1 (P1) stocks.
(b) As an alternative/complementary method, the rescue cells
may be gently trypsinized using Trypsin–EDTA (0.25%)
and overlaid directly onto Vero CCL81 cells. Overlaid
rescue cells will continue to produce virus that will infect
the Vero CCL81 cells, resulting in passage 1 (P1) stocks.
4. Monitor infected Vero CCL81 cells daily for widespread cyto-
pathic effect and/or reporter gene expression. Upon observation
of cytopathic effect and/or reporter gene expression, harvest
30 Griffin Haas and Benhur Lee

supernatant from Vero CCL81 cells and remove cell debris by

gentle centrifugation (2000× g for 5 min at 4 °C). Transfer
clarified P1 virus to screw-cap tubes and store at -80 °C.
5. Quantify P1 stocks by the desired method (plaque assay,
TCID50, etc.).

4 Notes

1. Lowering the growth temperature for bacteria reduces plasmid

copy number and may improve plasmid stability. This is partic-
ularly useful for large plasmids, as well as for plasmids that may
encode unstable/toxic sequences. Generally, we culture all
competent cells containing full-length viral cDNA plasmids at
30°C to minimize toxicity or spontaneous recombination
events. The accessory plasmids (T7, N, P, and L) can often be
grown above 30 °C.
2. Sequence-validation of the paramyxovirus cDNA plasmid is
extremely important, as even a single-nucleotide deletion or
insertion anywhere throughout the viral sequence can abrogate
rescue. Diagnostic digest alone is not sensitive enough to
detect such issues. If the cDNA sequence does not fulfill the
rule of six (e.g., if a single-nucleotide deletion spontaneously
occurs in a noncoding region, rendering the cDNA sequence
length indivisible by 6), then rescue will not be successful.
Validation of all plasmid sequences prior to rescue can save
the lab reagent, time, and effort.
3. The ratio of viral cDNA plasmid and accessory rescue plasmids
should be optimized prior to the rescue attempt. This can be
accomplished using monocistronic minireplicon/minigenome
systems. However, closely related paramyxovirus species can
often be rescued using common plasmid ratios. Thus, we rec-
ommend first attempting rescue by employing previously vali-
dated ratios specific to members of a respective genus, as
summarized in Table 1.
4. Excessive mixing of the transfection mixture can be detrimental
to transfection complex formation. We recommend pipetting
up and down the entire volume very gently, no more than
twice. Mixing should occur immediately following addition of
the transfection reagent (within 60 s). Once the transfection
complexes have formed, further mixing should be avoided.
Care should be taken to handle the tubes gently. We have
optimized the amounts of Lipofectamine LTX and PLUS
reagent required for numerous members of Paramyxoviridae
[23].
 Reverse Genetics Systems for Diverse Paramyxoviruses 31

5. If a specific virus rescues slowly (and concomitantly if the over-

crowding of cells is a concern), rescue cells may be grown at a
slightly lower temperature, and/or in DMEM supplemented
with 2% FBS (instead of 10% FBS). These conditions will slow
cell proliferation and may prolong cell viability, providing addi-
tional time for rescue. If required, transfected rescue cells may
be gently trypsinized and transferred into a new vessel for
co-culture with a permissive cell line.

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 Chapter 3

Rescue of Recombinant Newcastle Disease Virus

Expressing Heterologous Genes
Arantza Cobela-Garcı́a , Ignacio Mena , and Adolfo Garcı́a-Sastre

Abstract
Reverse genetics allows for the generation of recombinant infectious viruses from viral sequences or
complete viral genomes cloned into plasmids. Using reverse genetics, it is then possible to introduce
changes in the genome of infectious viruses for multiple applications.
Newcastle disease virus (NDV) is a non-segmented, negative-sense RNA virus that has been amenable to
manipulation by reverse genetics for more than two decades. Since then, recombinant NDVs have been
extensively used as viral vectors to express heterologous proteins. We describe the key steps required to
design and introduce an additional transcription unit in the genome of the Newcastle disease virus for the
efficient expression of a heterologous gene.

Key words Reverse genetics, Mononegavirales, Recombinant Newcastle disease virus, Virus-vectored
vaccine, Codon optimization

1 Introduction

Newcastle disease virus (NDV, order Mononegavirales, family Para-

myxoviridae [1]) is an avian pathogen that has been extensively used
as a model for the study of the replication and transcription of the
non-segmented, negative-stranded, RNA viruses. The successful
rescue of negative-stranded RNA viruses by reverse genetics was
complicated, mainly due to the need to introduce in susceptible
cells not only the complete viral genome but also the protein
components of the viral ribonucleoprotein (proteins N, P, and L).
The first member of the order Mononegavirales to be rescued was
the rabies virus in 1994 [2], and many other members followed in
the next few years [3]. With some variations (reviewed in [4]), the
rescue of non-segmented, negative-sense, RNA viruses nowadays
follows the same general approach described in 1994 (Fig. 1a).
Briefly, suitable cells are transfected with a plasmid that contains
the full-length cDNA of the viral genome, under the control of the

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

37
38 Arantza Cobela-Garcı́a et al.

A) vTF7-3

RV full length cDNA (+ sense) Rescued

recombinant RV
T7 RNA
N polymerase

P helper
plasmids

B) unique
restriction site T7 terminator
Hepatitis delta
SacII Ribozyme

NP P M F HN L

Fig. 1 (a) Basic steps followed for the rescue of a recombinant rabies virus (RV) in 1994. A similar approach is
still used nowadays. See text for details. (Not to scale). (b) Main features of the pNDV rescue plasmid. (Not to
scale)

promoter of a DNA-dependent RNA polymerase. By far, the most

used is the promoter of the RNA polymerase from the phage T7,
oriented to transcribe the antigenome (positive-sense) strand. It is
important that the transcribed RNA has the correct 5′ and 3′ ends
of the viral genome, without additional nucleotides from the plas-
mid. This is achieved by the correct positioning of the promoter
and transcription termination signals and by the use of self-cleaving
ribozyme sequences (Fig. 1b). In addition, members of the Para-
myxoviridae family (such as NDV) must comply with the “rule of
six”: The nucleotide size of the genome must be an exact multiple
of 6 for successful rescue [5].
Using the T7 promoter requires the co-expression of T7 RNA
polymerase in the cells, which is usually achieved by infection with a
recombinant poxvirus (such as MVA-T7), but expression plasmids,
or cell lines constitutively expressing T7 RNA polymerase, can also
be used. In addition to the plasmid containing the full-length
genomic cDNA, cells must be co-transfected with three helper
plasmids expressing the viral proteins N, P, and L. The helper
plasmids might express the viral proteins under the control of the
T7 promoter or a strong RNA polymerase II promoter. In the case
of NDV, the rescued virus is usually amplified by inoculating the
supernatant of the transfected cells into the allantoic cavity of
embryonated chicken eggs.
 Recombinant NDV Expressing a Foreign Gene 39

GE+IG+GS GE+IG+GS

Gene 1 Gene 2 Gene 3

Fig. 2 Schematic representation of functional paramyxovirus transcription units. Each open reading frame
(white boxes) is flanked by conserved non-translated sequences (gray boxes): GE gene end, IG intergenic, GS
gene start

Since the first descriptions of the rescue of infectious NDV by

reverse genetics in 1999 [6, 7], many groups have rescued recom-
binant NDVs with genetic modifications. A commonly used modi-
fication is the insertion of an additional transcription unit encoding
a foreign gene. This approach takes advantage of the well-studied
transcription strategy of the Mononegavirales. In the viral genome,
each open reading frame is flanked by regulatory sequences called
gene start (GS), gene end (GE), and intergenic (IG) regions, which
are recognized by the viral polymerase to transcribe the messenger
RNAs (Fig. 2). Adding these regulatory sequences to a coding
sequence inserted in the genome creates a functional additional
transcription unit that will be expressed by the viral polymerase.
The additional transcription unit can be inserted at any intergenic
position of the viral genome, and that position will drastically
influence the expression levels. Mononegavirales genes are
expressed in a transcriptional gradient, with the gene positioned
in the 3′ side (the N gene) expressed at high levels and the gene
positioned at the 5′ side (the L gene) expressed at the lowest level.
We and others [8, 9] have described that insertion at the intergenic
region between the P and M genes results in an optimal combina-
tion of viral growth, protein expression, and immunogenicity.
The two main general applications of recombinant NDVs
expressing heterologous genes are as viral-vectored vaccines [10]
and to improve the natural oncolytic properties of NDV [11]. It is
important to mention that for both applications, there are currently
candidates undergoing advanced phases of clinical trials. As a vac-
cine vector, recombinant NDVs can be used in avian and in mam-
malian hosts. Since Newcastle disease is a very important disease in
poultry, recombinant NDVs act as dual vaccines in birds, protecting
simultaneously against virulent NDV and against a second avian
pathogen. In fact, recombinant NDVs expressing influenza hemag-
glutinin of the H5 subtype are being used in several countries as
dual vaccines against NDV and highly pathogenic avian influenza
(HPAI). In mammals, NDV-vectored vaccines have several advan-
tageous features: since NDV is an avian pathogen, there is no
preexisting immunity against the vector. In addition, NDV cannot
block the innate immune response in mammalian cells as efficiently
as it does in avian cells, which results in both additional attenuation
and stronger immune responses.
40 Arantza Cobela-Garcı́a et al.

Here, we describe the steps to follow to design and insert an

additional transcription unit in the genome of a lentogenic
(non-virulent) NDV in order to efficiently express an immunogenic
protein. Using as example the H5 of a HPAI from the outbreak that
affected several states of the USA in 2014, we introduce modifica-
tions to increase safety, expression levels, and immunogenicity, as
described in [12]. The approach is very similar to the one recently
used by Sun and colleagues to obtain a rNDV expressing the spike
protein of SARS-CoV2 that is currently undergoing clinical trials
both as inactivated vaccine and as live-attenuated vaccine against
Covid-19 [13].

2 Materials

When not indicated otherwise, follow the conditions recom-

mended by the manufacturers.

2.1 Design and 1. Software to perform simple sequence analysis such us translat-
Cloning of the ing nucleotide sequences into amino acid sequences or identi-
Additional fying restriction sites. We find BioEdit (https://bioedit.
Transcription Unit software.informer.com/7.2/) and SnapGene Viewer
(https://www.snapgene.com/snapgene-viewer/) very conve-
nient and easy to use, but there are many other options.
2. Rescue plasmid that contains the full-length cDNA of
the genome of the NDV vaccine strain LaSota, flanked by the
sequence of the promoter of the T7 RNA polymerase on the 5′
side (to transcribe the positive-sense antigenome) and by the
sequence of the ribozyme of the hepatitis delta virus and the T7
terminator on the 3′ side. The cDNA contains a unique restric-
tion site SacII engineered between the stop codon and the gene
end (GE) regulatory sequence of the P gene (Fig. 1b).
3. Reagents for cloning: restriction enzyme SacII (NEB, cat.
#R0157S) to linearize the vector. CloneAmp HiFi PCR Premix
(TaKaRa, cat. #639298). Specific primers to amplify the insert
by PCR and to confirm the insert by sequencing. Equipment
and reagents to run agarose gel electrophoresis. In-Fusion
Snap Assembly Master Mix (TaKaRa, cat. #638948). Stellar
competent bacteria and provided SOC medium (Takara Bio,
cat. #636766). Luria–Bertani (LB) agar plates with ampicillin
(100 μg/mL). Kits for DNA purification from agarose gel
bands (we use Omega Bio-Tek cat.#D2500-01) and for plas-
mid preparation (we use Omega Bio-Tek cat.#D6942-01 and
Invitrogen cat.#K210005).
4. Equipment for growing bacteria at 37 °C (incubator and
shaker).
 Recombinant NDV Expressing a Foreign Gene 41

2.2 Rescue of the Rescue of lentogenic (avirulent) NDV, such as the vaccine strain
Recombinant NDV LaSota, may be performed in Biosafety Level 2 (BSL-2) conditions.
Expressing a Codon- Recombinant viruses with enhanced infectivity or pathogenicity
Optimized Influenza and virulent (mesogenic or velogenic) NDVs will require additional
Hemagglutinin biosafety and biocontainment measures.
All the tissue culture and egg inoculation work must be per-
formed in sterile conditions.
1. HEp2 cells.
2. CO2 incubator and biosafety cabinet.
3. Complete DMEM: Dulbecco’s modified Eagle medium
(DMEM) supplemented with 5% fetal bovine serum (FBS)
and antibiotics (penicillin (100 UI/mL)/streptomycin
(100 mg/mL)).
4. Phosphate-buffered saline (PBS) and PBS supplemented with
0.3% bovine serum albumin and antibiotics (PBS + BSA + P/
S).
5. Recombinant modified vaccinia virus Ankara expressing the T7
RNA polymerase (MVA-T7).
6. Opti-MEM, reduced serum medium.
7. Set of rescue plasmids: pNDV-H5 (containing the full-length
cDNA of the NDV genome with an additional transcription
unit encoding a codon-optimized H5 protein. Generated in
Subheading 3.1). Helper plasmids pTM1-N, pTM1-P, and
pTM1-L, expressing the viral proteins N, P, and L under the
control of the T7 promoter.
8. Mirus TransIT-LT1 Transfection Reagent.
9. Pathogen-free, 8–10-day-old, embryonated chicken eggs
(Charles River).
10. 0.5 mL syringes with 25G × 5/8″ needles (insulin syringes).
11. Turkey or chicken whole blood, diluted in PBS to 0.5% red
blood cells, to perform hemagglutination assay (HA).

3 Methods

3.1 In Silico Design 1. Download the coding sequence of the protein to be expressed
and Insertion into the by the recombinant NDV. As an example, we will use the
NDV Genome of an hemagglutinin (HA) from A/chicken/Iowa/04-20/2015
Additional (H5N2) (GenBank accession number: KR492974.3). Figure 3
Transcription Unit summarizes the modifications made to the original sequence.
Encoding a Chimeric 2. In the amino acid sequence, identify the multibasic cleavage
Influenza site (PQRERRRKR/GLF) and replace it by the low patho-
Hemagglutinin genic cleavage site from the same lineage (PQRETR/GLF)
(see Note 1).
42 Arantza Cobela-Garcı́a et al.

Addition of NDV
regulatory signals and
Kozak sequence (K)
codon optimization
GE+IG+GS

K HPAI H5 ectodomain L TM
SacII SacII
Flanking sequences Highly pathogenic cleavage
for cloning site replaced by low Transmembrane and cytoplasmic
pathogenic cleavage site domains (TM) of the HA replaced
by those of NDV F protein,
separated by a flexible linker (L)

Fig. 3 Modifications to the wild-type sequence of an avian influenza hemagglutinin gene in order to increase
safety, expression, and immunogenicity of the heterologous gene

3. Using in silico analysis (see Note 2), identify the transmem-

brane and cytoplasmic (TM + cyto) regions of the HA and
replace them by the TM + cyto from the F protein of the LaSota
strain of NDV. Place a flexible linker GGGGS between the
ectodomain and the TM + cyto. This is the amino acid
sequence that we want to codon-optimize.
4. Obtain the codon-optimized nucleotide sequence for the
desired species (see Note 3).
5. Before the initiation codon (ATG), add the noncoding
sequences required to obtain a functional NDV
transcription unit: gene end (GE: TTAGAAAAAA), intergenic
(IG: T), and gene start (GS: ACGGGTAGAA) as well as a
Kozak sequence (CCGCCACC) for optimal translation. After
the stop codon, add any number of nucleotides required to
make the size of the insert a multiple of 6.
6. Add the flanking sequences required for cloning: SacII restric-
tion sites and 15 nucleotides compatible with InFusion cloning
into the SacII-digested plasmid (these 15 nucleotides are typi-
cally added from the primer during the PCR amplification, but
having them already in the template will make the annealing
more specific).
7. After completing the design and before ordering the sequence,
it is important to double-check that the open reading frame
does not contain stretches of 5xA (or longer), since the viral
polymerase will interpret them as early termination signals
(gene end) and that the complete size of the insert is a multiple
of 6 (rule of six). If using restriction digestion to ligate the
insert, also check that there are not undesired restriction sites
(in our example, SacII).
 Recombinant NDV Expressing a Foreign Gene 43

cells
+
supernatant
MVA-T7
pNDV-H5
infection
pTM1-N 3 days
transfection Harvest
pTM1-P HA assay
characterization
pTM1-L

day 1 day 2 day 5

Fig. 4 Rescue of a recombinant NDV expressing influenza H5. On day 1, cells are infected with a recombinant
poxvirus expressing T7 RNA polymerase and transfected with the rescue plasmid containing the full-length
cDNA and the three helper plasmids (N, P, and L) all under the control of the T7 promoter. On day
2, supernatant and cells are inoculated into embryonated chicken eggs. On day 5, the allantoic fluid is
collected, and hemagglutination-positive samples are further analyzed

8. Order the synthesis of the designed sequence from the provider

of your choice (we usually order our DNA synthesis from
Azenta).
9. Insert the synthesized sequence into the SacII-digested pNDV
plasmid by InFusion or restriction–ligation, following the con-
ditions recommended by the manufacturer (see Note 4). After
screening for colonies containing the correct plasmid, always
confirm the complete sequence of the insert. Use sequencing
primers that anneal about 200–300 nt upstream (forward
primer) and downstream (reverse primer) of the SacII site.

3.2 Use the In this section, we describe a basic protocol to rescue a recombinant
Sequence-Confirmed NDV from plasmids and amplification in embryonated chicken
Rescue Plasmid pNDV- eggs (Fig. 4). Detailed protocols can also be found in [14, 15].
H5 to Rescue a rNDV 1. The day before infection/transfection, plate HEp-2 cells in a
Expressing a Chimeric, 6-well plate. Cells should be around 80–90% confluent at the
Codon-Optimized H5 time of transfection.
2. Wash the cells with sterile PBS and infect with MVA-T7 diluted
in 0.5 mL of PBS + BSA + P/S, at a multiplicity of infection of
5 PFU/cell. Incubate 1 h at 37 °C, rocking the plate every
10–15 min. During this time, prepare the transfection mix.
3. Prepare the transfection mix: for each well in a sterile Eppen-
dorf tube, add 250 μL of Opti-MEM + 1 μg of pNDV-H5
rescue plasmid + 0.5 μg of pTM1-N + 0.25 μg of pTM1-
P + 0.25 μg of pTM1-L + 6 μL of TransIT-LT1 Transfection
Reagent. Mix well and incubate at room temperature for about
20 min (until the end of the 1 h MVA-T7 infection).
44 Arantza Cobela-Garcı́a et al.

4. Aspirate the virus inoculum, wash the cell monolayer with

complete DMEM, and add 1 mL of complete DMEM. Add
the transfection mix dropwise and mix by gently rocking the
plate.
5. Incubate the infected/transfected cells for 24 h at 37 °C in a
CO2 incubator.
The following day, transfected cells and supernatant are
inoculated in the allantoic cavity of 8–10-day-old embryonated
chicken eggs.
6. Transilluminating the egg from the top, identify the limit
between the air chamber and the allantoic cavity (this is called
to “candle” the egg). With a piercing instrument, carefully
make a dent in the eggshell, right above that limit, to facilitate
the insertion of the needle.
7. Resuspend the cells by vigorous pipetting. Using an insulin
syringe, inoculate 200–300 μL per egg of cells+supernatant
into the allantoic cavity and seal the hole with warm paraffin
or nail polish.
8. Incubate the eggs for 2–3 days at 37 °C and then transfer them
to 4 °C for at least 3 h.
9. In sterile conditions, collect the allantoic fluid (8–10 mL is
typically obtained) and identify successfully infected eggs by
hemagglutination assay.
10. HA-positive rescued fluids must be further analyzed to confirm
the presence of viral RNA with the desired inserted sequence
(by RT-PCR) and to confirm the expression of the heterolo-
gous protein (by immunofluorescence, Western blot, flow
cytometry, and Coomassie staining, depending on the reagents
and techniques available).

4 Notes

1. Even though the recombinant NDV will use preferentially its

own glycoproteins (F and HN) for cell entry, it has been
described that rNDVs expressing an additional viral entry pro-
tein may use it as an alternative way to enter cells [16]. Chang-
ing the cleavage site to a low pathogenic cleavage site eliminates
the risk of changing the tissue tropism of the rNDV-H5.
A convenient table with the most common multibasic
cleavage sites and the corresponding low pathogenic cleavage
sites can be found in the website from the OIE: https://www.
offlu.org/wp-content/uploads/2022/01/Influenza-A-Cleav
age-Sites-Final-04-01-2022.pdf.
 Recombinant NDV Expressing a Foreign Gene 45

2. To identify the transmembrane region in amino acid sequences,

we use the following site: https://services.healthtech.dtu.dk/
service.php?TMHMM-2.0).
3. Several biotech companies offer free codon optimization tools
in their websites, or as a service along with DNA synthesis.
Each one uses different algorithms and the resulting optimized
sequences can be surprisingly different. Since it is impossible to
determine easily which version is really the “optimal,” we rou-
tinely use a very simple codon optimization tool online: http://
www.encorbio.com/protocols/Codon.htm, with good
results.
4. The codon-optimized sequence can be inserted into the SacII-
digested pNDV plasmid by classical SacII digestion followed by
ligation or by InFusion. We prefer the second option for several
reasons: in our hands, InFusion is very efficient—if done cor-
rectly, most colonies will contain the desired plasmid. By liga-
tion, the fragment can be inserted in any orientation, while
InFusion will always result in the correct orientation. Using
InFusion, it does not matter if the insert contains additional
SacII sites.

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 Chapter 4

Use of Reverse Genetics for the Generation of Recombinant

Influenza Viruses Carrying Nanoluciferase
C. Joaquin Caceres, L. Claire Gay, Flavio Cargnin Faccin,
and Daniel R. Pérez

Abstract
Influenza A (FLUAV) and influenza B (FLUBV) viruses are human and/or animal pathogens widely
studied due to their importance to public health and animal production. Both FLUAV and FLUBV possess
a genome composed of eight viral gene segments. For reverse genetics of influenza viruses, transcription of
the mRNA for the viral proteins is typically done from a plasmid encoding an RNA polymerase II (pol II)
promoter element upstream of cloned viral cDNA and expressed like host mRNA. On the other side, the
synthesis of the negative-sense, single-stranded, uncapped vRNAs can be accomplished by the host’s RNA
polymerase I (pol I). The reverse genetics for influenza has allowed the manipulation of influenza genomes
incorporating heterogeneous sequences into different segments of the influenza genome, such as reporter
genes. In this chapter, we outline the protocol from the generation of reverse genetic plasmid that can be
applied for the cloning of any of the segments of FLUAV or FLUBV. Furthermore, we describe a protocol
for generating FLUAV or FLUBV recombinant viruses carrying Nanoluciferase (NLuc) in the PB1 gene
using reverse genetics. Finally, we delineate a microneutralization protocol using FLUAV-NLuc or
FLUBV-NLuc viruses optimized for the use of antibodies from different sources (mice, ferrets, avian,
etc.), which provides a more sensitive, reliable, and avidity-independent method to assess the presence of
neutralizing antibodies against FLUAV or FLUBV.

Key words Influenza, Reverse genetics, NanoLuc, Microneutralization assays

1 Introduction

Influenza viruses are classified within the family Orthomyxoviridae

as segmented, single-stranded, negative-sense RNA viruses
[1]. Influenza A (FLUAV) and influenza B (FLUBV) viruses are
human and/or animal pathogens widely studied due to their
importance to public health and animal production. FLUAV can
infect a broad host range of avian and mammalian species, while
FLUBV is primarily a human pathogen [2, 3]. Both FLUAV and
FLUBV possess a genome composed of eight viral gene segments
that encode for at least 12 viral proteins [4–6]. Influenza viruses

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_4,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

47
48 C. Joaquin Caceres et al.

contain two surface glycoproteins, the hemagglutinin (HA) and the

neuraminidase (NA) [7], which are the main antigenic determi-
nants that elicit neutralizing antibodies and play a key role in
infection and propagation within the host [8–11]. FLUAV are
classified by their HA and NA into subtype combinations in
which currently, 18 HA and 11 NA subtypes have been described
[12–14]. Although FLUAV and FLUBV have similar genome
structures, they diverge in the lengths of proteins and noncoding
regions, the presence of accessory proteins, and antigenic proper-
ties [3, 6, 15, 16]. Despite the structural and molecular differences
between FLUAV and FLUBV, both can be generated by reverse
genetics through similar approaches [17, 18].
For reverse genetics of influenza viruses, transcription of the
mRNA for the viral proteins is typically done from a plasmid
encoding an RNA polymerase II (pol II) promoter element
upstream of cloned viral cDNA and expressed in a manner similar
to host mRNA [19]. Synthesis of the negative-sense, single-
stranded, uncapped vRNAs can be accomplished by the host’s
RNA polymerase I (pol I) [19]. The RNA pol I complex produces
uncapped ribosomal RNA. The transcription mediated by pol I is
terminated with the (murine) RNA polymerase I terminator (T-1),
to produce vRNAs with defined start and stop sites, respectively.
Bidirectional vectors have been made that contain pol II and pol I
promoters in opposite directions to drive the expression of both
mRNA and vRNA species from the same plasmid, thus consolidat-
ing the viral rescue system into eight plasmids (Fig. 1) [20, 21]. The
development of reverse genetics for FLUAV and FLUBV has also
allowed the manipulation of influenza genomes for the incorpora-
tion of heterogeneous sequences into different segments of the
influenza genome, such as reporter genes, allowing the assessment
of neutralizing antibodies using luciferase as a readout [22, 23]. In
this chapter, we describe the use of reverse genetics for FLUAV and
FLUBV using the eight-plasmid system with bidirectional promo-
ters as originally described by Hoffman et al. [20]. Furthermore,
this chapter describes the development of influenza viruses carrying
the NanoLuc (NLuc) reporter in the PB1 segment of FLUAV and
FLUBV (Fig. 1).
We also provided a detailed protocol for using these viruses to
assess the levels of neutralizing antibodies from serum samples that
can be applied to multiple samples-origin (mice, swine, avian, etc.)
as previously described [24, 25]. It is important to note that the
protocol described is independent of the avidity of the HA for red
blood cells as it is commonly used in microneutralization assays,
providing a more reliable, avidity-independent way of determina-
tion of neutralizing antibodies.
 Reverse Genetics for Influenza NanoLuc Viruses 49

Fig. 1 Schematic representation of the rescue of recombinant influenza viruses expressing NLuc using reverse
genetics. Eight plasmids (one per influenza segment) are transfected in a co-culture of 293T/MDCK cells.
Transfection supernatants are collected and used to amplify influenza in embryonated chicken eggs or MDCK
cells, generating recombinant influenza viruses expressing NLuc. Influenza-NLuc viruses can be subsequently
used to evaluate the presence of neutralizing antibodies. The NLuc activity is used as a readout of the viral
replication and is negatively correlated to the presence of neutralizing antibodies in the sample evaluated.
(Figure created with BioRender.com)

2 Materials

2.1 Influenza A and B 1. QIAamp Viral RNA kit (QIAGEN).

Viral RNA (vRNA) 2. 200 proof ethanol (molecular grade).
Extraction
3. 1.5 mL microcentrifuge tubes.
4. Nuclease-free water (i.e., QIAGEN).
5. RNaseZap or other solution to remove RNases (Invitrogen,
ThermoFisher Scientific).
6. Pipets to dispense from 1 to 1000 μL and corresponding filter
pipette tips.
7. Laboratory equipment: pipettes and tips of appropriate sizes,
vortex, microcentrifuge with a rotor capable of reaching up to
12,000×g, spectrophotometer (NanoDrop or similar).
50 C. Joaquin Caceres et al.

2.2 Amplification of 1. Influenza A primer set for cloning the eight gene segments into
Influenza A and B Gene pDP2002 bidirectional reverse genetic plasmid vector
Segments from vRNA (Table 1) (see Note 1).
2. Influenza B primer set for cloning the eight gene segments into
pDP2002 bidirectional reverse genetic plasmid vector
(Table 1).
3. Superscript III One-Step RT-PCR System with Platinum Taq
DNA Polymerase (Thermo Fisher Scientific).
4. Nuclease-free water (i.e., QIAGEN).
5. Agarose, LE analytical grade (i.e., Promega).
6. SYBR® Safe gel staining (Invitrogen).
7. 1 kb ladder (Thermo Scientific).
8. QIAquick gel extraction kit (QIAGEN).
9. 1.5 mL microcentrifuge tubes (i.e., USA Scientific).
10. 250 μL PCR tubes (i.e., USA Scientific).
11. Ice or cooling tube racks.
12. Laboratory equipment: pipettes and tips of appropriate sizes,
vortex, microcentrifuge with a rotor capable of reaching up to
12,000×g, thermocycler, DNA gel electrophoresis system,
transilluminator, water bath or heating block capable of reach-
ing 50 °C, scale capable of determining weight in milligrams,
NanoDrop (or similar spectrophotometer).

2.3 Bidirectional Plasmid pDP2002 is a derivative of pHW2000 [20]. A 444 nt

Reverse Genetic spacer sequence was incorporated between two BsmBI sites to
pDP2002 Plasmid monitor and more efficiently obtain a double-digested vector for
Vector the purpose of cloning full-length influenza cDNAs. The pDP2002
plasmid contains two transcription units in opposite orientations.
The first unit contains the human polymerase I promoter (hpol I)
and the murine polymerase I transcription terminator (tI) for the
synthesis of vRNAs. The second unit drives viral mRNA transcrip-
tion from the polymerase II-driven cytomegalovirus promoter
(pCMV) and the bovine growth hormone polyadenylation signal
(aBGH). Influenza viral cDNAs are cloned into the reverse genetic
vectors using restriction sites engineered at the 5′ and 3′ ends of
each segment in a manner compatible with the reverse genetic
vector. Plasmids are prepared using commercially available plasmid
preparation kits, following the manufacturer’s recommendations,
and are best stored at -20 °C at a concentration of 0.25–1.0 mg/
mL. We recommend that plasmid stocks be diluted into working
stocks at a concentration of 100 ng/μL before transfection to
minimize pipetting errors and standardize pipetting volumes for
each plasmid.
Table 1
Primers used to clone FLUAV and FLUBV into pDP2002

Forward Reverse
Gene primer primer
FLUAV
PB2 Bm-PB2 1F TATTCGTCTCAGGGAGCGAAAGCAGGTC Bm-PB2 ATATCGTCTCGTATTAGTAGAAACAAGGTCG
2341R TTT
PB1 Bm-PB1 1F TTATTCGTCTCAGGGAGCGAAAGCAGGCA Bm-PB1 ATATCGTCTCGTATTAGTAGAAACAAGGCA
2341R TTT
PA Bm-PA-1F TATTCGTCTCAGGGAGCGAAAGCAGGTAC Bm-PA 2233R ATATCGTCTCGTATTAGTAGAAACAAGGTAC
TT
HA Bm-HA 1F TATTCGTCTCAGGGAGCAAAAGCAGGGG Bm-NS 890R ATATCGTCTCGTATTAGTAGAAACAAGGGTG
TTTT
NP Bm-NP-1F TATTCGTCTCAGGGAGCAAAAGCAGGGTA Bm-NP 1565R ATATCGTCTCGTATTAGTAGAAACAAGGGTA
TTTTT
NA Bm-NA 1F TATTCGTCTCAGGGAGCAAAAGCAGGAGT Bm-NA 1413R ATATCGTCTCGTATTAGTAGAAACAAGGAG
TTTTTT
M Bm-M-1F TATTCGTCTCAGGGAGCAAAAGCAGGTAG Bm-M 1027R ATATCGTCTCGTATTAGTAGAAACAAGGTAG
TTTTT
NS Bm-NS 1F TATTCGTCTCAGGGAGCAAAAGCAGGGTG Bm-NS 890R ATATCGTCTCGTATTAGTAGAAACAAGGGTG
TTTT
FLUBV
PB2 Bm-PB2b 1F TATTCGTCTCAGGGAGCAGAAGCGGAGCG Bm-PB2b ATATCGTCTCGTATTAGTAGAAACACGAGCA
TTTTCAAGATG 2396R TT
Reverse Genetics for Influenza NanoLuc Viruses

PB1 Bm-PB1b 1F TATTCGTCTCAGGGAGCAGAAGCGGAGCC Bm-PB1b ATATCGTCTCGTATTAGTAGAAACACGAGCC

TTTAAGATG 2369R TT

(continued)
51
 52
Table 1
(continued)

Forward Reverse
Gene primer primer
PA Bm-PAb-1F TATTCGTCTCAGGGAGCAGAAGCGGTGCG Bm-PAb ATATCGTCTCGTATTAGTAGAAACACGTGCA
TTTGA 2308R TT
HA MDV-B 5′ TATTCGTCTCAGGGAGCAGAAGCAGAGCA MDV-B 3′ ATATCGTCTCGTATTAGTAGTAACAAGAGCA
Bm-HA TTTTCTAATATC Bm-HA TTTTTC
NP MDV-B 5′ TATTCGTCTCAGGGAGCAGAAGCACAGCA MDV-B 5′ ATATCGTCTCGTATTAGTAGAAACAACAGCA
C. Joaquin Caceres et al.

Bm-NP TTTTCTTGTG Bm-NP TTTTTTAC

NA Bm-NAb 1F TATTCGTCTCAGGGAGCAGAAGCAGAGCA Bm-NAb ATATCGTCTCGTATTAGTAGTAACAAGAGCA
1557R TTTT
M MDV-B 5′ TATTCGTCTCAGGGAGCAGAAGCACGCAC MDV-B 3′ ATATCGTCTCGTATTAGTAGAAACAACGCAC
Bm-M TTTCTTAAAATG Bm-M TTTTTCCAG
NS MDV-B 5′ TATTCGTCTCAGGGAGCAGAAGCAGAGGA MDV-B 3′ ATATCGTCTCGTATTAGTAGTAACAAGAGGA
Bm-NS TTTGTTTAGTC Bm-HA TTTTTAT
Primer sequences used to amplify the eight gene segments from FLUAV and FLUBV for cloning into pDP2002. In bold is the primer sequence that encodes the influenza
nucleotides on the 5′ and 3′ end of the cDNA. Upstream of the bold region is the sequence for BsmBI recognition
 Reverse Genetics for Influenza NanoLuc Viruses 53

2.4 Cloning of 1. BsmBI-v2 restriction endonuclease (New England Biolabs).

Influenza A and B 2. BsaI-HF v2 restriction endonuclease (New England Biolabs).
Virus Gene Segments
3. AarI restriction endonuclease (Thermo Fisher Scientific).
into pDP2002
4. Antarctic phosphatase (New England Biolabs).
5. MinElute PCR Purification Kit (QIAGEN).
6. Quick ligation kit (New England Biolabs).
7. TOP10 chemically competent cells (Thermo Fisher Scientific).
8. Super Optimal broth with Catabolite repression (SOC)
medium (Thermo Fisher Scientific).
9. BD Difco Luria–Bertani (LB) broth supplemented with 50 μg/
mL of ampicillin solution (Difco Luria–Bertani broth; Fisher
Scientific).
10. LB agar plates supplemented with 50 μg/mL of ampicillin
solution (BD Difco Luria–Bertani agar; Fisher Scientific).
11. Agarose, LE analytical grade (Promega).
12. SYBR® Safe gel staining (Invitrogen).
13. QIAquick gel extraction kit (QIAGEN).
14. GoTaq® Green Master Mix (Promega).
15. QIAprep Spin Mini kit (Qiagen).
16. HiSpeed Plasmid Maxi kit (Qiagen).
17. 1.5 or 2 mL microcentrifuge tubes (USA Scientific).
18. 250 μL PCR tubes (USA Scientific).
19. Ice or cooling tube racks.
20. Laboratory equipment: pipettes and tips of appropriate sizes,
vortex, microcentrifuge with a rotor capable of reaching up to
12,000×g, water bath or heating block able to reach 42 °C,
thermocycler, DNA gel electrophoresis system, transillumina-
tor, NanoDrop (or similar spectrophotometer), steady and
shaker microbiological incubators set at 37 °C.

2.5 Cell Culture 1. T75 tissue culture flasks, canted neck (VWR).
Growth and 2. Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose
Maintenance (1×) liquid; with L-glutamine; sodium pyruvate, and sodium
bicarbonate (Sigma).
3. 100× antibiotic–antimycotic solution (100 units/mL penicil-
lin G, 0.1 mg/mL streptomycin sulfate, and 0.25 μg/mL
amphotericin B) (VWR).
4. L-Glutamine solution 200 mM (Sigma).
5. Phosphate-buffered saline (Thermo Fisher).
6. Fetal bovine serum (FBS), heat-inactivated, sterile-filtered
(Sigma).
54 C. Joaquin Caceres et al.

7. Trypsin–EDTA (Sigma).
8. Individually wrapped serological pipettes of the appropriate
size as needed.
9. Sterile tubes or bottles appropriate to prepare and store media
and reagents.
10. Laboratory equipment: cell culture incubator set at 37 °C with
5% CO2, water bath set at 37 °C, pipettor for serological
pipettes, biosafety cabinet class II.

2.6 Generation of 1. Madin–Darby canine kidney (MDCK) cells (ATCC, CCL-34)

Influenza A and B and human embryonic kidney 293T cells (HEK293T) (ATCC,
NanoLuc Viruses by CRL-3216) (see Note 2).
Reverse Genetics 2. Influenza A and B pDP2002 plasmid sets encoding for the
Using the pDP2002 eight gene segments for each virus. The PB1 plasmid carries
Plasmid Sets the Nanoluciferase (NLuc) reporter in each case.
3. Transit-LT1 transfection reagent (VWR).
4. Opti-MEM Reduced Serum Medium (Fisher Scientific).
5. Trypsin L-1-tosylamide-2-phenylethyl chloromethyl ketone
(TPCK-trypsin) (Worthington Biochemical Corporation).
6. 100× antibiotic–antimycotic solution, cell culture grade
(100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate,
and 0.25 μg/mL amphotericin B) (VWR).
7. Individually wrapped 6-well tissue culture plates (VWR).
8. Sterile 1.5 ml screw-cap tubes (Genesee Scientific).
9. Sterile conical tubes (15–50 mL).
10. Single-channel pipettes capable of dispensing from 1 to
1000 μL.
11. Individually wrapped sterile serological pipettes (5–10 mL) as
needed.
12. Laboratory equipment: pipettes and filtered tips of appropriate
sizes, cell culture incubator set at 35 °C or 37 °C with 5% CO2,
water bath set at 37 °C, hemocytometer or another cell
counter, inverted microscope, pipettor for serological pipets,
tabletop minicentrifuge, biosafety cabinet class II, ultra-freezer
set at -80 °C.

2.7 Propagation of 1. Cleared infectious cell culture supernatant containing the res-
Rescued Influenza A cued FLUAV or FLUBV.
and B Viruses in MDCK 2. MDCK cells (ATCC, CCL-34).
Cells
3. T75 (or T175) tissue culture flasks, canted neck (VWR).
4. Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose
(1×) liquid; with L-glutamine; sodium pyruvate, and sodium
bicarbonate (Sigma).
 Reverse Genetics for Influenza NanoLuc Viruses 55

5. Phosphate-buffered saline (PBS) (Thermo Fisher).

6. Fetal bovine serum (FBS), heat-inactivated, sterile-filtered
(Sigma).
7. Opti-MEM Reduced Serum Medium (Fisher Scientific).
8. TPCK-trypsin (Worthington Biochemical Corporation).
9. 100× antibiotic–antimycotic solution (100 units/mL penicil-
lin G, 0.1 mg/mL streptomycin sulfate, and 0.25 μg/mL
amphotericin B) (VWR).
10. Single-channel pipettes capable of dispensing from 1 to
1000 μL.
11. Filtered pipette tips.
12. Sterile serological pipets (5–10 mL) as needed.
13. Centrifuge conical tubes (15–50 mL).
14. Laboratory equipment: pipettes and filtered tips of appropriate
sizes, cell culture incubator set at 35 °C with 5% CO2, water
bath set at 37 °C, hemocytometer or another cell counter,
inverted microscope, pipettor for serological pipets, tabletop
minicentrifuge, biosafety cabinet class II, refrigerated centri-
fuge capable of containing 15 and 50 mL conical tubes set at
4 °C ultra-freezer set at -80 °C.

2.8 Propagation of 1. Cleared infectious cell culture supernatant containing the res-
Rescued Influenza A cued FLUAV-NLuc or FLUBV-NLuc.
and B NanoLuc Viruses 2. 9- to 11-day-old specific pathogen-free (SPF) embryonated
in Embryonated chicken eggs (ECEs).
Chicken Eggs 3. 70% ethanol
4. Egg puncher (i.e., Dremel Multipro 7.2 V, model 770) or
16G × 1.5″ needle with robber stopper.
5. 1 mL syringes with 25G × 5/8″ needles.
6. 10 mL syringes.
7. 18G × 1″ needles.
8. Glue to seal the eggs (i.e., Elmer’s glue).
9. 0.5% turkey or chicken red blood cells .
10. Phosphate-buffered saline (PBS) (Thermo Fisher).
11. 96-well V-bottom plates.
12. Multichannel pipette able to dispense 50 μL with
corresponding pipette tips.
13. Laboratory equipment: egg candler, egg incubator set at 33 °C
or 35 °C, tabletop minicentrifuge, biosafety cabinet class II,
refrigerated centrifuge capable of containing 15 and 50 mL
conical tubes set at 4 °C, ultra-freezer set at -80 °C.
56 C. Joaquin Caceres et al.

2.9 Titration of 1. FLUAV-NLuc or FLUBV-NLuc virus of interest.

FLUAV-NLuc and 2. MDCK cells (ATCC, CCL-34) seeded the day before in a
FLUBV-NLuc by Tissue 96-well cell culture plate.
Culture Infectious
3. Empty 96-well cell culture plate (VWR).
Dose 50 (TCID50)
4. 70% ethanol.
5. Opti-MEM Reduced Serum Medium (Fisher Scientific).
6. TPCK-trypsin (Worthington Biochemical Corporation).
7. 100× antibiotic–antimycotic solution (100 units/mL penicil-
lin G, 0.1 mg/mL streptomycin sulfate, and 0.25 μg/mL
amphotericin B) (VWR).
8. Single-channel pipettes capable of dispensing from 1 to
1000 μL.
9. Multichannel pipettes capable of dispensing from 1 to 300 μL.
10. Filtered pipette tips.
11. Sterile serological pipets (5–10 mL) as needed.
12. Centrifuge conical tubes (15–50 mL).
13. 0.5% turkey red blood cells.
14. Sterile disposable reagent reservoirs (Fisher Scientific).
15. Laboratory equipment: pipettes and filtered tips of appropriate
sizes, cell culture incubator set at 35 °C with 5% CO2, water
bath set at 37 °C, inverted microscope, pipettor for serological
pipets, and biosafety cabinet class II.

2.10 Use of FLUAV- 1. Nano-Glo Luciferase Assay System (Promega).

NLuc and FLUBV-NLuc 2. 70% ethanol.
Viruses for the
3. Receptor-destroying enzyme (RDE, VWR).
Assessment of
Neutralizing 4. 50% and 0.5% turkey or chicken red blood cells.
Antibodies 5. Black 96-well plates for luciferase assays (Thermo Fisher
Scientific).
6. Single-channel pipettes capable of dispensing from 1 to
1000 μL.
7. Multichannel pipettes capable of dispensing from 1 to 300 μL.
8. Filtered pipette tips.
9. Sterile serological pipets (5–10 mL) as needed.
10. Sterile disposable reagent reservoirs (Fisher Scientific).
11. Rotational shaker for 96-well plates.
12. Aluminum foil.
13. Phosphate-buffered saline (PBS) (Thermo Fisher).
 Reverse Genetics for Influenza NanoLuc Viruses 57

14. 100× antibiotic–antimycotic solution (100 units/mL penicil-

lin G, 0.1 mg/mL streptomycin sulfate, and 0.25 μg/mL
amphotericin B) (VWR).
15. Luciferase reader for 96-well formal.
16. Laboratory equipment: pipettes and filtered tips of appropriate
sizes, cell culture incubator set at 35 °C with 5% CO2, water
bath set at 37 °C, inverted microscope, pipettor for serological
pipets, and biosafety cabinet class II.

3 Methods

3.1 vRNA Extraction Dedicated equipment, supplies, and reagents solely for RNA work
from Influenza A and B are highly recommended; see Notes 3 and 4 for the precautions and
Viruses recommendations to be followed during the RNA extraction pro-
cedure. Below we describe the RNA extraction procedure using the
QIAamp Viral RNA kit (QIAGEN).
1. Equilibrate buffer AVE to room temperature.
2. Prepare buffer AW1 and Buffer AW2 according to manufac-
turer’s instructions.
3. Dispense 560 μL buffer AVL into a 1.5 mL microcentrifuge
tube. If the sample volume is >140 μL, then increase AVL
buffer proportionally.
4. Add 140 μL of the sample to buffer AVL and pulse-vortex for
15 s.
5. Incubate at room temperature for 10 min.
6. Briefly centrifuge to remove droplets from the lid.
7. Add 560 μL of ethanol (96–100%) and pulse-vortex for 15 s.
Briefly centrifuge to remove droplets from the lid.
8. Add 630 μL of the solution from step 5 to the column (within
a 2 mL collection tube provided with the kit). Close the cap
and centrifuge at 6000×g for 1 m. Transfer the column into a
clean 2 mL collection tube and discard the previous tube.
9. Repeat step 8 until all the lysate has been passed though the
column.
10. Add 500 μL of buffer AW1 to the column. Close the cap and
centrifuge at 6000×g for 1 min.
11. Transfer the column into a clean 2 mL collection tube and
discard the previous tube.
12. Add 500 μL of buffer AW2 to the column. Close the cap and
centrifuge at 6000×g for 1 min.
13. Transfer the column into a clean 2 mL collection tube and
centrifuge at full speed for 1 min.
58 C. Joaquin Caceres et al.

14. Transfer the column into a clean 1.5 mL microcentrifuge tube

and add 60 μL of room temperature buffer AVE (to elute the
RNA). Close the cap and incubate at room temperature for
1 min.
15. Centrifuge at 6000×g for 1 m. The RNA is in the flowthrough.
16. Store RNA at -80 °C if it cannot be used immediately.

3.2 FLUAV or FLUBV 1. Amplification of FLUAV and FLUBV gene segments from
RT-PCR Amplification vRNA can be achieved with any RT-PCR system; however, we
recommend the use of Superscript III One-Step RT-PCR System
with Platinum Taq DNA Polymerase (Thermo Fisher Scientific)
as it has rendered the most robust results, and the protocol
described below has been optimized for the use of this
RT-PCR system. See Table 1 for gene-specific primers to be used.
2. In a PCR tube, add 2.5 μL of the RNA previously extracted and
keep the tubes on ice.
3. In a 1.5 mL microcentrifuge tube, prepare reaction mix 2 as
follows (adjust volume accordingly based on number of reac-
tions needed. 25 μL reactions can be prepared by cutting in half
the volumes for each reagent):

(a) Nuclease-free water 8.5 μL

(b) 2× reaction mix 12.5 μL
(c) Primer forward (10 μM) 0.5 μL
(d) Primer reverse (10 μM) 0.5 μL
(e) SuperScript III RT/Platinum TaqMix 0.5 μL

4. Add 22.5 μL of mix 2 per each tube containing your mix 1.

5. Mix well and briefly spin down to collect the contents at the
bottom of the tube. Always keep on ice until placed into the
thermocycler.
6. Set the thermocycler with the following temperature
conditions:

Step Temperature Time No. of cycles

Retro transcription 55 °C 30 m 1
Initial denaturation 94 °C 2m 1
Denaturation 94 °C 15 s 30
Annealing 56 °C 30 s
Extension 72 °C 5–7 min
(see Note 5)
Final extension 72 °C 10 min 1
Hold 4 °C 1
 Reverse Genetics for Influenza NanoLuc Viruses 59

7. Preheat thermocycler to the initial denaturation temperature

before placing the reaction tubes.
8. Once hot, place the reaction tubes in the thermocycler and
start the run.
9. Analyze PCR products by standard 1% agarose gel electropho-
resis using SYBR® at 100 volts for 20 min. SYBR® is 1000×, so
dilute accordingly.
10. Purify the PCR products from the agarose gel using QIAquick
gel extraction kit (QIAGEN) or gel extraction kit of your
choice following the manufacturer’s instructions.
11. Elute DNA with 30 μL of the provided elution buffer or
nuclease-free water.
12. Quantify DNA using a NanoDrop or spectrophotometer of
your choice.
13. Store DNA at -20 °C until needed if it cannot be used
immediately.
Up to this point, up to eight PCR products containing full-
length FLUAV or FLUBV segments should have been pro-
duced. Each of these segments is flanked by restriction sites
compatible with the pDP2002 reverse genetic vector.

3.3 Cloning of FLUAV 1. Using the purified PCR products from step 11, Subheading
and FLUBV Gene 2.2, set up the enzymatic digestions as follows:
Segments into
pDP2002 (a) Purified PCR product 26 μL
(b) 10× restriction endonuclease buffer 3 μL
(c) Nuclease-free water 20 μL
(d) Endonuclease (BsmBI, BsaI, or AarI) 1 μL

2. Perform digestions at 37 °C (BsaI and AarI) or 55 °C (BsmBI)

for 4 h to overnight in a thermocycler or other device able to
reach the required temperatures, but some considerations need
to be considered (see Note 6).
3. Next, purify the digested fragment using the MinElute PCR
Purification Kit. Digested product is eluted using 20 μL of
elution buffer.
4. Measure the concentration using the spectrophotometer of
your choice. Gel purification of PCR product after digestion
is not necessary. DNA can be stored at -20 °C if not used
immediately.
60 C. Joaquin Caceres et al.

5. Set digestion reaction to digest the vector, in this case

pDP2002:

(a) pDP2002 (~100 ng/μL stock) 30 μL

(b) 10× restriction endonuclease buffer 6.5 μL
(c) Endonuclease (BsmBI) 1 μL
(d) Nuclease-free water 33 μL

6. Set to incubate at 55 °C overnight in the thermocycler.

7. Run digested product through gel electrophoresis in a 1%
agarose gel (using SYBR® Safe gel staining) at 100 volts for
20 min. Two bands will be easily identifiable, one of 447 base
pairs (bp) and a larger one of 2958 bp. Excise the larger band
from the gel, and place into a 2 mL microcentrifuge tube.
8. Purify the DNA using the QIAquick gel extraction kit follow-
ing manufacturer’s instructions and elute DNA in 30 μL of
elution buffer.
9. Measure the concentration using a NanoDrop. Store DNA
at -20 °C if not used immediately.
10. Dephosphorylate the purified vector fragment using the
Antarctic phosphatase (NEB) following manufacturer’s
conditions.

(a) pDP2002 digested (up to 1 μg) X μL

(b) 10× reaction buffer 2 μL
(c) Antarctic phosphatase 1 μL
(d) Nuclease-free water To complete 20 μL

11. Incubate at 37 °C for 1 h and then stop the reaction by heat

inactivation for 2 min at 80 °C.
12. Purified the digested and phosphatase-treated plasmid using
the MinElute PCR Purification Kit. Digested and phosphatase-
treated plasmid is eluted using 30 μL of elution buffer. Measure
the concentration using the spectrophotometer of your choice.
DNA can be stored at -20 °C if not used immediately.
13. Proceed with ligation reactions following an insert/vector
ratio of 3:1 (see Note 7).
14. Calculate the insert/vector ratio using the following formulas:

Vector size in bp
= molecular ratio∴
Insert size in bp
 Reverse Genetics for Influenza NanoLuc Viruses 61

50 ng of vector
= 1 part of insert in ng × 3
molecular ratio
= 3 parts of insert in ng
15. Calculate the volume of insert and vector needed based on the
concentrations obtained in steps 3 and 11 and the calculations
from step 13.
16. Set ligation reactions as follows for a total reaction volume of
15 μL (see Note 8):

(a) Digested PCR product x μL (from step 3)

(b) Digested pDP2002 (50 ng total) x μL (from step 13)
(c) 2× Quick Ligase reaction buffer 7.5 μL
(d) Nuclease-free water Up to 15 μL
(e) Quick Ligase 1 μL

17. Mix well, quick spin, and set to incubate at room temperature
for 5 min and then place the reaction in ice. Ligation reactions
can be stored at -20 °C if it cannot be used for transformation
on the same day.
18. Transform ligation reactions into TOP10 cells following the
manufacturer’s instructions.
19. Spread 100 μL of transformation rxn onto LB agar plates
supplemented with 50 μg of ampicillin.
20. Set to incubate overnight at 37 °C. Transformants can be
screened by colony PCR (cPCR) the following day.

3.4 Screening of After 16–18 h of incubation at 37 °C, plates from step 15, Sub-
Transformant Colonies heading 3.3, should have colonies with potential positive reverse
by cPCR genetic (RG) clones carrying the desired FLUAV and FLUBV
gene segments. It is recommended to screen between five and
ten transformant colonies. To screen for RG clones carried into
the pDP2002 vector, we recommend using the following
primer set: T7Seq (5′-TAATACGACTCACTATAGG-3′) and
SeqPol1 (5′-GAGGTATATCTTTCGCTCCG-3′).
1. Before starting, pre-warm an LB agar plate (with 50 μg/mL of
ampicillin). Draw a grid and label the compartments with the
corresponding colony ID numbers. This plate will serve to save
a copy of the colonies selected for screening.
2. In a 1.5 mL microcentrifuge tube, prepare cPCR reactions as
follows (amounts disclosed below are per reaction; adjust
depending on the number of reactions to be performed):
62 C. Joaquin Caceres et al.

(a) Nuclease-free water 5.9 μL

(b) 2× GoTaq master mix 7.5 μL
(c) T7seq (100 ng/μL stock) 0.3 μL
(d) SeqPol1 (100 ng/μL stock) 0.3 μL

3. Aliquot 14 μL of reaction mix into 200 μL PCR tubes.

4. With a sterile 10 μL pipette tip or a sterile toothpick, collect the
selected colonies one at a time; perform this procedure under
the flame to keep sterility. Then touch the pre-warmed LB plate
(see step 1) to inoculate the bacteria on the surface of the agar
(within the designated compartment). Set plate to incubate at
37 °C overnight.
5. Next, immerse the same bacteria-containing tip into the
corresponding cPCR mix tube and shake vigorously.
6. Mix well and briefly spin down to collect contents at the
bottom of the tube. Always keep reactions on ice until they
are loaded in the thermocycler.
7. Set thermocycler with the following temperature conditions:

Step Temperature Time No. of cycles

Initial denaturation 95 °C 5m 1
Denaturation 95 °C 1m 35
Annealing 56 °C 45 s
Extension 72 °C 5–7 m (see Note 5)
Final extension 72 °C 10 m 1
Hold 4 °C 1

8. Place the reaction tubes in the thermocycler and run at the

specified temperature conditions.
9. Analyze cPCR products by gel electrophoresis in a 1% agarose
gel stained with SYBR® Safe gel staining and select potential
positive candidates for sequencing based on the cPCR results.
10. To isolate the plasmids from the selected clones, prepare 15 mL
conical tubes containing 5 mL LB broth supplemented with
50 μg/mL of ampicillin.
11. Retrieve the LB plate containing grown copies of the screened
clones. Using a sterile 10 μL pipette tip, pick the desired colony
and inoculate the corresponding tube with LB broth.
12. Set cultures to incubate overnight (~16 h) at 37 °C/225 rpm
in a shaker incubator.
 Reverse Genetics for Influenza NanoLuc Viruses 63

13. The next day, pellet the bacterial cultures at 3000 rpm/10 m,
discard the supernatant, and proceed with plasmid purification
using the QIAprep Spin Mini kit (QIAGEN). Follow manu-
facturer’s instructions (see Note 9). Measure the concentration
using the spectrophotometer of your choice. DNA can be
stored at -20 °C if not used immediately.
The purified plasmids are now ready for sequencing. In
addition to cPCR screening, plasmids need to be sequenced to
confirm the presence of unexpected mutations in either the
gene segment or the plasmid vector, as well as the presence of a
complete FLUAV or FLUBV gene segment and its orientation. The
following primer set is recommended for Sanger sequencing:
pCMVF (5′-ATTACATGTTGCCAAGTACGCCCCCTATT
GACG-3′), T7Seq (5′-TAATACGACTCACTATAGG-3′), Seq-
Pol1 (5′-GAGGTATATCTTTCGCTCCG-3′) and BGH-R (5′-T
AGAAGGCACAGTCGAGG-3′).

3.5 Cell Culture If cells have been kept in liquid nitrogen, perform fast thawing
Growth and following source’s instructions. For general growth and care, fol-
Maintenance low the recommendation listed below (volumes adjusted for T75
flask).
1. After extracting a vial of cells from the liquid nitrogen, thaw
quickly in 37 °C water bath.
2. Dispense the vial’s contents into a T75 flask containing 10 ml
of pre-warmed DMEM supplemented with 10% FBS and 1×
penicillin/streptomycin solution, and 2 mM L-glutamine
(complete media).
3. Place the cells into a cell culture incubator set at 37 °C/5%
CO2.
4. The next day, remove the media; wash cells with 5 mL of
pre-warmed, sterile 1× PBS; and add 10 mL of fresh
pre-warmed complete media.
5. When the cells have reached confluence (about 48 h), remove
and discard the media.
6. Wash cells with 2 mL of pre-warmed, sterile 1× PBS.
7. Add 2 mL (MDCK) or 1 mL (HEK293T) of 0.25% trypsin–
EDTA solution and incubate it for 10 min (MDCK cells) or
3 min (HEK293T) at 37 °C (in the cell culture incubator) until
the cells have completely detached. You may tap the sides of the
flask to help with detachment. Incubation time can be
extended up to 20 min for MDCK cells if needed.
8. Add 8 mL of complete media to stop the trypsinization and
wash the bottom of the flask by pipetting up and down (see
Note 10).
64 C. Joaquin Caceres et al.

9. Pipette up and down a few times to resuspend the cells. Add

back to the flask 1–2 mL of cells and transfer the rest into a
conical tube or appropriate size (15–50 mL).
10. Adjust the volume to 12 mL with complete media.
11. Place the flask into the cell culture incubator under the same
conditions as above.
12. Pass cells every three days following steps 5–10.

3.6 Rescue of Before starting a transfection, it is recommended to prepare high-

FLUAV-NLuc and quality plasmid preparations (see Note 10) using the HiSpeed
FLUBV-NLuc Using Plasmid Maxi kit (QIAGEN) or other similar kits of your choice.
pDP2002 Plasmid Sets The following procedure is very simple but also prone to cross-
contamination if you are not careful. Use extreme care when
handling cells, transfection reagents, plasmids, and media. The
procedure described below is for performing transfections using a
6-well plate format and HEK293T/MDCK cell co-cultures. It is
also amenable for scaling down to 12- and 24-well plate formats (see
Note 11). This protocol for the generation of recombinant NLuc
viruses has been optimized for the use of PB1-NLuc from A/
Puerto Rico/08/34 (H1N1) for FLUAV and PB1-NLuc from
B/Brisbane/60/2008 for FLUBV. The rest of the genes of interest
should be cloned into a reverse genetic vector and used together
with the PB1-NLuc plasmids for the rescue of FLUAV-NLuc or
FLUBV-NLuc viruses. For higher efficiency, we recommend pair-
ing the respective PB1-NLucs with the remaining internal genes of
A/Puerto Rico/08/34 (H1N1) or B/Brisbane/60/2008, respec-
tively. The system is amenable for the rescue of 2 + 6 viruses (HA
+NA of interest) or 1 + 7 (HA of interest + NA of the respective
backbones)
1. Centrifuge the cells obtained in Subheading 2.5 at 450×g for
5 min at 4 °C.
2. Resuspend pelleted cells in 5 mL of Opti-MEM supplemented
with 1× antibiotic–antimycotic solution (Opti-MEM-AB/AM)
and proceed to count manually or with an automated
Cellometer.
3. Based on cell count results, calculate the volume needed from
each cell suspension to seed 9 × 105 HEK293T cells and
1.5 × 105 MDCK cells/well (6-well plate). Bring co-culture
cells’ suspension to a volume large enough to add 3 mL of cell
suspension/well.
4. Rock plate(s) to evenly distribute cells in the wells and set to
incubate at 37 °C/5% CO2 overnight (~16 to 24 h).
5. The next day, when the co-cultures have reached ~80% to 90%
confluency, prepare the cocktail of seven plasmids (one plasmid
per influenza viral RNA gene segment excluding the HA of
 Reverse Genetics for Influenza NanoLuc Viruses 65

interest) in a 1.5 mL microcentrifuge tube. Please note that the

use of filter tips is recommended for this and subsequent steps
to avoid potential contaminations. If a working stock of each
plasmid has been prepared at 100 ng/μL, pipette 10 μL of each
plasmid (1 μg of each plasmid, 8 μg pDNA) and set aside.
Distribute the mix in as many reactions as you will perform.
Always consider a negative control where one segment (HA) is
missing.
6. In a separate tube, mix 104 μL of plain Opti-MEM and add
16 μL of TransIT-LT1 (2 μL/μg of plasmid DNA). It is easier
to calculate the total amount of Opti-MEM/TransIT-LT1
needed for all the transfections and prepare a cocktail with the
total volume (plus extra ~10% for both).
7. Vortex the Opti-MEM/TransIT-LT1 cocktail and add 120 μL
to the plasmid DNA mixture and mix well. It is essential to
change tips before going into the next sample to avoid cross-
contamination.
8. Incubate the mixture at room temperature for approximately
45 min, and then, very slowly, add 800 μL of plain Opti-MEM.
Mix gently.
9. Remove the medium from the cells’ monolayers and add the
transfection mixture to the corresponding wells. Washing the
cells prior to addition of the transfection mixture is not neces-
sary as no FBS was added to the seeding media. Use caution
when adding transfection mixture onto the cells. It is best to
place the pipette tip as close as possible to the cell monolayer
and pipette down dropwise or deposit by the wall of the well to
prevent aerosol formation.
10. Incubate the cells overnight (~16 h) at 35 °C/5% CO2.
11. After incubation time has been completed, tilt the plate, and
carefully remove the transfection mixture from cells by posi-
tioning the pipette tip at the corner of the well where the liquid
has accumulated. Discard in 10% bleach solution.
12. Then, add 1 mL of Opti-MEM-AB/AM to the cells and incu-
bate at 35 °C/5% CO2 until 24-h post-transfection (hpt) has
been completed.
13. At 24 hpt, add 1 mL of Opti-MEM-AB/AM containing 2 μg/
mL TPCK-trypsin to each transfected well (1 μg/mL final
concentration per well).
14. Monitor cells daily for cytopathic effect (CPE), particularly in
the MDCK cells. Small foci of dead cells are indicative of active
influenza virus replication which may become apparent for
some, but not all, successful virus rescues starting at 72 hpt.
15. If no CPE is observed at 72 hpt (or later if desired, up to
120 hpt), a blind passage in MDCK cells may be performed.
Add an additional 1 mL of Opti-MEM/AB/AM+ 3 μg/mL
66 C. Joaquin Caceres et al.

TPCK-trypsin (1 μg/mL final concentration per well) at

72 hpt if decided to prolong the incubation passing this time
point.
16. To perform the blind passage, withdraw the cell culture super-
natant into the appropriate centrifuge tube and centrifuge at
5000 rpm, at 4 °C, for 5 min.
17. Without disturbing the cell pellet, transfer the supernatant to a
fresh tube.
18. Remove the media from a fresh MDCK cell monolayer
prepared the day before (6-well plate seeded with 1.5 × 105
cells/well) and add 500 μL of cleared supernatant to the
corresponding well.
19. Set to incubate for 1 h at 35 °C/5% CO2, rocking the plate
every 15 min.
20. Next, discard the supernatant and add 2 mL of Opti-MEM-
AB/AM+1 μg/mL TPCK-trypsin per well and monitor daily
for CPE and/or HA assay at 48–72 hpi.
21. CPE and/or HA-positive samples can be either stored at
-80 °C for later or further propagated in either MDCK cells
or 9–11-day-old chicken embryonated eggs.

3.7 Propagation of 1. The day before infection, wash and treat MDCK cells with
FLUAV-NLuc and trypsin as described in Subheading 3.5. Resuspend cells with
FLUBV-NLuc in MDCK 10 mL of complete growth media (DMEM supplemented with
Cells 10% FBS and 1× penicillin/streptomycin solution, 2 mM
L-glutamine).
2. Into a new T75 flask, add 2 mL of cell suspension and 10 mL of
complete media.
3. Set cells to incubate at 37 °C/5% CO2 overnight (~16 to 24 h).
4. Once the cells have reached ~80% to 90% confluency, using
plain Opti-MEM, make serial dilutions of your samples (rang-
ing between 1/100 and 1/10,000) from the FLUAV-NLuc/
FLUBV-NLuc transfection or blind passage supernatant (from
Subheading 3.6), based on the CPE severity and of HA titer.
We recommend the propagation of each virus from two differ-
ent dilutions.
5. Wash cells 2× with 5 mL of 1× PBS.
6. Next, inoculate cells (in the T75 flask) with 3 mL of the
FLUAV-NLuc/FLUBV-NLuc dilutions and incubate for 1 h
at 35 °C/5% CO2, rocking the plate every 15 min.
7. Afterward, remove the inoculum and add 10 mL of Opti-
MEM-AB/AM+1 μg/mL TPCK-trypsin per well and monitor
daily for CPE and/or HA assay at 48–72 hpi. CPE may start
appearing at 48 hpi. Incubation may be prolonged beyond
72 hpi (up to 120 hpi) if deemed appropriate to maximize
 Reverse Genetics for Influenza NanoLuc Viruses 67

the amount of virus recovered from the culture. TPCK should

be added 48 hpi if incubation is prolonged.
8. Determine the hemagglutination titer by performing HA assay.
For each sample, harvest the dilution with the higher HA units.
9. Collect the cell culture supernatant into a 15 mL conical tube
and clear by centrifugation at 2500 rpm for 10 min at 4 °C.
10. Transfer the cleared supernatant into a new 15 mL conical tube
(set in ice), homogenize by pipetting up and down, and pre-
pare aliquots of the desired volume.
11. Store aliquots at -80 °C.

3.8 Propagation of 1. Candle embryos and mark the limit between the air chamber
FLUAV-NLuc and and the allantoid cavity.
FLUBV-NLuc in ECEs 2. Label eggs accordingly.
3. Prepare tenfold dilutions of the virus starting by mixing 100 μL
of transfection or blind passage supernatant (from Subheading
3.6) with 900 μL of 1xPBS (supplemented with 1× antibiotic–
antimycotic solution) and keep in ice until needed.
4. Decontaminate eggshell with 70% ethanol or ethanol/iodine
solution.
5. Drill/punch a hole on the shell a few millimeters (~3 mm) from
the mark between the air chamber and the allantoid cavity.
6. Using 1 mL syringes with a needle (25G × 5/8″), inoculate
each dilution into three to five embryos with 100 μL/embryo.
Three different dilutions (ranging from 10-2 to 10-7) are
recommended.
7. Seal the hole with glue.
8. Set embryos to incubate at 33 °C (FLUBV-NLuc) or 35 °C
(FLUAV-NLuc) for 48 h candling daily to remove unspecific
mortality. No virus-induced mortality is expected from
embryos inoculated with low pathogenic FLUAV or FLUBV.
9. Once incubation has been completed, chill embryos overnight
at 4 °C.
10. Using 1 mL syringes with needle, collect 100 μL of allantoic
fluid (AF) from each chilled embryo into tubes or a 96-well
plate.
11. Prepare 96-well V-bottom plates (as many as needed) by add-
ing 50 μL of 1× PBS to each well.
12. Add 50 μL of AF to individual wells of column 1.
13. Perform twofold dilutions from columns 1 through 12.
14. Next, add 50 μL of 0.5% turkey red blood cells to each well.
15. Tap or shake the plate to homogenize and set to incubate for
30 min at room temperature.
68 C. Joaquin Caceres et al.

16. Select the highest dilution at which all the inoculated embryos
give the highest HA titer to avoid viral interfering particles.
17. Into a single 50 mL conical tube (set in ice), collect as much AF
as possible from each egg from the selected dilution.
18. Next, clear the AF by centrifugation at 2500 rpm for 10 min at
4 °C.
19. Use the cleared AF to prepare aliquots of the desired volume.
20. Store aliquots at -80 °C.

3.9 Titration of 1. The day before infection, prepare a 96-well plate with 1.5 × 104
FLUAV-NLuc and MDCK cells/well. Use a total volume of 100 μL of Opti-
FLUBV-NLuc by Tissue MEM-AB/AM.
Culture Infectious 2. Set cells to incubate at 37 °C/5% CO2 overnight (~16 to 24 h).
Dose 50 (TCID50) 3. Once the cells have reached ~80% to 90% confluency, use a new
96-well plate to prepare the virus dilutions. For this, prepare
tenfold serial dilutions of virus dispensing 216 μL of Opti-
MEM-AB/AM containing 1 μg/mL TPCK-trypsin to
each well.
4. Thaw an aliquot of the virus to be used on ice.
5. Add 24 μL of virus to the first four wells of the 96-well plate
(A1, B1, C1, and D1) prepared in step 3. If two viruses are
being titrated, repeat the same procedure with wells E1, F1,
G1, and H1.
6. Pipette up and down between seven and ten times to properly
mix the contents of the first column (A1–H1) and aspirate
24 μL into the next column (A2–H2). Do not pipette up and
down after the sample has been deposited into the next col-
umn. Ensure to change tips with each dilution.
7. Once the virus dilutions are completed, remove the media from
the cells prepared the day before. Aspirate 200 μL from the
dilution plate starting from column 12 and deposit the volume
into the plate containing cells. Repeat the same process with
each column until dilution -1 (Column A) has been added
(there is no need of changing tips).
8. Incubate cells at 35 °C/5% CO2 for 72 h.
9. After 72 h, remove plates from the incubator and aspirate 50 μL
of supernatant starting from the highest dilution into an HA
assay plate.
10. Next, add 50 μL of 0.5% turkey or chicken red blood cells.
11. Tap HA assay plate gently and incubate for 30 min at room
temperature.
12. Proceed to virus titer calculation using the Reed–Muench
method [26].
 Reverse Genetics for Influenza NanoLuc Viruses 69

3.10 Use of FLUAV- 1. The day before performing the assay, prepare a 96-well plate
NLuc and FLUBV-NLuc with 1.5 × 104 MDCK cells/well. Use a total volume of 100 μL
Viruses for the of Opti-MEM-AB/AM. If sera from the mammalian origin will
Assessment of be evaluated, treat 1 volume of sera with 3 volumes of a fresh
Neutralizing aliquot of receptor-destroying enzyme (RDE, VWR) at 37 °C
Antibodies overnight.
2. The following day, stop the RDE activity by incubating the
samples at 56 °C in a water bath for 30 min. Allow the samples
to reach room temperature. Generate a 1/10 dilution of the
sera adding PBS-AB/A.
3. If sera from avian origin will be evaluated, treat the day of the
assay 1 volume of sera with 1 volume of a solution of chicken
red blood cells at 50%. Complete with PBS to make a 1/10
dilution of sera. Centrifuge at 1000 rpm for 1 min and repeat
the same process with the supernatant. Once the process is
repeated, collect the supernatant, and proceed with the assay.
4. Prepare a viral stock of concentration 100 TCID50 per 50 μL.
It is recommended to do 1/10 serial dilutions in PBS-AB/AM
of the stock until you reach the 100 TCID50 per 50 μL.
5. In a different 96-well plate, add 100 μL of serum sample to the
first column.
6. Next, add 50 μL of PBS-AB/AM to all the wells from columns
2–12. Then make serial dilutions aspirating 50 μL from column
1 into the next column. Do not pipette up and down after the
sample has been deposited into the next column. Ensure to
change tips with each dilution.
7. Once dilutions are completed, add 50 μL of FLUAV-NLuc or
FLUBV-NLuc virus (adjusted to 100 TCID50/50 μL) start-
ing from the highest dilution.
8. Incubate at 35 °C/5% CO2 for 1 h.
9. After the incubation is complete, remove the media from the
96-well MDCK plate prepared in step 1 and transfer the serum
dilutions to the MDCK plate.
10. Incubate for 15 min at 4 °C, followed by 45 m at 35 °C.
11. Discard the serum dilutions and add 200 μL Opti-MEM-AB/
AM containing 1 μg/mL TPCK-trypsin to each well.
12. Incubate at 35 °C/5% CO2 for 48 h.
13. Once incubation for 48 h is completed, proceed to the NLuc
activity reading using the Nano-Glo® Luciferase Assay System
(Promega).
14. Set the assay buffer to that until it reaches room temperature.
15. Turn on the 96-well luciferase reader that will be used to
record the luciferase data.
70 C. Joaquin Caceres et al.

16. Remove the supernatant, keeping 50 μL of it on the plate. To

accomplish this, remove 150 μL from the total 200 μL added in
Item 11 in Subheading 2.10.
17. Turn off the lights in the biosafety cabinet. It is recommended
to turn off the light in the laboratory if possible.
18. Prepare the mixture of assay buffer + Nano-Glo luciferase
substrate. For one 96-well plate, mix 5 mL of assay buffer
with 100 μL of Nano-Glo luciferase substrate.
19. Add 50 μL of Nano-Glo reaction mix per well to the plate
containing the 50 μL of cell supernatant. Cover the plate with
aluminum foil.
20. Shake the plate on a rotational shaker at 600 rpm for 3 min. It
is important to keep the plate covered with aluminum foil.
21. Once the shaking is finished, incubate for 7 min at room
temperature without shaking.
22. Transfer 100 μL into a flat 96-well black plate (Thermo Fisher
Scientific) and read the luciferase using the 96-well plate
reader.
23. Record the luciferase values of each sample. Transfer the data
to an Excel sheet or similar for posterior analysis.

3.11 Analysis of 1. For the data analysis and generations of graphs, we recommend
Luciferase Expression the use of the software PRISM (https://www.graphpad.com)
and Determination of (Fig. 2).
the Effective Serum 2. Export the luciferase data of your mock-infected control (virus
Dilution 50 (ESD50) in the absence of antibodies) and the samples of interest.
3. Plot the data showing the dilution factor of the serum of
interest in the X-axis and the luciferase values in log10 scale
on the Y-axis as shown in Fig. 2.
4. To calculate the serum dilution where 50% of neutralization is
observed (ESD50), analyze the data through a nonlinear
regression and obtain the EC50, which is equivalent to the
ESD50.

4 Notes

1. Other ambisense/bidirectional plasmids, such as pDZ [27] or

pHW2000 [28], can be used for reverse genetics of FLUAVs or
FLUBVs with appropriate amplification primers.
2. MDCK/human embryonic kidney (HEK293T) co-cultures
could be used to rescue influenza viruses when using the spol
I reverse genetic system. However, HEK293T is not an
approved cell line for vaccine development. In addition,
 Reverse Genetics for Influenza NanoLuc Viruses 71

Fig. 2 Microneutralization assays using FLUAV- or FLUBV-NLuc viruses. (a–c) Mice antisera were used to
assess the presence of neutralizing antibodies using recombinant (a) FLUAV-NLuc (H1N1) against two different
homologous vaccines. (b) FLUAV-NLuc viruses (H3N1) carrying the HA of A/Hong Kong/1/68 (H3N2) (HK68) or
A/turkey/Ohio/313053/04 (H3N2) (OH04) were against homologous or heterologous antisera. (c) FLUBV-NLuc
viruses (homologous and heterologous virus) against sera raised after LAIV vaccination. (d, e) Different ferret
antisera were evaluated against (d) FLUAV-NLuc (H3N1) carrying the HA of A/Texas/1/77 (H3N2) or (e) the HA
of A/Switzerland/9715293/13 (H3N2). (f) Quail antisera raised against A/Guinea Fowl/Hong Kong/WF10/99
(H9N2) (WF10) was evaluated against FLUAV-NLuc (H9N1) carrying the homologous HA or phylogenetically
different HAs. (Figure created with BioRender.com)

HEK293T cells are less efficient for the rescue of FLUAV and
FLUBV than PK-15 cells when employing the spol I reverse
genetic system [21].
3. Dedicated equipment, supplies, and reagents solely for RNA
work are highly recommended. Disposable gloves must be
always worn while performing the RNA extraction procedure
and while handling RNA samples to avoid RNA degradation.
Extracted RNA samples should be always kept on ice and,
preferably, be used immediately for RT-PCR assays. The
remaining RNA can be kept frozen at -80 °C. Consult the
Kit’s manufacturer’s instructions for more details about
storage.
4. The vRNAs are extracted using silica-based (i.e., QIAamp Viral
RNA kit) or organic-based (TRIzol reagent, Invitrogen)
reagents or any other available RNA extraction method follow-
ing procedures recommended by the manufacturer. RNA extrac-
tion using TRIzol reagent is preferred when extracting RNA
from samples with low viral loads. We recommend the QIAamp
Viral RNA kit, which has provided consistent and reliable results,
with adequate vRNA purity and devoid of contaminants and
72 C. Joaquin Caceres et al.

ribonucleases. Viral titers of ≥104 TCID50 (≥105 EID50) are

typically required for efficient preparation of vRNAs and
subsequent RT-PCR amplification. Virus isolation from clinical
samples, tissue-cultured MDCK cells, or 9–11-day-old chicken
embryonated eggs are recommended to obtain appropriate
quantities of vRNA for downstream procedures. Extracted
vRNAs are eluted and resuspended in RNase-free ddH2O.
5. For optimal amplification of FLUAV and FLUBV segments,
use an extension time of 5 min when amplifying the HA, NP,
NA, M, and NS gene segments. When amplifying the PB2,
PB1, and PA, use an extension time of 7 min.
6. If using a water bath, please note the potential for excessive
evaporation resulting from prolonged incubation periods.
7. The recommended insert/vector ratio for the ligation reaction
depends on the size of both the insert and the vector. When the
vector is larger than the insert, a 3:1 ratio is recommended. If
the insert is of equal or larger size than the vector, a 1:1 ratio
would be more appropriate.
8. The final volume of the ligation reaction can be adjusted if the
vector and/or insert concentrations are too low that require
the use of larger volumes. Just scale up the rection buffer and
scale down the nuclease-free water volumes accordingly to
maintain the proper concentrations in the reaction.
9. Plasmids are purified using commercially available plasmid prep-
aration kits, following the manufacturer’s recommendations,
and are best stored at -20 °C at a concentration of
0.25–1.0 mg/mL. It is recommended to prepare high-quality
plasmids for transfection experiments as impurities may interfere
with the transfection efficiency. Measure DNA and RNA con-
centrations at an absorbance of 260 nm using a NanoDrop or
similar spectrophotometer. The 260/280 ratio is used to esti-
mate sample purity. A plasmid preparation with a 260/280 nm
ratio of ≥1.8 is highly recommended. We also recommend that
plasmid stocks are diluted into working stocks at a concentration
of 100 ng/μL prior to transfection to minimize pipetting errors
and to standardize pipetting volumes for each plasmid. The
integrity of the plasmid DNA should be assessed by electropho-
resis in agarose gels before each transfection.
10. The minimum volume of complete media to be added to stop
the trypsinization must equal the volume of trypsin that was
added. One can always add more trypsin to ensure proper wash
of the attachment surface and decrease bubble and foam
formation.
11. Although transfection can be performed in smaller well plate
formats, the efficiency of virus rescue decreases with smaller
wells due to the fewer number of cells susceptible to
transfection.
 Reverse Genetics for Influenza NanoLuc Viruses 73

Acknowledgments

DRP’s research is funded by a subcontract 75N93021C00014

from the Centers for Influenza Research and Response (CEIRR)
from the National Institute of Allergy and Infectious Diseases
(NIAID). Additional funds were obtained by DRP under
GRANT12901999, Proposal 2019-05890, Accession Number
1022658 from the National Institute of Food and Agriculture
(NIFA), US Department of Agriculture. DRP receives additional
support from the Georgia Research Alliance and the Caswell
S. Eidson endowment funds from the University of Georgia.

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 Chapter 5

Reverse Genetics of Bat Influenza A Viruses

Susanne Kessler, Adolfo Garcı́a-Sastre, Martin Schwemmle,
and Kevin Ciminski

Abstract
New World fruit bats were recently found to harbor two distinct and previously unknown influenza A
viruses (IAVs) of the subtypes H17N10 and H18N11. Although viral genome sequences were detected in
the liver, intestine, lung, and kidney of infected bats and the complete genome sequences have been isolated
from their rectal swab samples, all attempts to isolate an infectious virus from bats in nature have failed. The
lack of an infectious bat IAV isolate was overcome by reverse genetic approaches that led to the generation
of an infectious virus in vitro. Using such synthetic bat IAVs enabled the identification of their unconven-
tional cell entry via major histocompatibility complex II (MCH-II) molecules and their ability to replicate in
mice, ferrets, and bats. Importantly, we also showed that these synthetic recombinant bat IAVs are not able
to reassort with conventional IAVs, preventing the acquisition of enhanced transmission properties in
non-bat species by reassortment with conventional IAVs. As authentic viruses are key for understanding the
molecular biology of bat IAVs, in this chapter, we describe our recently established reverse genetics protocol
for generating H17N10 and H18N11 in vitro. This step-by-step protocol starts with cloning of cDNA
copies of the viral RNA segments into reverse genetics plasmids, followed by the generation of a highly
concentrated stock and finally a method to determine viral titers.

Key words New World bat, H17N10, H18N11, Bat influenza A virus, Reverse genetics, cDNA,
HEK293T cells, RIE1495 cells

1 Introduction

Influenza viruses belong to the family of Orthomyxoviridae and are

divided into the four genera: influenza virus types A, B, C, and
D. Of these, influenza A viruses (IAVs) possess the most pro-
nounced host ecology and the highest antigenic complexity
[1, 2]. To date, 18 different hemagglutinin (HA) and 11 neuramin-
idase (NA) antigenic subtypes have been described [1]. Impor-
tantly, while wild waterfowls are considered to be the natural
reservoir for IAVs of the subtypes H1–16 and N1–9, the subtypes
H17–H18 and N10–N11 have been exclusively found in fruit bats
in Central and South America [3–5].

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_5,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

75
76 Susanne Kessler et al.

Bat IAVs (H17N10 and H18N11) structurally resemble con-

ventional IAVs (Fig. 1). Moreover, the genome comprises eight
single-strand viral RNA (vRNA) segments in negative orientation,
each encapsidated by numerous copies of the viral nucleoprotein
(NP) and terminally bound by the heterotrimeric RNA-dependent
RNA polymerase (PB2, PB1, and PA), forming the viral ribonu-
cleoprotein complex (vRNP). The open reading frame (ORF) of
each vRNA segment is flanked by so-called noncoding regions
(NCRs) at the terminal 3′ and 5′ genome ends, which are highly
conserved between different IAV strains and function as a regu-
latory site for the viral polymerase. It is believed that NCRs
together with adjacent parts of the vRNA coding region are essen-
tial for the correct packaging of the genomic content into viral
particles by forming RNA–RNA and/or RNA–protein interactions
[6–9]. The segmented nature of the IAV genome facilitates a
process called reassortment which is the exchange of genome seg-
ments between two strains within one co-infected cell. However, a
prerequisite for reassortment is the functional compatibility of the
parental viruses on RNA and protein level to ensure both sufficient
genome packaging and viral fitness [10, 11]. In the past, reassort-
ment of conventional IAVs from multiple hosts preceded the emer-
gence of a pandemic virus [12].
Previous studies have shown that H17N10 and H18N11 can
exchange their genomic segments [13], but fail to reassort with
conventional IAVs [13–16]. Moreover, the receptor-binding sur-
face glycoproteins H17 and H18 HA are unable to bind sialic acid
residues [4, 17, 18], the canonical receptor of all conventional
IAVs. Instead, H17 and H18 HA utilize major histocompatibility
complex class II (MHC-II) molecules for cell entry [19]. Because
of this alternative receptor usage, the protocol to recover recombi-
nant viruses in vitro differs from conventional IAVs and is more
delicate.
Here we describe an 8 + 4-plasmid rescue protocol for
bat-derived IAVs that has been established by our lab [20]
(Fig. 2) and is based on the original protocol by Hoffmann et al.
for the generation of IAVs [21]. The eight genomic bat IAV
cDNAs are individually inserted between the human RNA poly-
merase I (hPol-I) promoter and the murine terminator sequence of
the pHW2000 rescue plasmid. The hPol-I promoter and termina-
tor elements are flanked by the RNA polymerase II (Pol-II)-depen-
dent immediate-early promoter of the human cytomegalovirus and
a polyadenylation signal [21]. While the hPol-I-controlled gene
cassette generates negative-sensed genomic vRNAs, the Pol-II cas-
sette allows the synthesis of positive-stranded mRNAs. To increase
viral rescue titers of bat IAVs, we modified the original rescue
protocol by co-transfecting pCAGGS expression plasmids coding
for the four ribonucleoprotein genes (PB2, PB1, PA, NP). For this,
the ORFs of these genes are individually cloned between the
 Reverse Genetics of Bat Influenza A Viruses 77

Fig. 1 Structure of a bat IAV virion. The eight gene segments of a bat IAV are packed inside the viral particle
and code for at least ten different proteins. The mRNA transcripts of the M segment and the NS segment have
alternative splice sites allowing their processing into the splice products M2 and nuclear export protein (NEP),
respectively. A host-derived lipid-bilayer forms the viral envelope that is stabilized by a subjacent layer of M1
proteins. The three transmembrane proteins HA, NA and M2 are embedded into the viral membrane. HA
mediates cell entry by receptor binding and membrane fusion. The ion channel M2 induces a pH-shift
releasing the genomic content. The function of bat IAV NA (N10 and N11), however, is still unknown

chicken Pol-II β-actin promoter and the rabbit β-globin polyade-

nylation signal. Additionally, we provide detailed protocols for virus
stock generation and titration of H17N10 and H18N11.

2 Materials

2.1 Reagents and 1. TRIzol reagent (Invitrogen).

Kits 2. Ethanol absolute (Sigma-Aldrich).
3. Direct-zol RNA kit (Zymo Research).
4. OneStep RT-PCR kit (QIAGEN).
5. Zymoclean Gel DNA Recovery kit (Zymo Research).
6. ZymoPURE Plasmid Miniprep kit (Zymo Research).
7. Penicillin–streptomycin (Gibco).
8. TPCK-treated trypsin (Sigma-Aldrich).
78 Susanne Kessler et al.

Fig. 2 Schematic workflow for generation of bat IAV stocks. HEK293T cells are transfected with eight different
pHW2000 plasmids, each coding for one gene segment of the virus. Additionally, pCAGGS expression
plasmids coding for the vRNP components PB2, PB1, PA, and NP are transfected to increase the rescue
efficiency. At 48 h post transfection, the supernatant is propagated in RIE1495 cells pretreated with DEAE in a
T75 flask. Subsequently, the supernatant from the T75 flask is collected 60 h post infection and viral particles
are concentrated via ultracentrifugation

2.2 Primers Primers used for cDNA synthesis (see Subheading 3.2) should be
designed based on the highly conserved noncoding regions of the
bat influenza vRNAs shown in Table 1. In general, primers should
be between 25 and 40 nucleotides in length and comprise some
random nucleotides (lower case), a restriction site (italics; see Note
1), the conserved terminal nucleotides (bold), and segment-specific
nucleotides (underlined):
gatcCGTCTCAGGGAGCAGAAGCAGGTCAGAGATTGTC
Primers for cloning the ORFS of PB2, PB1, PA, and NP into
pCAGGS plasmids should be between 25 and 40 nucleotides in
length and comprise some random nucleotides, followed by a
restriction site and the gene-specific nucleotides of the 5′ or 3′
end of the ORF.

2.3 Cells and Media 1. Human embryonic kidney 293T (HEK293T) cells (American
Type Culture Collection, ATCC, CRL-3216), Madin–Darby
canine kidney cells II (MDCK II), and canine RIE1495 cells
are grown in culture medium: Dulbecco’s modified Eagle’s
medium (DMEM; Gibco, Thermo Fisher Scientific) supple-
mented with 10% fetal calf serum (FCS), 100 U/mL penicillin,
and 100 mg/mL streptomycin. According to cell confluency,
cells were split two to three times per week at a ratio of 1:10.
2. HEK293T cells are transfected in Opti-MEM medium and
subsequently maintained in rescue medium: DMEM supple-
mented with 0.2% bovine serum albumin (BSA), 100 U/mL
penicillin, and 100 mg/mL streptomycin.
Table 1
Overview of the 5′ and 3′ terminal ends of the A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10) and A/flat-faced bat/Peru/033/2010
(H18N11) genome segments

Segment Subtype 5′ end 3′ end

PB2 H17N10 AGCAGAAGCAGGTCAGAGATTGTCAAT CAATACTAAAAATGACCTTGTTTCTACT
H18N11 AGCAGAAGCAGGTCAAAGATTGTCAAA AATCACTAAAAATGACCTTGTTTCTACT
PB1 H17N10 AGCAGAAGCAGGCAAACTATTTTAAA GGGTCATTAGTTGTTGAAACGAAAAAATGCCTTGTTTCTACT
H18N11 AGCAGAAGCAGGCAAACTATTTTGAA GGATCATTAGCTATCAATGCGAAAAAATGCCTTGTTTCTACT
PA H17N10 AGCAGAAGCAGGTACTTAAACA AGAACTCTATCAGCTTTGATTCAATGTAAGAAAAAAGTACCTTG
TTTCTACT
H18N11 AGCAGAAGCAGGTACTTAGACA ATAACTCTATCAGTTTTAGTTCAATATAAGAAAAAAGTACCTTG
TTTCTACT
HA H17N10 AGCAGAAGCAGGGTCACTATTACTCTGTGC AACGGTGGAATTAACCTTGTCATTCAGAAAAGCAAAAAAGACCC
TACT TTGTTTCTACT
H18N11 AGCAGAAGCAGGGTGATTATTATTCAGA GGCTGTGGTGTTAGCTAATGTCAATCTATTA
TTGCAAAAAACACCCTTGTTTCTACT
NP H17N10 AGCAGAAGCAGGGTTAATAATCACATTG GCAAAAAATACCCTTGTTTCTACT
TGACATTTAAAG
H18N11 AGCAGAAGCAGGGTTAATAATCACATTG GAAAAAATACCCTTGTTTCTACT
TGACATTTAAAG
NA H17N10 AGCAGAAGCAGGAGTTTTTAATA CCAATGGACAGCGAATGAAAAAACTCCTTGTTTCTACT
H18N11 AGCAGAAGCAGGAGTTTTTCATA GTATATGATTACATTCATATTTTAAATGGATGTATAAGAAAAAAC
TCCTTGTTTCTACT
M H17N10 AGCAGAAGCAGGCATTATCCAAA AATGAAAAAATGCCTTGTTTCTACT
H18N11 AGCAGAAGCAGGCATTATTCAAA AATGAAAAAATGCCTTGTTTCTACT
NS H17N10 AGCAGAAGCAGGGTATCTAAAGACATA TTAAAAAATACCCTTGTTTCTACT
Reverse Genetics of Bat Influenza A Viruses

H18N11 AGCAGAAGCAGGGTATCTAAAGACATA TTAAAAAATACCCTTGTTTCTACT

Sequences highly conserved between the terminal ends of all bat influenza A virus genomes are shown in bold. Segment-specific sequences are underlined
79
80 Susanne Kessler et al.

3. MDCK II and RIE1495 cells are infected in infection medium

(see Note 2): DMEM supplemented with 0.2% BSA, 100 U/
mL penicillin and 100 mg/mL streptomycin, and 1 μg/mL of
TPCK-treated trypsin.

3 Methods

3.1 Extraction of Bat The genomic material of influenza viruses is RNA that is highly
Influenza A vRNA sensitive to degradation through RNAses found on work surfaces
and the skin. Therefore, it is highly recommended to wear dispo-
sables gloves and use sterile equipment and conditions while
handling RNA material. To further avoid RNA degradation, sam-
ples should be kept on ice during the entire process of RNA
isolation. Viral RNA (vRNA) is extracted from virus-containing
supernatant, swab samples, or organ tissue using TRIzol reagent
and then purified using the Direct-zol RNA kit (Zymo Research) or
a comparable kit according to the manufacturer’s instructions.
Purified RNA material can be stored at -80 °C until cDNA
synthesis.

3.2 cDNA Synthesis Reverse transcription (RT) of the purified vRNA (see Subheading
of Bat Influenza A 3.1) into complementary DNA (cDNA) is performed using the
Virus vRNA QIAGEN OneStep RT-PCR kit or a similar product. To generate
the cDNA of each of the eight genomic vRNA segments, an indi-
vidual RT-PCR must be done using segment-specific primers (see
Subheading 2.2). Mix the components and run the RT-PCR
according to the protocol listed in Table 2. Synthesized cDNA
can be stored at -20 °C until further use.

3.3 Cloning of Bat Bat influenza A viruses are recovered from cloned cDNA (see Sub-
Influenza A Virus heading 3.2) in vitro using bidirectional pHW2000 rescue plasmids
cDNAs into pHW2000 together with pCAGGS protein expression plasmids. We recom-
Rescue Plasmids and mend the additional use of pCAGGS helper plasmids for the rescue
pCAGGS Helper protocol as it increases viral yields. For this, the ORF of the cDNA
Plasmids sequences of the viral ribonucleoprotein components PB2, PB1,
PA, and NP are individually inserted into the pCAGGS plasmid
backbone. Similarly, the cDNA sequences of the eight viral gene
segments are inserted into the pHW2000 backbone.
1. Perform an enzymatic digest of the PCR products and the
vector plasmid in a reaction tube by mixing the following
components (see Note 3) and incubating them for 2 h at 37 °C:

cDNA PCR product/vector (pHW2000, pCAGGS) 1 μg/2 μg

Restriction enzyme (e.g., BsmBI, SalI, ApaI) 1–4 μL
10× buffer 2 μL
Sterile H2O To 20 μL
 Reverse Genetics of Bat Influenza A Viruses 81

Table 2
Components required for the OneStep RT-PCR and the conditions used for
the thermal cycler

Component Volume
5× buffer 10 μL
dNTP mix (10 mM) 1 μL
Primer A forward 2 μL
Primer B reverse 2 μL
OneStep RT-PCR enzyme mix 2 μL
RNase inhibitor 2 μL
Total RNA 1–10 μL (0.1–2 μg)
Sterile RNase-free H2O To 50 μL
Reverse transcription 30 min 50 °C
Initial PCR activation step 15 min 95 °C
Three-step cycle (32 cycles)
Denaturation 1 min 94 °C
Annealing 1 min 58 °C
Extension 1–2 min 72 °C
Final extension 10 min 72 °C

2. Prevent relegation of the linearized vector by adding 5 U of

FastAP alkaline phosphatase (Thermo Scientific) to the vector
restriction digest and incubate for 15 min at 37 °C.
3. Purify the digested products by agarose gel electrophoresis and
extract the respective bands using the Zymoclean Gel DNA
Recovery kit (Zymo Research) or any other comparable gel
extraction kit according to the manufacturer’s instructions.
4. Ligate the digested PCR products into the vector by mixing the
following components and incubating them for 15 min at
room temperature:

5× rapid ligation buffer 2 μL

T4 DNA Ligase (5 U) 1 μL
Vector 1 μL
PCR product 6 μL
82 Susanne Kessler et al.

5. Amplify the ligated plasmids by transforming competent E.coli

DH5α cells. For this purpose, mix 50 μL bacterial cells
together with 5 μL of the ligation mix and incubate for
30 min on ice. Plate the bacterial cells onto LB–agar plates
containing 100 μg/mL ampicillin, invert the plates and incu-
bate them overnight at 37 °C.
6. The next day, pick individual bacterial colonies and transfer
them from the plate to test tubes containing 4 mL liquid LB
medium with 100 μg/mL ampicillin. Let bacterial cells grow in
a shaker for 10 h at 37 °C.
7. Extract amplified plasmid DNA by using the ZymoPURE Plas-
mid Miniprep kit (Zymo Research) or any other comparable
plasmid DNA isolation kit according to the manufacturer’s
instructions.
8. Confirm that sequences of your plasmid DNA preparations are
devoid of inadvertent mutations by sequencing.

3.4 Rescue of Bat The rescue procedure is exemplarily described for one transfection
Influenza A Virus by reaction (one well) in a 6-well plate and can be easily adjusted to
Reverse Genetics more reactions and a different format (12-well or 24-well). One day
prior to transfection, 5 × 105 HEK293T cells are seeded per well of
a 6-well plate in 2 mL culture medium and are grown at 37 °C with
5% CO2.
1. Bring Opti-MEM® medium to room temperature before
transfection.
2. For one transfection reaction, prepare 100 μL of Opti-MEM®
medium containing 8 μL Lipofectamine 2000 transfection
reagent (2 μL/μg DNA) and incubate for 10 min at room
temperature. Meanwhile, proceed preparing the plasmid trans-
fection mixture.
3. For the plasmid transfection mixture, prepare 100 μL of Opti-
MEM® medium containing 300 ng of each of the eight
pHW2000 rescue plasmids and 400 ng of each of the four
pCAGGS expression plasmids in a 1.5 mL Eppendorf tube.
4. Add the Opti-MEM® medium–Lipofectamine 2000 mixture
to the plasmid transfection mixture, mix gently, and incubate
for 20 min at room temperature.
5. Meanwhile, replace the culture medium of the HEK293T cells
with 1.5 mL Opti-MEM® medium.
6. Carefully pipette the transfection mixture (200 μL/well) onto
the HEK293T cells in small droplets, then gently sway the
plate, and place it at 37 °C with 5% CO2.
7. At 6 to 8 h post transfection, replace the cell supernatant with
2 mL rescue medium.
 Reverse Genetics of Bat Influenza A Viruses 83

8. At 48 h post transfection, collect the virus-containing cell

culture supernatant and remove the cell debris through centri-
fugation at 4000×g for 10 min. Transfer the supernatant in a
fresh tube, add 2 μL of TPCK-treated trypsin (1 μg/mL), and
immediately proceed with virus propagation. Alternatively,
store at -80 °C for later usage (see Note 4). The expected
rescue efficiency is 5 × 103 to 1 × 104 FFU/mL.

3.5 Propagation of One day prior to infection, 1 × 106 RIE1495 cells are seeded in a
Bat Influenza A T-75 flask in 10 mL culture medium and are grown at 37 °C with
Viruses In Vitro 5% CO2 to reach 80–90% confluency. The following paragraph
exemplarily describes infection of a single T-75 flask. However,
to produce sufficient viral stock titers, it is recommended
to concentrate the virus-containing supernatant of multiple flasks
(see Note 5).
1. Remove culture medium from the cells and pretreat cells with
5 mL PBS containing 0.2% BSA and 1 mg/mL DEAE-dextran
for 15 min at room temperature (see Note 6). Occasionally,
sway the flask gently to prevent the cells from drying.
2. Wash cells with PBS containing 0.2% BSA.
3. Infect cells with 4 mL virus-containing rescue supernatant in
10 mL infection medium and incubate the flask at 37 °C with
5% CO2.
4. At 48 h post infection, 7 mL fresh infection medium was added
onto the cells.
5. At 60 h post infection, harvest the cell culture supernatant and
remove cell debris through centrifugation at 4000×g for
10 min at room temperature. Transfer the supernatant in a
fresh tube.
6. Prepare an ultracentrifugation tube by pipetting a 5 mL 30%
sucrose cushion, then carefully layer the 17 mL cell superna-
tant, and concentrate the propagated virus through ultracen-
trifugation at 25,000 rpm for 2 h at 4 °C (Beckman coulter,
Optima L-90K Ultracentrifuge, SW 32 Ti rotor).
7. After ultracentrifugation, carefully remove the culture medium
and the sucrose layer and resuspend the virus pellet in 1–2 mL
infection medium.

3.6 Titration of Bat Viral titers are determined as endpoint titers by immunofluores-
Influenza A Viruses cence assay. It is recommended to titrate virus stocks at least in
triplicates. One day prior to infection, 1 × 104 MDCK II cells per
well of a 96-well plate are seeded in 100 μL culture medium and are
grown at 37 °C with 5% CO2 to reach 90–100% confluency.
84 Susanne Kessler et al.

1. Remove culture medium from the cells and pretreat with

100 μL PBS containing 0.2% BSA and 1.5 mg/mL DEAE-
dextran for 30 min at room temperature.
2. Meanwhile, prepare a tenfold dilution series of the virus stock
in infection medium in a separate 96-well plate.
3. Remove the inoculum and infect cells with 90 μL of the serially
diluted virus stock. Incubate the cells for 48 h at 37 °C with 5%
CO2.
4. At 48 h post infection, remove the inoculum and fix cells with
100 μL of 4% PFA per well for 15 min at room temperature.
5. Remove fixation solution and wash cells three times with
100 μL PBS.
6. Permeabilize cell membranes by using 100 μL of 0.5% Triton
X-100 per well for 5 min at room temperature. Wash cells three
times with 100 μL PBS.
7. Prepare primary antibody solution by diluting antibodies spe-
cific to bat influenza A virus antigens (e.g., NP) in PBS contain-
ing 5% NGS. Incubate cells in 50 μL of the primary antibody
solution for 1 h at room temperature.
8. Remove antibody solution and wash cells three times with
100 μL PBS.
9. Prepare fluorophore-conjugated secondary antibody solution
in PBS containing 5% NGS. Incubate cells in 50 μL of the
secondary antibody solution for 45 min at room temperature.
10. Remove antibody solution and wash cells three times with
100 μL PBS (see Note 7).

4 Notes

1. Note, while we described primers containing the BsmBI

restriction sites, alternative restriction sites may be used to
avoid internal digestion of the segment. It is thus recom-
mended to check the plasmid map before primer design and
cloning.
2. A list of cells supporting H18-mediated entry was published
previously [20]. For virus stock production, MDCK HLA-DR
cells [19], which stably overexpress the human HLA-DR com-
plex, can be used as an alternative to RIE1495 cells.
3. The amount of enzyme and the type of digestion buffer depend
on the manufacturer’s instructions.
4. Although rescue supernatant can be stored at -80 °C, it is
highly recommended to immediately proceed with virus prop-
agation for stock generation as freezing decreases viral titers.
 Reverse Genetics of Bat Influenza A Viruses 85

5. Bat IAVs are highly prone to mutations in their surface glyco-

protein genes [22, 23]. It is therefore urgently recommended
to sequence virus stocks.
6. Alternatively, cells can be pretreated with 2 mL of 200 mU/mL
sialidase for 1 h at 37 °C.
7. Although not essential, we suggest to perform the penultimate
washing step with 100 μL PBS containing DAPI, Hoechst
33342, or a comparable dye (according to the manufacturer’s
instructions) to visualize cellular nuclei.

Acknowledgments

Figures were created with Biorender.com. This work was supported

by grants from the European Research Council (ERC) to
M.S. (NUMBER 882631—Bat Flu). This work was also partly
funded by CRIPT (Center for Research on Influenza Pathogenesis
and Transmission), an NIAID-funded Center of Excellence for
Influenza Research and Response (CEIRR, contract number
75N93021C00014) to A.G.-S.

Disclosures The A.G.-S. laboratory has received research support

from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex,
Johnson & Johnson, Dynavax, 7 Hills Pharma, PharmaMar,
ImmunityBio, Accurius, nanoComposix, Hexamer, N-fold LLC,
Model Medicines, Atea Pharma, Applied Biological Laboratories,
and Merck, outside of the reported work. A.G.-S. has consulting
agreements for the following companies involving cash and/or
stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex,
Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological
Laboratories, PharmaMar, Paratus, CureLab Oncology, CureLab
Veterinary, Synairgen, and Pfizer, outside of the reported work. A.
G.-S. has been an invited speaker in meeting events organized by
Seqirus, Janssen, Abbott, and AstraZeneca. A.G.-S. is the inventor
on patents and patent applications on the use of antivirals and
vaccines for the treatment and prevention of virus infections and
cancer, owned by the Icahn School of Medicine at Mount Sinai,
New York, outside of the reported work.

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 Chapter 6

Rescue of Infectious Salmon Anemia Virus (ISAV) from

Cloned cDNA
Daniela Toro-Ascuy, Matı́as Cárdenas, Yesseny Vásquez-Martı́nez,
and Marcelo Cortez-San Martı́n

Abstract
The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important
emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV,
which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization
and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish
promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and
mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral
RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of
the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious
rISAV in salmon cells.

Key words Infectious salmon anemia virus, ISAV, Viral RNA, Reverse genetics, Salmon cells, Trans-
fection, ITS-1, RNA polymerase I, RNA polymerase II

1 Introduction

ISAV belongs to the Orthomyxoviridae family, which is an envel-

oped, pleomorphic virus and has a viral particle size range from
90 to 140 nm [1]. Its genome consists of eight single-stranded,
negative-sensed RNA segments ((-)ssRNA) that encode at least
ten proteins. The detailed study of other members of the family
Orthomyxoviridae, such as influenza viruses A and B, has been
possible, thanks to the development of reverse genetics systems,
which allow the manipulation of the viral genome [2]. The reverse
genetics systems for influenza viruses are based on cloned cDNA
copies of each segment into plasmids that allow transcription of the
genomic viral RNAs from an RNA polymerase I promoter (pol-I)
and viral protein expression from an RNA polymerase II promoter
(pol-II), respectively (Fig. 1) [3–5]. An important obstacle in ISA

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_6,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

87
88 Daniela Toro-Ascuy et al.

Fig. 1 Schematic diagram of the reverse genetics system for ISA virus rescue. ASK cells are co-transfected
with 12 plasmids, 8 pSS-URG plasmids encoding each of the eight vRNA gene segments, and 4 plasmids
expressing the viral polymerase complex and the nucleoprotein (NP). After transcription of the vRNA segments
in the nucleus by the RNA polymerase I, they associate with the polymerase complex and NP to form viral
ribonucleoprotein (vRNP) complexes leading to de novo generation of ISA virus

virus rescue by reverse genetics has been the lack of an efficient

promoter for the RNA polymerase I, which was not previously
described for the Atlantic salmon. As all the promoters for RNA
polymerase I are species-specific [6], they do not have a clear
genetic structure and are found in the extensive intergenic spacer
(IGS) region of ribosomal RNAs [7]. Identifying the sequences
corresponding to the RNA polymerase I promoter and its enhan-
cers is difficult since the IGS region varies between 15 and 23 kb in
length in the Salmo genus [8, 9].
Understanding the virulence factors and pathogenic mechan-
isms of ISAV is essential to understand its molecular biology and
developing better vaccines. Thus, we sought to develop plasmid-
based reverse genetics system to generate recombinant ISAV
(rISAV) as a tool to further understand this virus. Due to the
need of a salmon species-specific promoter with the characteristics
of RNA polymerase I, we evaluated the use of a 571-bp-long region
of the internal transcribed spacer region 1 (ITS-1) of the Salmo
salar genome [9]. Flatworm ITS-1 has been described to contain
genetic elements such as a transcription promoter and regulator
 Infectious Salmon Anemia Virus Reverse Genetics 89

motifs with characteristics of functional ancestral sequences

[10]. Recently, we showed that ITS-1 promoters drive the RNA
synthesis of ISAV vRNA, thus acting as an RNA polymerase I
promoter [11]. In addition, to avoid the generation of vRNA
molecules containing additional nucleotides in both RNA ends
that affect the correct interaction between the viral genome and
the viral polymerase, we added the hammerhead and the hepatitis δ
virus ribozymes sequences, flanking the 5′ and the 3′ end of each
vRNA, respectively (Fig. 2). Finally, to ensure the completion of
transcription, the sequence of the rabbit β-globin terminator was
added to complete the design of the cassette [12]. With the strate-
gic incorporation of all these elements into the plasmid system,
Atlantic salmon kidney (ASK) cells did not require any element in
trans to generate recombinant ISAV. Therefore, this system is able
to express intact genomic vRNA, allowing the full rescue of infec-
tious rISAV.

2 Materials

2.1 Viral RNA 1. Commercial E.Z.N.A. total RNA kit I (Omega, Bio-Tek, Inc.).
Extraction Add β-mercaptoethanol to TRK buffer before use: 20 μL
β-mercaptoethanol per 1 mL of TRK buffer.
2. RNase Away.
3. 1.5 and 2 mL RNase-free tubes.
4. Molecular biology grade ethanol (100%).
5. RNase-free, DEPC-treated water.
6. Microcentrifuge.

2.2 cDNA Synthesis 1. PCR Nucleotide Mix (dNTP).

and PCR 2. SuperScript III One-Step RT-PCR System with Platinum® Taq
DNA Polymerase (Thermo Fisher).
3. Primers for RT-PCR of each genomic segment for ISAV
(Table 1).
4. RNase-free 0.2 mL PCR tubes.
5. RNase-free, DEPC-treated water.
6. Temperature/PCR cycler.
7. Analytic grade agarose (Lonza).
8. Wizard SV Gel and PCR Clean-Up System (Promega).

2.3 Cloning ISAV 1. SapI restriction enzyme.

cDNA 2. DNA T4 ligase.
3. pSS-URG plasmid.
90 Daniela Toro-Ascuy et al.

Fig. 2 Generation of the universal reverse genetics plasmid for ISAV. (a) Schematic of the design of the
pSS-URG cassette. The universal vector contains the sequence of the Salmo salar promoter (RNA pol I pro), the
hammerhead ribozyme (HH ribozyme), the ribozyme of the hepatitis δ virus (HδV ribozyme), and the
transcription terminator of the rabbit β-globin (transcription Ter). (b) The schematic diagram illustrates the
two SapI restriction site sequences in the pSS-URG, which are cut twice by SapI, yielding the vector
fragments. The SapI sites in the vector are eliminated and the ISAV viral cDNAs can be inserted in only one
orientation by T4 DNA ligase

Table 1
Primers used for the amplification of the eight genomic segments of ISAV via RT-PCR

Primer F (forward) Primer R (reverse)

Segment 5′-3′ 5′-3′
1a AGCTAAGAATGGACTTTATATCA AACCTTCGAAGCCAAACAGATAG
GAAAACACG
1b CAATATCAAGTCCGTTCGAC GTGG AGTAAAAAATGGACATTTTATTGATTAAA
AGTATCGTC
2 AGCAAAGAACGCTCTTTAATAACC AGTAAAAAATGCTCTTTTACTTATTAAAAA
T
3 AGCAAAGATTGCTCAAATCCC AGTTAAAATTGCTCTTTTCTTTATTTG
4 AGCTAAGATTGGCTGTTTCAAGA AGTAAAAATTGGCTTTTTGGAAAA
5 AGTTAAAGATGGCTTTTCTAACA AGTAAAAATTGGCTATTTATACAATTAA
ATTTT TAATG
6 AGCAAAGATGGCACGATTCA AGTAAAAAATGCACTTTTCTGTAAACG
7 AGCTAAGATTCTCCTTCTACAA AGTAAAAATTCTCCTTTTCGTTTTAAA
TGGA
8 AGCAAAGATTGGCTATCTACCA AGTAAAAAAAGGCTTTTTATCTTTTG
Segment 1 was amplified in two fragments using both 1a and 1b pair primers
 Infectious Salmon Anemia Virus Reverse Genetics 91

4. Temperature/PCR cycler.
5. Analytic grade agarose.
6. Wizard SV Gel and PCR Clean-Up System.
7. RNase-free, DEPC-treated water.
8. Chemocompetent Escherichia coli Novablue cells.
9. Luria–Bertani media.
10. Ampicillin 1 μg/mL.
11. Pure Yield TM Plasmid Miniprep system.
12. Pure Yield TM Plasmid Midiprep system.

2.4 Cloning of 1. Protein expression vector pTriex-3 (Novagen) or pCI-neo

Expression Plasmids (Promega) (see Note 1).
2. Primers that amplify the PB2, PB1, PA, and NP ORFs of
ISAV901_09 (Table 2).
3. Platinum Pfx DNA Polymerase.
4. PCR Nucleotide Mix (dNTP).
5. RNase-free, DEPC-treated water.
6. Restriction enzymes NcoI and XhoI for ORF PB2 and PA,
XhoI and SmaI for ORF PB1, MluI and XbaI for ORF of NP.
7. DNA T4 ligase.
8. Analytic grade agarose.
9. Wizard SV Gel and PCR Clean-Up System.
10. RNase-free, DEPC-treated water.
11. Chemocompetent Escherichia coli Novablue cells.
12. Luria–Bertani media.
13. Ampicillin 1 μg/mL.
14. Pure Yield TM Plasmid Miniprep system.
15. Pure Yield TM Plasmid Midiprep system.

2.5 Transfection of 1. Regular growth medium: Leibovitz medium (L-15), 10% fetal
Salmon Cells bovine serum, 50 μg/mL gentamicin, 40 μM
β-mercaptoethanol, and 6 mM L-glutamine.
2. Transfection medium: Medium L-15, 40 μM
β-mercaptoethanol, and 6 mM L-glutamine.
3. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4.
4. 12-well plates.
5. Fugene 6 (Promega).
6. Atlantic salmon kidney cells (ASK, ATCC_CRL-2747).
92 Daniela Toro-Ascuy et al.

Table 2
Primers used for the amplification that amplifies the PB2, PB1, PA, and NP ORFs of ISAV901_09 via
RT-PCR

Primer forward Primer reverse

PB2 NcoI-ATGCCATGGACTTTATATCAGAAAA XhoI-CCGCTCGAGAACACCATATTCA
CACGATCAGCG TCCATAGG
PB1 SmaI-TCCCCCGGGAAACTCTAGTAGGTG XhoI-CCGCTCGAGAACACGCTTTTTC
TTCTTAATCAC
NP MluI-CGACGCGTCATGGCCGATAAAGG XbaI-CGCTCTAGATCAAATGTCAGT
TATGAC GTCTTCCTC
PA NcoI-CATGCCATGGATAACCTCCGT XhoI-CCGCTCGAGTTGGGTACTGACT
GAATGCATAAACC GCAATTTTC

7. pSS-URG/S1-S8 plasmids and protein expression plasmids

(PB2, PB1, PA, and NP).
8. Incubator at 18 °C.

2.6 Isavirus Growth 1. Infection medium: Leibovitz medium (L-15),

50 μg/mL gentamicin, 40 μM β-mercaptoethanol, and
6 mM L-glutamine.
2. Regular growth medium.
3. Incubator at 18 °C.
4. Atlantic salmon kidney cells (ASK, ATCC_CRL-2747).
5. 6- or 12-well plates.
6. PBS.

3 Methods

The generation of ISAV viruses can be divided into the following

steps: (1) Extraction and amplification of the ISAV viral RNAs.
(2) Sequence analysis of the viral RNAs. (3) Cloning of the viral
cDNAs into the appropriate plasmid vectors for vRNA (pSS-URG)
or protein synthesis. (4) Transfection of cells with reverse genetics
plasmids. For efficient ISAV generation, it is essential to use cell
lines susceptible to replication of ISAV (i.e., ASK cells) and to
optimize the parameters of transfection in this cell line. (5) Amplifi-
cation of the generated viruses.

3.1 Viral RNA 1. Mix 350 μL of virus stock with 350 mL of buffer TRK. The
Extraction protocol outlined here is based on the E.Z.N.A. total RNA kit
I; however, other RNA extraction kits or procedures may be
used (see Note 2).
 Infectious Salmon Anemia Virus Reverse Genetics 93

2. Add 700 μL of cold EtOH 70% in sterile DEPC-treated water.

3. Transfer lysed virus solution onto a HiBind RNA column
placed in a 2 mL collection tube.
4. Centrifuge at 10,000×g for 1 min at room temperature.
5. Wash the HiBind RNA column with 500 μL of RNA wash
buffer I.
6. Centrifuge at 10,000×g for 1 min at room temperature and
discard the flow-through.
7. Add 500 μL of RNA wash buffer II directly into the HiBind
RNA column.
8. Centrifuge at 10,000×g for 1 min at room temperature and
discard the flow-through.
9. Repeat steps 7 and 8.
10. Centrifuge the HiBind RNA column at maximum speed for
2 min to completely dry the HiBind RNA column.
11. Pipette 40 μL of RNase-free water onto membrane.
12. Centrifuge for 1 min at 10,000×g to elute RNA.
13. Store RNA at -80 °C until further use.

3.2 cDNA Synthesis The following protocol is based on SuperScript™ III One-Step
and PCR RT-PCR System with Platinum® Taq DNA Polymerase. Other
commercially available enzymes or kits for reverse transcription
and PCR may be used.
1. Mix the following components on ice:

Extracted RNA 8 μL
Forward primer (10 μM) 0.5 μL
Reverse primer (10 μM) 0.5 μL
dNTPs (10 μM) 1.5 μL
RNase-free water 1 μL

2. Incubate at 65 °C for 5 min.

3. After incubation, immediately place reaction tubes on ice.
4. Add the following components:

2× buffer (provided with enzyme) 12.5 μL

SuperScript III RT/Platinum Taq mix (Invitrogen) 1 μL

5. Program the thermocycler for RT-PCR:

94 Daniela Toro-Ascuy et al.

RT cycle 50 °C for 30 min

One 94 °C for 2 min
cycle
35 cycles 94 °C for 15 s
52 °C (segments 1 and 6) or 49 °C (segments 2–5 and 7–8) for
30 s
68 °C for 2 min and 15 s (segments 1–4) or 1 min and 30 s
(segments 5–8)
One 68 °C for 5 min
cycle

6. Visualize the PCR products in a 0.8% agarose gel (w/v), and

then purify the PCR products from the gel using the Wizard SV
Gel and PCR Clean-Up System according to the manufac-
turer’s instructions.
7. Sequence the PCR products obtained for each genomic seg-
ment of ISAV.

3.3 Cloning of Viral The eight genomic segments of ISAV901_09 were synthesized by
cDNAs Genscript Co in the cloning vector pUC57 with each of its ends
containing a SapI restriction site. Alternatively, the pSS-URG plas-
mids containing each viral segment and the previously mentioned
elements that allow vRNA synthesis, and the expression plasmids,
can be synthesized by Genscript [15]. If that is the case, proceed
according to step 7.
For cloning of viral cDNAs into pSS-URG vector (see Note 3),
digest with restriction enzyme SapI (see Note 4).
1. 2 Digest 2 μg of each pUC57 vector containing the genomic
segments ISAV (cDNA) or the cDNA produced in the previous
step (Subheading 3.2) with the restriction enzyme SapI (5 U
enzyme/μg of DNA).
2. Digest 1 μg of pSS-URG vector with the restriction enzyme
SapI (5 U enzyme/μg of DNA).
3. Incubate digestion for 2 h at 37 °C.
4. Visualize the digestion products in a 0.8% agarose gel (w/v),
and then purify from the gel with the Wizard SV Gel and PCR
Clean-Up System according to the manufacturer’s
instructions.
5. Ligate the digested fragments using the following ligation mix:

DNA ligase-T4 (5 U/μL) 1 μL

10× DNA ligase T4 buffer 1 μL
Insert: linear plasmid Molar ratio 5:1
RNase-free water q.s to 10 μL
 Infectious Salmon Anemia Virus Reverse Genetics 95

6. Incubate for 16 h at 4 °C.

7. Use 10 μL of ligation mix to transform 100 μL of chemically
competent E. coli Novablue cells. If using synthetic plasmids,
use 1 ng of plasmid to transform 100 μL of chemically compe-
tent E. coli Novablue cells.
8. Incubate for 30 min on ice, 45 s at 42 °C, and 5 min on ice.
9. Add 900 μL of medium LB without ampicillin.
10. Incubate for 1 h at 37 °C with agitation.
11. Plate the bacterial cells on LB–agar with 1 μg/mL ampicillin.
12. Incubate for 16 h at 37 °C.
13. Analyze 20 bacterial clones for rapid analysis of colonies
[13]. If using synthetic plasmids, proceed directly to plasmid
purification as described in step 16.
14. Purify positive clones using the Pure Yield TM Plasmid Mini-
prep system according to the manufacturer’s instructions.
15. Sequence recombinant plasmids for each segment using pri-
mers specific for the Hδ and HH ribozyme sequences.
16. Purify positive clones (without spurious mutations by sequenc-
ing) using Pure Yield TM Plasmid Midiprep system according
to the manufacturer’s instructions.

3.4 Cloning of 1. In addition to the eight pSS-URG plasmids for the transcrip-
Expression Plasmids tion of the eight ISAV viral RNA segments, helper plasmids for
the expression of the ISAV viral proteins PB2, PB1, PA, and NP
must be prepared. As an alternative, pCDNA3.1 plasmids con-
taining the ORF of each protein can be directly purchased from
Genscript.
2. Design oligonucleotides that amplify the PB2, PB1, PA, and
NP open reading frames.
3. As a template for PCR amplification, use the respective (a) viral
cDNAs or (b) pSS-URG plasmids.
4. Clone PCR products into a protein expression vector, such as
pTriex-3 or pCI-neo or pCDNA3.1.
5. Sequence the resulting constructs to confirm the absence of
unwanted mutations introduced by PCR.

3.5 Transfection of 1. Seed 2.5 × 104 ASK cells/cm2 in a 12-well plate.

Salmon Cells 2. Incubate cells for 24–48 h at 18 °C in a regular growth
medium. Cells should be 80% confluent at the time of
transfection.
3. Premix DNAs for transfection (9 μg total): Use 250 ng each of
the PB2, PB1, PA, and NP protein expression plasmids and
1 μg of each pSS-URG plasmids for the transcription of ISAV
viral RNA. Total premix DNA volume should be ≤100 μL.
96 Daniela Toro-Ascuy et al.

4. Prepare the following transfection cocktail:

– Add 94 μL of transfection medium to an Eppendorf tube.
– Add 13.5 μL of transfection reagent (Fugene 6) per premix
of DNA (3:2 ratio Fugene:DNA).
– Incubate for ~5 min at room temperature.
– Add premixed DNAs.
– Vortex for 1 min and spin down.
– Incubate transfection mixture for 30 min at room
temperature.
5. Transfection:
– Growth medium needs to be discarded and cells need to be
washed before transfection.
– Wash cells twice with PBS; add 1 mL of PBS each time.
– Remove PBS and add the transfection cocktail dropwise
onto the cells.
– After adding the transfection mixture, add 300 μL of addi-
tional transfection medium dropwise onto the cells.
6. Incubate for 4 h at 18 °C.
7. Remove supernatant and add 1 mL of growth medium.
8. Controls should include the following:
– Untreated ASK cells.
– ASK cells treated with transfection reagent to monitor cyto-
toxic effects of the transfection reagent.
– ASK cells transfected with plasmids for vRNA synthesis, but
not plasmids for protein synthesis; no virus should be recov-
ered from this control.
– ASK cells transfected with plasmids for protein synthesis,
but not plasmids for vRNA synthesis; no virus should be
recovered from this control.
9. Incubate cells for 7 days at 15 °C; typically, no cytopathic effect
(CPE) is observed (see Note 5).
10. Collect virus-containing supernatant from transfected cells.
11. Optional:
– Spin down supernatant for 5 min at room temperature to
pellet ASK cells.
– Transfer supernatant to a new tube.
12. Store virus-containing supernatant at -80 °C until further use
(ideally store sample in aliquots).
 Infectious Salmon Anemia Virus Reverse Genetics 97

3.6 Isavirus Growth 1. Seed 2.5 × 104 ASK cells/cm2 in a 6- or 12-well plate in a
in ASK Cells regular growth medium.
2. Incubate cells for 1–2 days at 18 °C. Cells should be 80–90%
confluent at the time of infection.
3. Prepare tenfold dilutions of virus-containing supernatant col-
lected from transfected ASK cells. Dilute supernatant in infec-
tion medium.
4. Wash ASK cells twice with PBS.
5. Add 500 μL of (un)diluted virus-containing supernatant to
ASK cells. Infect ASK cells for 4 h at 15 °C.
6. Wash ASK cells three times with PBS to remove the inoculum.
7. Incubate cells with a regular growth medium.
8. Observe cells daily for CPE:
– CPE is indicative of virus replication.
– For undiluted virus-containing supernatant, CPE typically
appears within 7 days of infection.
– If no CPE is observed, analyze sample by RT-PCR in super-
natant (see Note 6).
9. When ~80% of ASK cells are lysed (typically 7 days post infec-
tion), harvest virus-containing supernatant.
10. Spin down supernatant for 5 min at 4 °C to pellet floating cells.
11. Transfer virus-containing supernatant to a new tube.
12. Store virus-containing supernatant at -80 °C until further use
(ideally store samples in aliquots).
13. Before the virus is used for further studies, its sequence should
be confirmed.
14. Before the virus is used for further studies, virus stocks should
be grown, and their titers should be determined by qPCR or
plaque assay [14].

4 Notes

1. pCDNA3.1 expression plasmids can be directly purchased from

Genscript if cloning needs to be avoided [15].
2. A minimum of 1 mL of supernatant is required for RNA
extraction and RT-PCR performed on the eight genome
segments.
3. The pSS-URG vector was designed to incorporate any geno-
mic segment of any ISAV strain.
98 Daniela Toro-Ascuy et al.

4. The incorporation of a restriction site for SapI allows

nucleotide-specific fusion of any two DNA segments without
the introduction of mutations or unwanted nucleotides.
5. If the virus cannot be rescued, or the rescue efficiency is low,
the following modifications can be attempted:
– Test different amounts and/or different ratios of protein
expression plasmids.
– Test different amounts and/or ratios of different transfec-
tion reagents.
– Incubate transfected cells for 10–14 days.
6. Use qRT-PCR to quantify the vRNA from segment 8 to deter-
mine the copy numbers from all of the cell culture supernatants
[15, 16].

Acknowledgments

We thank Prof. Daniel R. Perez for technical and scientific support

during ISAV reverse genetics development. We acknowledge finan-
cial support from Fondecyt 11110212, Fondecyt 1161006, and
VIU 14E025. M.C.-S.M. thanks to DICYT-USACH and
DGT-USACH. D.T.-A. thanks Conicyt-Chile for a PhD fellow-
ship. M.C. thanks DICYT-USACH 021943CSM-POSTDOC
fellowship.

References
1. Dannevig BH, Falk K, Namork E (1995) Iso- 7. Comai L (2004) Mechanism of RNA polymer-
lation of the causal virus of infectious salmon ase I transcription. Adv Protein Chem 67:123–
anaemia (ISA) in a long-term cell line from 155
Atlantic salmon head kidney. J Gen Virol 76 8. Castro J et al (1997) Molecular analysis of a
(Pt 6):1353–1359 NOR site polymorphism in brown trout
2. Garcia-Sastre A, Palese P (1993) Genetic (Salmo trutta): organization of rDNA inter-
manipulation of negative-strand RNA virus genic spacers. Genome 40(6):916–922
genomes. Annu Rev Microbiol 47:765–790 9. Reed KM, Hackett JD, Phillips RB (2000)
3. Fodor E et al (1999) Rescue of influenza A Comparative analysis of intra-individual and
virus from recombinant DNA. J Virol 73(11): inter-species DNA sequence variation in salmo-
9679–9682 nid ribosomal DNA cistrons. Gene 249(1–2):
4. Hoffmann E, Webster RG (2000) Unidirec- 115–125
tional RNA polymerase I-polymerase II tran- 10. Van Herwerden L, Caley MJ, Blair D (2003)
scription system for the generation of influenza Regulatory motifs are present in the ITS1 of
A virus from eight plasmids. J Gen Virol 81 some flatworm species. J Exp Zool B Mol Dev
(Pt 12):2843–2847 Evol 296(1):80–86
5. Neumann G et al (1999) Generation of influ- 11. Toro-Ascuy D et al (2015) Development of a
enza A viruses entirely from cloned cDNAs. reverse genetic system for infectious salmon
Proc Natl Acad Sci U S A 96(16):9345–9350 anemia virus: rescue of recombinant fluores-
6. Zobel A, Neumann G, Hobom G (1993) RNA cent virus by using salmon internal transcribed
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viral cDNA. Nucleic Acids Res 21(16): ron Microbiol 81(4):1210–1224
3607–3614
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12. Lanoix J, Acheson NH (1988) A rabbit beta- and titration in salmon ASK cells. J Fish Dis
globin polyadenylation signal directs efficient 37(11):989–995
termination of transcription of polyomavirus 15. Cárdenas M, Michelson S, Pérez DR,
DNA. EMBO J 7(8):2515–2522 Montoya M, Toledo J, Vásquez-Martı́nez Y,
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Molecular cloning: a laboratory manual. Cold anemia virus infectivity is determined by multi-
Spring Harbor Laboratory, Cold Spring ple segments with an important contribution
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14. Castillo-Cerda MT et al (2014) Development doi.org/10.3390/v14030631
of plaque assay for Chilean infectious salmon 16. Munir K, Kibenge FS (2004) Detection of
anaemia virus, application for virus purification infectious salmon anaemia virus by real-time
RT-PCR. J Virol Methods 117(1):37–47
 Chapter 7

Reverse Genetics System for Rift Valley Fever Virus

Breanna Tercero and Shinji Makino

Abstract
Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifes-
tations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In
ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant
animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed
vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of
reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools
for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be
rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus–
host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we
will describe the experimental procedures for the implementation of RVFV reverse genetics.

Key words Recombinant Rift Valley fever virus, Reverse genetics, T7 polymerase-driven rescue
system, Transfection of cDNAs, Plaque assay, Virus amplification

1 Introduction

Rift Valley fever virus (RVFV) is a mosquito-borne virus that is

maintained in nature in sub-Saharan Africa. RVFV can cause severe
disease in both humans and ruminants, who can become infected
by bites from infected mosquitos or direct contact with infected
animal tissue or blood [1]. In majority of cases, patients experience
an acute febrile illness but can generally recover. However, a small
percentage of patients develop a severe form of the illness charac-
terized by ocular damage, vision loss, late-onset encephalitis, and
hemorrhagic fever [1–14]. In ruminants, RVFV can cause high
mortality rates in young animals and high rates of abortion in
pregnant animals called “abortion storms,” resulting in an enor-
mous negative impact on the economy of affected regions [1]. RVF
has already spread outside continental Africa to Saudi Arabia,
Yemen, and Madagascar, highlighting its potential to spread to
any area of the world, including North America [15–

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_7,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

101
102 Breanna Tercero and Shinji Makino

17]. Furthermore, studies have shown that mosquito populations

in the USA support RVFV replication [15, 18, 19]. Considering
the health and economic concerns, the lack of availability of
licensed vaccines and anti-RVFV reagents for human or animal
use is of great concern.
RVFV, belonging to genus Phlebovirus, family Phenuiviridae,
and order Bunyavirales, is an enveloped, single-stranded, seg-
mented RNA virus, containing three negative-sense or ambisense
genomic RNA segments, termed L, M, and S (Fig. 1). The RNA
segments are encapsidated by the nucleocapsid, N protein, and
bound by the RNA-dependent-RNA polymerase, L protein, form-
ing the ribonucleoprotein (RNP) complexes (Fig. 1a). Upon entry
into cells, the incoming virus releases the RNPs into the cytoplasm,
where viral replication and transcription, mediated by N and L
proteins, occur [20–23]. Each RNA segment contains noncoding
regions (NCRs) flanking the open reading frames (ORF)

Fig. 1 RVFV virion, coding strategy, and viral RNA species produced during infection. (a) RVFV virion structure:
RVFV envelope consists of a lipid bilayer and two glycoproteins, Gn (dark blue) and Gc (light blue). The core of
the virus consists of three viral RNA segments (black lines) encapsidated by the nucleocapsid, N protein
(yellow), and bound by the RNA-dependent RNA polymerase, L protein (purple), forming ribonucleoprotein
complexes. Created by Biorender.com. (b) RVFV coding strategy: RVFV carries a single-stranded, negative-
sense, tri-segmented RNA genome, consisting of the large (L), medium (M), and small (S) viral RNA segments.
The genomic strands of L and M segments are used as templates to transcribe L and M mRNAs, respectively.
The L mRNA encodes the L protein, and the M mRNA encodes the Gn/Gc, NSm, and 78 kDa proteins. The S
segment has an ambisense coding strategy, wherein the N mRNA, encoding the N protein, is transcribed from
the genomic RNA, and the NSs mRNA, encoding the NSs protein, is transcribed from the antigenomic RNA. IGR
intergenic region
 Reverse Genetics System for Rift Valley Fever Virus 103

[24]. RVFV RNA segments form a panhandle structure due to the

presence of complementary sequences in the 3′ and 5′ terminal
ends of NCRs [25]. The transcriptional promoters and termination
sequences for the synthesis of viral mRNAs reside within the NCRs
[26]. The 3′ NCRs of antigenomic L and M segments carry tran-
scription termination signals for L mRNA and M mRNA, respec-
tively [27]. The S segment carries an intergenic (IGR) region,
which harbors the transcription terminal signals for N and NSs
mRNA, between the N and NSs ORFs (Fig. 1b) [27–29].
The genomic L and M RNAs serve as templates for the synthe-
sis of their cognate antigenomic RNAs and mRNAs (Fig. 1). The L
segment encodes the L protein, and the M segment encodes a
polyprotein precursor that is cleaved to produce the two envelope
glycoproteins Gn/Gc, a nonstructural protein NSm, and an acces-
sory protein, 78 kDa. The S RNA utilizes an ambisense coding
strategy for gene expression. The genomic S RNA segment serves
as a template for the synthesis of N mRNA, encoding the N
protein, while the antigenomic S RNA segment serves as the tem-
plate for the synthesis of NSs mRNA, which encodes the virulence
factor, NSs protein [24] (Fig. 1). RVFV mRNAs contain a host-
derived cap structure at the 5′ end, obtained by a cap-snatching
mechanism employed by the N and L proteins, and do not contain
poly (A) sequences at the 3′ end [30, 31]. The RVFV envelope
glycoproteins, Gn/Gc, mediate the attachment of virus to cell
receptors followed by endosomal membrane fusion, and drive
viral assembly, which occurs at the Golgi compartment [32].
The establishment of reverse genetics systems for bunyaviruses
has provided powerful experimental approaches for investigating
virus replication, virus–host interactions, and mechanisms of path-
ogenesis. More importantly, reverse genetics has been used to
generate recombinant infectious viruses carrying mutations within
their genomes that can serve as live-attenuated vaccine candidates.
RVFV has been successfully rescued from cloned cDNAs using
both the RNA polymerase I/II system and the bacteriophage T7
RNA polymerase system, the latter being the system described in
this chapter [33–35]. In this system, plasmids, encoding the viral
RNA segments under the control of the T7 promoter, are
co-transfected with supporter plasmids, expressing N and L pro-
teins, into cells where the T7 RNA polymerase is provided in trans
using either eukaryotic expression plasmids or T7 RNA polymerase
stable-expressing cell lines. The expressed N and L proteins drive
the replication and transcription of viral RNA segments, which are
produced from the T7 polymerase-driven plasmids in the cyto-
plasm. This results in the accumulation of all the viral genomic
RNA segments and viral proteins necessary for the recovery of
infectious virus particles. In addition, a plasmid expressing the Gn
and Gc glycoproteins can also be co-transfected into these cells but
is not an absolute requirement for virus rescue [35].
104 Breanna Tercero and Shinji Makino

Rescue of the highly pathogenic RVFV strains ZH548 and

ZH501 requires the use of an enhanced BSL-3 or a BSL-4 facility,
making it difficult for researchers to handle and study these viruses.
However, a live-attenuated RVFV vaccine strain, MP-12, can be
handled in a BSL-2 laboratory, allowing researchers to study RVFV
without the constraints of working in a high containment labora-
tory. The MP-12 virus was generated by serial passages of the
RVFV ZH548 strain in the presence of a chemical mutagen,
5-fluorouracil, resulting in 23 mutations in the three genome seg-
ments and rendering the virus highly attenuated [36, 37]. In this
chapter, we will focus on a reverse genetics technique, based on
BSR-T7/5 cells that stably express T7 polymerase, for the genera-
tion of the attenuated RVFV strain, MP-12 [35, 38]. We describe
the procedures to rescue infectious MP-12 virus as well as the
methods for the amplification and titration of MP-12 in Vero E6
cells. It is to be noted that this method has also been used to rescue
RVFV strain ZH501 in a BSL-4 laboratory, although the use of the
plasmid expressing the Gn and Gc glycoproteins was not
included [39].

2 Materials

2.1 RVFV Reverse 1. pProT7-L (+), pProT7-M (+), and pProT7-S (+) [35]: The
Genetics Plasmids full-length antigenomic sense (+) L, M, and S RNA segments
were cloned into pProT7 RNA expression plasmids, wherein
2.1.1 Viral RNA
each viral RNA segment was placed between the T7 promoter
Expression Plasmids
and the hepatitis delta virus (HDV) ribozyme, followed by the
T7 terminator sequence. The HDV ribozyme is used to cleave
the viral RNAs and obtain authentic viral RNA 3′ ends. These
plasmids direct the synthesis of L, M, and S antigenomic RNAs
(see Notes 1 and 2).

2.1.2 Viral Protein 1. pT7-IRES-vL and pT7-IRES-vN [40]: pT7-IRES contains an

Expression Plasmids encephalomyocarditis virus (EMCV) internal ribosome entry
site (IRES) sequence that is placed between the T7 promoter
and the inserted ORF to drive the IRES-mediated translation
of the encoded ORFs. pT7-IRES plasmids encoding the nucle-
ocapsid (vN) or the polymerase (vL) ORFs are used to produce
N and L proteins, respectively, which are required for RVFV
RNA replication and transcription (see Notes 2 and 3).
2. pCAGGS-vG [35]: pCAGGS contains the chicken polymerase
II-driven β-actin promoter. The entire ORF of the M segment
in pProT7-M (+) was cloned into the multiple cloning site of
pCAGGS protein expression plasmid resulting in the expres-
sion of 78 kDa, NSm, and Gn/Gc envelope glycoproteins. This
plasmid is used for the synthesis of RVFV envelope glycopro-
teins, which are required for the generation of virus particles
(see Note 2).
 Reverse Genetics System for Rift Valley Fever Virus 105

2.2 Cell Lines 1. For virus rescue: BSR-T7/5 cells stably expressing T7 RNA
polymerase (see Note 4): BSR-T7/5 cells are a clone of the
baby hamster kidney (BHK-21) cell line that stably express the
T7 RNA polymerase under selection in media containing G418
(Geneticin) [38].
2. For virus amplification and titration: Vero E6 (African green
monkey kidney epithelial) cells.

2.3 Tissue Culture 1. BSR-T7/5 cell growth media: Glasgow’s minimal essential
Media medium (GMEM) supplemented with 10% fetal bovine
serum (FBS), 10% tryptose phosphate broth (TPB), 1 mg/
mL G418, 1X penicillin/streptomycin (100 unit/mL of peni-
cillin, 100 μg/mL of streptomycin), 1X MEM amino acid
solution, and sodium bicarbonate.
2. BSR-T7/5 cell transfection media: Same as growth media
without antibiotics.
3. Opti-MEM I (Gibco).
4. Vero E6 growth media: Dulbecco’s modified minimum essen-
tial medium (DMEM) containing 5% FBS and 1X penicillin/
streptomycin (100 unit/mL of penicillin, 100 μg/mL of
streptomycin).
5. Modified Eagle Media (MEM) 2X containing 10% FBS and
10% TPB.
6. Virus dilution media: DMEM without FBS and containing 1X
penicillin/streptomycin (100 unit/mL of penicillin, 100 μg/
mL of streptomycin).

2.4 Reagents and 1. 60 mm cell culture dish.

Supplies 2. TransIT-LT1 (Mirus) transfection reagent: A LT1:DNA ratio
of 3:1 is used for virus rescue (see Note 5).
3. 1.7 mL Eppendorf microcentrifuge tubes.
4. 15 mL conical tube.
5. 2 mL cryotubes.
6. 6-well cell culture plate.
7. 96-well deep-well plate.
8. 1.2% Noble agar, prepared in autoclaved distilled water.
9. Neutral red solution.
10. Phosphate-buffered saline (PBS).
11. 100 mm cell culture dish.
106 Breanna Tercero and Shinji Makino

3 Methods

3.1 RVFV MP-12 1. Seed ~8 × 105 BSR-T7/5 cells in a 60 mm dish approximately

Generation by Reverse 24 h prior to transfection.
Genetics 2. Incubate cells in BSR-T7/5 cell growth medium at 37 °C and
3.1.1 Preparation of 5% CO2. Cells should be 70–80% confluent at the time of
BSR-T7/5 Cells transfection (see Note 6).
3. Before transfection mix is added, remove growth media from
cells and add 5 mL of BSR-T7/5 cell transfection media lack-
ing antibiotics (see Note 7).

3.1.2 Transfection Mix 1. Table 1: Recommended amounts of Opti-MEM I, DNA, and

TransIT-LT1 transfection reagent for a 60 mm dish.
2. Premix DNAs for transfection: In a 1.7 mL microcentrifuge
tube, prepare the RVFV MP-12 DNA plasmid mixture using
the recommended amounts (Table 1).
3. Total amount of DNA to be transfected should be equal to
11 μg.
4. In a separate microcentrifuge tube, add 375 μL of Opti-MEM I
media and the premixed DNAs.
5. Add 33 μL of TransIT-LT1 reagent to the tube containing
Opti-MEM I and premixed DNAs.
6. Incubate the transfection mixture for 15 min at room temper-
ature (RT).

Table 1
Amount of plasmid DNA, TransIT-LT1, and media for RVFV rescue

Plasmid Concentration (60 mm dish)

pT7-IRES-vL 1.1 μg
pT7-IRES-vN 2.2 μg
pCAGGS-vG 1.1 μg
pProT7-L (+) 2.2 μg
pProT7-M (+) 2.2 μg
pProT7-S (+) 2.2 μg
Total DNA 11 μg
Reagent Volume
TransIT-LT1 33 μL
Opti-MEM I 375 μL
Cells 70–80% confluency
Recommended amounts of plasmids and TransIT-LT1 to generate recombinant MP-12
virus in a 60 mm dish are indicated
 Reverse Genetics System for Rift Valley Fever Virus 107

3.1.3 Transfection 1. Add the transfection mixture dropwise to cells.

2. Incubate the cells at 37 °C and 5% CO2.
3. After 24 h, remove the transfection media from cells.
4. Add BSR-T7/5 growth media, containing antibiotics, to cells.
5. Incubate the cells at 37 °C and 5% CO2 for an additional
4 days, which is a total of 5 days after transfection.

3.1.4 Virus Collection 1. Collect the tissue culture supernatant containing RVFV into a
and Clarification: P0 15 mL conical tube.
Rescue Stock 2. Remove cell debris by centrifugation (2500 × g, for 15 min at
4 °C).
3. Collect the supernatant containing the virus and discard the
cell pellet.
4. Aliquot the virus into cryotubes and store at -80 °C (see
Note 8).

3.2 Plaque Assay of It is possible that the RVFV titers from the initial rescue are low,
RVFV MP-12: P0 and, therefore, we recommend amplifying the virus to generate
Rescue Stock a working stock. Before the preparation of a working stock
of the virus (P1), the titer of the P0 rescue stock should
be determined by plaque assay. This can be used to evaluate
the transfection efficiency of the rescue system and to determine
the dilution of the P0 virus stock required for the generation
of the P1 working stock (see Note 9).
1. Seed ~5 × 105 Vero E6 cells/well in a 6-well plate in growth
medium approximately 24 h prior to infection.
2. Incubate cells at 37 °C and 5% CO2. Cells should be 95–100%
confluent at the time of infection (see Note 10).
3. Prepare a tenfold serial dilution of the P0 virus stock (10-1–
10-6) in duplicate wells: Add 450 μL of virus dilution media
into the wells of a 96-well deep-well plate and then add 50 μL
of P0 virus stock into the first dilution wells (10-1). Carefully
pipette up and down to mix and then discard the pipette tip (see
Note 11). Using a new pipette tip, transfer 50 μL of the first
dilution into the second dilution wells (10-2) and mix. Con-
tinue this process until the final dilution (10-6).
4. Remove media from Vero E6 cells.
5. Add 400 μL of the serially diluted virus stock to Vero E6 cells.
6. Incubate cells for 1 h at 37 °C and 5% CO2. Rock the plate
gently every 15 min to ensure the even distribution of the virus
inoculum on the cells.
108 Breanna Tercero and Shinji Makino

Fig. 2 Plaque formation by MP-12 in Vero E6 cells. Vero E6 cells were infected
with MP-12 virus and overlaid with medium containing Noble agar. Plaques were
stained with neutral red solution

7. During incubation, prepare the Noble agar and 2X MEM

media: Microwave 1.2% Noble agar and place it in a 42 °C
water bath until the virus incubation is complete. Warm the 2X
MEM media at 42 °C.
8. After incubation, aspirate the inoculum.
9. Prepare a mixture of Noble agar and 2X MEM media (1:1
ratio). Add 2 mL of the mixture to each well (see Note 12).
The final concentrations of the components in the mixture
should be 1X MEM, 0.6% Noble agar, 5% FBS, and 5% TPB.
Perform this step quickly to prevent the Noble agar/2X MEM
mixture from solidifying before adding to the wells.
10. Incubate the cells for 3 days at 37 °C and 5% CO2.
11. On day 3, add 0.5 mL of neutral red solution (10% neutral red
in PBS) to each well.
12. Incubate the cells for a minimum of 4 h at 37 °C and 5% CO2.
Plaques should become visible starting at 4 h (see Note 13).
13. Count plaques in wells in which the plaques can be clearly
distinguished (Fig. 2).
14. Calculate the plaque-forming units (PFU) per mL using the
following formula:
PFU/mL = number of plaques x dilution factor × 2.5.

3.3 Amplification of 1. Seed ~2 × 106 Vero E6 cells in a 100 mm dish in growth

RVFV MP-12: P1 medium approximately 48 h prior to infection.
Working Stock 2. Incubate cells at 37 °C and 5% CO2. Cells should be 90–100%
confluent at the time of infection.
 Reverse Genetics System for Rift Valley Fever Virus 109

3. Remove media.
4. Calculate the multiplicity of infection (MOI) for the genera-
tion of P1 working stock. We recommend an MOI of 0.01 for
the infection with P0 stock virus to generate P1 working stock
(see Note 14). MOI calculation: desired PFU of virus for
infection = number of cells at confluency × MOI 0.01.
5. Dilute P0 virus stock with the virus dilution media accordingly
to prepare the accurate PFU of virus for infection at the MOI of
0.01.
6. Add the diluted virus inoculum (2 mL) to Vero E6 cells.
7. Incubate cells for 1 h at 37 °C and 5% CO2. Rock the plate
gently every 15 min to ensure the even distribution of the virus
inoculum on the cells.
8. After incubation, wash cells with PBS or growth medium to
remove the virus inoculum.
9. Add growth medium (10 mL) to cells and incubate for
2–3 days at 37 °C and 5% CO2. Observe the formation of
cytopathic effect (CPE) daily (see Note 15).
10. Collect the supernatant containing virus when the CPE is
80–90%, and clarify the supernatant, as described in Subhead-
ing 3.1, step 4 (see Note 16).
11. Aliquot the P1 working stock of virus in cryotubes and store at
-80 °C.
12. Evaluate the titer of the P1 working stock by plaque assay, as
described in Subheading 3.2 (see Notes 17 and 18).

4 Notes

1. RVFV rescue was more efficient when the RNA expression

plasmids produced the antigenomic (+) sense viral RNAs.
Virus rescue using RNA expression plasmids that express the
genomic (-) sense viral RNAs resulted in unsuccessful virus
rescue or extremely low viral titers [35].
2. Plasmids for the generation of recombinant RVFV MP-12 can
be grown in One Shot TOP10 Chemically Competent E. coli
cells using Luria broth (LB) media in the presence of 100 μg/
mL of ampicillin. Plasmids can be purified using Nucleobond
Xtra Midiprep kit following manufacturer’s recommendations
and stored at -20 °C. pProT7 and pT7-IRES plasmids are
low-copy number plasmids; thus, purification of these plasmids
should use the Nucleobond Xtra low-copy recommended midi
protocol. pCAGGS plasmid is a high-copy number plasmid and
therefore the Nucleobond Xtra protocol for high-copy midi
should be followed. Alternatively, a commercial maxiprep kit
(Qiagen) can also be used for plasmid DNA purification.
110 Breanna Tercero and Shinji Makino

3. It is important to note that the inclusion of a plasmid expres-

sing NSs protein did not provide any advantage for the rescue
of RVFV. In fact, the recovery of RVFV in the presence of NSs
protein was unsuccessful in previous rescue attempts [35].
4. Alternatively, BHK/T7-9 cells that stably express T7 RNA
polymerase can be used for RVFV rescue, if BSR-T7/5 cells
are not available [35, 41].
5. TransIT-LT1 is the most suitable transfection reagent for
RVFV reverse genetics system.
6. A high amount of DNA is transfected into BSR-T7/5 cells
during virus rescue; therefore, the confluency must be around
70–80%. Cells transfected at 40–50% confluency will result in
cell death and unsuccessful virus rescue.
7. To prevent cytotoxicity and low transfection efficiency,
antibiotic-free growth media is used during transfection.
8. Virus stock should be aliquoted into small volumes to prevent
multiple freeze–thaw cycles, which can reduce RVFV titers.
9. The titer of the P0 rescue stock should be determined prior to
the amplification for the generation of the P1 working stock.
This will enable the calculation for infection with the P0 virus
stock at a low MOI, which reduces the likelihood of generation
of defective interfering (DI) particles in the resulting P1 work-
ing stock. The titers of the P0 virus usually range from 104 to
106 PFU/mL, depending on the transfection efficiency.
10. Monolayers with lower cell confluency could lead to inaccurate
virus titer measurements compared to highly confluent
monolayers.
11. Always change pipette tips before transferring to the next
dilution to prevent carryover contamination.
12. The mixture of Noble agar and 2X MEM media should not be
hot when it is added to the cells because the cells will die and
detach from the plate, rendering the plaque assay ineffective.
13. Alternatively, after neutral red staining, the plates can be
wrapped in foil and incubated overnight at RT before counting
plaques the next day.
14. DI RNAs are deletion mutants of the parent virus that are
replication-competent and can be packaged into virus particles
[42]. DI particles can appear spontaneously in cell culture due
to serial passages of virus at a high MOI. Due to their smaller
size, relative to full-length viral RNA, DI RNAs replicate more
efficiently, resulting in a decrease in the parent virus replication.
To reduce the chances of DI RNA generation in the P1 work-
ing virus stock, we recommend generating P1 virus at a
low MOI.
 Reverse Genetics System for Rift Valley Fever Virus 111

15. CPE induced by RVFV infection in Vero E6 cells is character-

ized by rounding of the cells and disruption of the cell mono-
layer. By 2 days post-infection, a significant number of dead
cells will be floating in the supernatant. We recommend col-
lecting the supernatant 2–3 days post-infection when the CPE
is 80–90%.
16. The titers of the P1 working virus stock will be lower if the
CPE is 100% (all cells dead and floating) at the time of virus
collection.
17. It is possible that the P0 and P1 virus stocks carry truncated
RNAs, which are generated from the L RNA segment during
P0 virus rescue or P1 virus amplification. We recommend
checking for the presence of truncated RNAs or DI particles
in the virus stocks by Northern blot analysis, as these RNAs
may interfere with viral replication and growth.
18. P1 working stock virus titers range from 106 to 107 PFU/mL.

Acknowledgments

This study was supported by Public Health Service grant AI148763

from the National Institutes of Health and pilot grants from the
Institute for Human Infections and Immunity at the University of
Texas Medical Branch. BT was supported by the James
W. McLaughlin Postdoctoral Fellowship Fund at the University
of Texas Medical Branch.

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 Chapter 8

Plasmid-Based Lassa Virus Reverse Genetics

Luis Martı́nez-Sobrido, Chengjin Ye, and Juan Carlos de la Torre

Abstract
Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public
health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is
estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of
Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are
available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin
whose efficacy remains controversial. The development of reverse genetics approaches has provided inves-
tigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of
mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-
acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has
facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to
rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined muta-
tions in their genomes to investigate virus–host interactions and mechanisms of viral pathogenesis. Reverse
genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus
detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise,
reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing
mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without
the need of high security biocontainment laboratories. In this chapter, we describe the experimental
procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.

Key words Lassa virus, Recombinant Lassa virus, Lassa virus reverse genetics, Lassa virus rescue
systems

1 Introduction

The family Arenaviridae consists currently of five genera: Anten-

navirus, Hartmanivirus, Inmovirus, and Reptarenavirus [1]. The
genus Mammarenavirus includes over 43 species, the genus
Reptarenavirus currently includes 5 species, the genus Hartmani-
virus includes 8 species, the genus Antennavirus currently includes
3 species, and the genus Inmovirus currently includes a single
species. Based on antigenic properties, mammarenaviruses have
been divided traditionally into two distinct groups. Old World

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_8,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

115
116 Luis Martı́nez-Sobrido et al.

(OW) mammarenaviruses (“Lassa–lymphocytic choriomeningitis

serocomplex”) include viruses indigenous to Africa and the ubiqui-
tous lymphocytic choriomeningitis virus (LCMV), whereas New
World (NW) mammarenaviruses (“Tacaribe serocomplex”) include
viruses indigenous to the Americas [1]. This classification is largely
consistent with phylogenetic data and muroid rodent host phylog-
eny, with OW mammarenaviruses infecting murid rodents primarily
in Africa and NW mammarenaviruses infecting cricetid rodents
primarily in the Americas [1]. Genetically, OW mammarenaviruses
constitute a single lineage, while NW mammarenaviruses are fur-
ther classified into clades A, B, A/B, and C [1]. Asymptomatically,
mammarenavirus-infected rodents move freely in their natural hab-
itat and may invade human dwellings. Humans are infected most
likely through mucosal exposure to aerosols, or by direct contact
between infectious materials and abraded skin [1]. Mammarena-
virus infections in humans are common and in some cases severe
[1]. Several mammarenaviruses, chiefly Lassa virus (LASV) in West-
ern Africa [1] and Junin virus (JUNV) in the Argentinean Pampas
[2], cause hemorrhagic fever (HF) disease in humans and represent
important public health problems in their endemic areas. More-
over, evidence indicates that the worldwide-distributed prototypic
mammarenavirus LCMV is a neglected human pathogen of clinical
significance [3] that poses a special threat to immunocompromised
individuals [4]. In addition, several mammarenaviruses, including
LASV, have features that make them credible biodefense
threats [5].
LASV is the mammarenavirus with the highest impact in public
health [6], estimated to infect several hundred thousand individuals
yearly in West Africa resulting in a high number of LF cases asso-
ciated with high morbidity and significant mortality [7]. Moreover,
increased travel from and into endemic areas has led to the impor-
tation of LF cases into the United States, Europe, Japan, and
Canada [8]. Recent studies indicate that LASV endemic regions
are expanding [9], and the recent identification of the mammar-
enavirus Lujo virus (LUJV) as responsible for a cluster of fatal cases
of HF disease in Southern Africa [10] has raised concerns about the
emergence of novel HF-causing OW mammarenaviruses outside
their current known endemic regions. Concerns caused by human
pathogenic mammarenavirus infections are further aggravated by
the lack of Food and Drug Administration (FDA)-licensed vaccines
and current antiviral drug treatment being limited to the use of
ribavirin whose efficacy remains controversial [11].
Mammarenaviruses are enveloped viruses with a bi-segmented
negative-sense, single-stranded, RNA genome (Fig. 1a) [1]. Each
genome segment encodes, using an ambisense coding strategy, two
viral proteins in opposite orientation separated by a noncoding
intergenic region (IGR) (Fig. 1a) [1]. The large (L, top) segment
encodes the viral RNA-dependent RNA polymerase (RdRp) or L
 LASV Reverse Genetics 117

A) B)
L vRNA
IGR
L
5’ Z 3’
UTR UTR

S vRNA
IGR
5’ GPC
NP 3’ GP1
UTR UTR L NP Z
GP2

Fig. 1 LASV genome replication and virion structure. (a) Genome organization: LASV has a bi-segmented
negative-sense single-stranded RNA genome. Each of the two viral (v)RNA genome segments uses an
ambisense coding strategy to direct the synthesis of two viral proteins in opposite orientation. The large (L,
top) vRNA segment (7.2 kb) encodes the viral L and Z proteins. The small (S, bottom) vRNA segment (3.5 kb)
encodes the viral NP and GPC. IGR intergenic region. UTR untranslated regions. (b) Virion structure: LASV are
enveloped viruses whose virion surface is decorated by the matured GP complex constituted of heterotrimers
of SSP/GP1/GP2. GP1 binds to cell surface receptor used by the virus and GP2 mediates the pH-dependent
fusion event at the late endosome resulting in release of the vRNP into the cell cytoplasm where it directs the
biosynthetic processes of replication and gene transcription of the viral genome. Underneath the viral
membrane is a layer made of the Z protein (orange). The core of the virus is made of two viral ribonucleopro-
tein (vRNP) complexes, composed of the viral genome segments (black lines) encapsidated by the viral NP
(red). NP-Z interaction mediates incorporation of the vRNPs into nascent LASV virions. Associated with the two
vRNP complexes is the viral RNA-dependent RNA polymerase L protein (green) that, together with NP, is the
minimal component required for LASV genome replication and gene transcription

protein (Fig. 1a, green) [12], and the matrix Z protein (Fig. 1a,
orange) [13], which is the major driving force of mammarenavirus
budding [13]. The small (S, bottom) segment encodes the viral
glycoprotein precursor (GPC) (Fig. 1a, blue) that is proteolytically
cleaved by signal peptidases and the proprotein convertase (PC)
subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) to yield a
stable signal peptide (SSP), the N-terminal GP1, and the
membrane-anchored GP2. The mature mammarenavirus GP com-
plex, made of SSP/GP1/GP2 heterotrimers, forms the spikes that
decorate the virion surface and mediate virus cell entry (Fig. 1b).
GP1 binds to cellular receptors, whereas the transmembrane GP2
mediates viral fusion [1]. Segment S also encodes the viral nucleo-
protein (NP) (Fig. 1a, red), which is responsible of encapsidating
the viral RNA (Fig. 1b), and, together with the L polymerase
protein and the viral RNA, forms the viral ribonucleoproteins
(vRNPs) that are the minimal trans-acting factors required for
mammarenavirus genome replication and gene transcription
[12, 14] (Fig. 2). In addition, mammarenavirus NP mediates the
incorporation of the vRNPs into mature infectious virions by inter-
acting with Z [15]. Furthermore, NP has also been shown to
counteract the host type-I interferon (IFN-I) [16–21] and
118 Luis Martı́nez-Sobrido et al.

Genomic RNA
IGR
UTR UTR
5’ 3’
NP/L
GP/Z

TRANSCRIPTION

REPLICATION
TRANSLATION
NP/L NP L

Antigenomic RNA

3’ 5’
GP/Z
NP/L

TRANSCRIPTION IGR
GP1
TRANSLATION
GP/Z Z
GP2

Fig. 2 LASV genome replication and gene transcription: The L polymerase

associated with each of the two vRNPs initiates transcription from the viral
promoter located within the untranslated region (UTR, black boxes) at the 3′
termini of the vRNAs. Primary transcription results in the synthesis of NP (S
segment) and L (L segment) mRNAs. Transcription termination is mediated by
the intergenic region (IGR), a secondary stem-loop structure found in both vRNA
segments between each of the two viral genes. Subsequently, the virus L
polymerase adopts a replicase mode and moves across the IGR to generate a
copy of the full-length antigenome S and L vRNAs. The S and L antigenomic
vRNAs serve as templates for the synthesis of GPC and Z mRNAs, respectively.
The antigenomic S and L vRNAs also serve as templates for the amplification of
the corresponding vRNA genome species. The entire replication cycle of LASV
takes place in the cytoplasm of infected cells

inflammatory [20, 22] responses during viral infection to facilitate

viral replication.
Mammarenaviruses enter cells via receptor-mediated endocyto-
sis and delivered to the late endosomes. Alpha-dystroglycan (αDG)
has been described as the main receptor for OW and NW clade C
mammarenaviruses [23]. However, clade A, B, and A/B NW
mammarenaviruses appear to use the human transferrin receptor I
(Tfr1) for viral entry [24]. The low pH of the late endosome
triggers a conformational change of the GP2 transmembrane
domain, leading to the fusion between viral and cell membranes,
which results in the release of the vRNP into the cell cytoplasm
where viral RNA replication and gene transcription take place
[1]. Mammarenavirus gene transcription is mediated by the viral
promoters located within the untranslated regions (UTRs) at the 3′
 LASV Reverse Genetics 119

termini of viral RNA (vRNA) and complementary RNA species

(cRNA) [1] (Fig. 2). NP and L are located at the 3′ end of the S
and L viral segments, respectively, and are translated from mRNAs
with antigenomic sense polarity transcribed directly from the
vRNAs and are the first proteins produced upon mammarenavirus
infection [1] (Fig. 2). The 3′ ends of the non-polyadenylated viral
mRNAs have been mapped to a predicted stem-loop structure
within IGR [1] (Fig. 2). GPC and Z proteins, located at the 5′
end of the S and L genome viral segments, respectively, are trans-
lated from mRNAs derived from antigenome RNA species (cRNA)
after replication of the vRNAs [1] (Fig. 2). The cRNA segments
also serve as templates for the synthesis of new vRNAs [1] (Fig. 2).
Newly synthesized vRNAs are encapsidated by the viral NP to form
the vRNP complexes and are packaged into progeny infectious
virions by interaction with the viral Z protein [15]. Mammarena-
virus assembly involves the interaction of the new vRNP complexes
with the SSP/GP1/GP2 complex present at the membrane of
infected cells, a process mediated by interaction with the Z protein
[25]. New virions bud from the plasma membrane of infected cells
in a process mediated by the viral Z protein [13, 26–28].
The development and implementation of mammarenavirus
reverse genetics systems have provided investigators with new and
powerful experimental approaches for the investigation of mam-
marenavirus molecular and cell biology. The use of cell-based mini-
genome approaches has allowed the study of the cis-acting
sequences and trans-acting factors required for replication and
transcription of the viral genome [15, 29]. Likewise, the use of
cell-based minigenome systems has allowed researchers to identify
novel anti-mammarenaviral candidate drugs without the need of
using live forms of the virus [29–32]. Reverse genetics techniques
have also allowed the generation of recombinant infectious mam-
marenaviruses entirely from plasmid DNA with predetermined
mutations in their genomes to examine their contribution in viral
replication using cell cultures, as well as in viral pathogenesis and
associated disease using validated animal models of infection
[17, 33]. Likewise, implementation of mammarenavirus reverse
genetics has allowed researchers to study mammarenavirus–host
interactions [17–19, 29, 34, 35] and potentiated the generation
of attenuated forms of mammarenaviruses as potential novel candi-
date live-attenuated vaccines (LAV) [34–39], as well as the devel-
opment of screening methods to identify and evaluate novel anti-
mammarenavirus drugs targeting specific steps of the virus life cycle
[31, 34, 37]. Reverse genetics approaches have also been used for
the generation of recombinant tri-segmented
(r3) mammarenavirus expressing foreign genes [34, 35, 38, 39]
to facilitate the identification of antivirals inhibiting different steps
of the mammarenavirus life cycle [31] and to develop vaccine vector
candidates [34, 35, 39]. Plasmid-based reverse genetics have been
120 Luis Martı́nez-Sobrido et al.

also used for the generation of recombinant bi-segmented mam-

marenavirus expressing reporter fluorescent genes from the locus of
the viral NP using the porcine Teschovius 2A [40, 41]. Mammare-
navirus reverse genetics have also been applied to generate single-
cycle infectious, reporter-expressing, recombinant mammarena-
viruses that are limited to replicate in GP-expressing complement-
ing cell lines [37, 42], allowing the study of some aspects of the
biology of highly pathogenic mammarenaviruses (e.g., neutralizing
antiviral responses and identification of inhibitors of GP-mediated
cell entry) without the use of high biosafety level (BSL) laboratory
conditions [37]. In this chapter, we will focus on plasmid-based
reverse genetics techniques used to generate infectious recombi-
nant rLASV.

2 Materials

2.1 LASV Reverse We have developed an RNA mPol-I /Pol-II reverse genetics system
Genetics Plasmids for the rescue of the highly pathogenic Josiah strain of LASV
lineage IV [43]. Plasmids used for the generation of rLASV
(Fig. 3) can be transformed and amplified in DH5α competent
bacteria (Invitrogen) using Luria broth (LB) media in the presence
of 100 μg/mL of ampicillin (Fisher Scientific) at 37 °C for 16–18 h
(h), with the exception of the mPol-I L plasmid (see Notes 1 and 2)
[43]. Plasmids can be purified using commercially available max-
iprep kits (e.g., Qiagen) following manufacturer’s recommenda-
tions and stored at -20 °C until being used (see Notes 3 and 4).
1. pCAGGS protein expression plasmids: The pCAGGS plasmids
use the polymerase II (Pol-II)-driven chicken β-actin promoter
and the rabbit β-globin polyadenylation (pA) signal sequence
to direct the synthesis of LASV NP and L, which are the
minimum viral components for LASV genome replication and
gene transcription (Fig. 3a) required for the generation of
rLASV (Fig. 4) [43] (see Note 5).
2. mPol-I vRNA expression plasmids: LASV reverse genetics use
the species-specific [34] mouse RNA polymerase I (mPol-I)
promoter to direct the synthesis of S and L genome or anti-
genome RNA species [43] and the mPol-I terminator to gen-
erate authentic LASV vRNA 3′ ends [43] (Fig. 3b) (see Notes
2 and 6). For the generation of rLASV (Fig. 4), two mPol-I
plasmids expressing the S (mPol-I S) and L (mPol-I L) geno-
mic or antigenomic vRNAs are used [43] (Figs. 3b and 4) (see
Notes 2 and 6).
 LASV Reverse Genetics 121

A) GP B) pCAGGS L L
mPol-I S NP

mPol-I L L
Z pCAGGS NP NP

polI polI Chicken -acn

promoter terminator pA
promoter

mPol-I plasmids pCAGGS plasmids

Ampr SV40 origin Ampr SV40 origin

Fig. 3 Schematic representation of the LASV reverse genetics plasmids. (a) vRNA expression plasmids: vRNA
expression plasmids under the control of the mouse polymerase I (mPol-I) promoter (gray arrow) and
terminator (gray box) sequences direct the synthesis of LASV vRNA S (top) and L (bottom) segments. (b)
Protein expression plasmids: Protein expression pCAGGS plasmids use the chicken β-actin promoter (black
arrow) and the rabbit b-globin polyadenylation (pA) signal (black box) sequences to direct the synthesis of
LASV L (pCAGGS L, top) and NP (pCAGGS NP, bottom). These plasmids are required to provide the minimal
proteins required to initiate LASV gene transcription and genome replication. Both vRNA mPol-I and protein
pCAGGS expression plasmids are ampicillin resistant (Ampr) and have the origin of replication of Simian virus
40 (SV40)

2.2 Cell Lines for the The reverse genetics system we used for the rescue of infectious
Generation of rLASV rLASV is based on the use of the mPol-I promoter to direct
intracellular synthesis of the S and L genome or antigenome RNA
species (Figs. 3 and 4) [43] (see Note 6). The activity of the mPol-I
promoter exhibits species specificity and therefore is restricted to
rodent cells [34]. We use baby hamster kidney (BHK-21) cells since
they are easy to maintain, have high transfection efficiencies, and are
able to produce high viral titers [34, 35, 38, 39]. BHK-21 cells are
available from the American Type Culture Collection (ATCC).
Vero (African green monkey kidney epithelial) cells (LASV amplifi-
cation and titration) are available from the ATCC. Vero cells are
used to amplify and titrate rLASV from cell culture supernatants
collected from transfected BHK-21 cells [43]. Both cell lines are
maintained in T75 flasks at 37 °C in a 5% CO2 atmosphere.

2.3 Cell Culture 1. Cell culture medium: DMEM supplemented with 10% fetal
Media and Reagents bovine serum (FBS) and 1% penicillin/streptomycin (PS).
This cell culture media is used for maintenance of both BHK-
21 and Vero cells. Mix 445 mL DMEM (Invitrogen), 50 mL of
FBS (Atlanta Biologics), and 5 mL of 100X PS (Invitrogen).
Store at 4 °C.
2. Opti-MEM I medium: Opti-MEM I is used in the transfection
protocol to rescue rLASV.
122 Luis Martı́nez-Sobrido et al.

A) NP L
Day 1
plasmid
pCAGGS NP pCAGGS L transfecon

L
Z GP
NP

mPol -I L mPol-I S Day 4

Cell
scale-up

Day 7
virus
B) detecon
Mock rLASV
α-NP

Fig. 4 Generation of rLASV: BHK-21 cells are transiently co-transfected, using LPF2000, with the pCAGGS
protein expression plasmids encoding LASV NP and polymerase L (required to initiate viral gene transcription
and genome replication) together with the mPol-I vRNA expression plasmids encoding the viral L and S
segments (required to provide LASV vRNAs to initiate viral gene transcription and genome replication) (a). At
72 h post-transfection, BHK-21 cells are trypsinized and scaled-up into T75 flasks (a). After an additional
72–96-h incubation period, cell culture supernatants are collected and presence of LASV is determined by
immunofluorescence using LASV-specific antibodies (b). The chicken β-actin promoter (black arrow) and the
rabbit β-globin polyadenylation (pA) signal are indicated in the pCAGGS protein expression plasmids. The
mPol-I promoter and terminator sequences in the mPol-I vRNA expression plasmids are indicated by gray
arrows and boxes, respectively. Viral untranslated (UTR, black boxes) and intergenic (IGR) regions in the mPol-I
vRNA expression plasmids are indicated

3. Infection and post-infection media: Mix (2:1) Opti-MEM I

and DMEM 10% FBS1% PS. Store at 4 °C (see Note 7).
4. 10X phosphate-buffered saline (PBS): Mix 80 g of NaCl, 2 g of
KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O
up to 1 L. Adjust pH to 7.3 and sterilize by autoclaving. 10X
PBS can be stored at room temperature (room temperature).
5. 1X PBS: Dilute (1: 9) 10X PBS with ddH2O. Sterilize by
autoclaving. Store at room temperature.
6. Bovine serum albumin (BSA) 2.5%: 2.5 g of BSA (Sigma) in
97.5 mL of 1X PBS. Store at 4 °C.
7. Lipofectamine 2000 (LPF2000): LPF2000 is used for plasmid
DNA transfection in BHK-21 cells for rescue of rLASV. We
recommend using a LPF2000:DNA ratio of 2.5:1 for LASV
rescue.
 LASV Reverse Genetics 123

8. Trypsin-EDTA: Trypsin-EDTA is used to detach monolayers

of BHK-21 or Vero cells from the cell culture T75 flasks, prior
to DNA transfection or for the passaging of transfected cells.

3 Methods

3.1 Biosafety Rescue of replication competent rLASV must be done in BSL4

Conditions to Generate following guidelines and standard operating procedures written in
rLASV the corresponding BSL4 Standard Operating Procedures Manual
that should meet the requirements outlined in 42 CFR
73 [43]. Samples that require further experimental steps outside
of BSL4 should be either irradiated, fixed in formalin, or treated
with TRIzol reagent following appropriated institutional biosafety
approved protocols. All disposable materials leaving the BSL4 facil-
ities are autoclaved and then placed in a biohazard container for
incineration, or non-disposable items are either autoclaved, fumi-
gated, or chemically disinfected prior to their relocation, in accor-
dance with the institutional biosafety approved protocols.

3.2 LASV Rescue 1. Preparation of LPF2000/DNA transfection mix: First, mix

System Experimental 250 μL of Opti-MEM I media with 10 μL of LPF2000 (1:2.5
Approach (Fig. 4) ratio of plasmid DNA/LPF2000) per transfection. LASV res-
cues are performed in 6-well plates (see Note 8). Incubate the
Opti-MEM I-LPF2000 mixture for approximately 5–10 min at
room temperature. During this incubation time, prepare the
Opti-MEM I-DNA plasmid mixture.
2. Opti-MEM I-DNA plasmid mixture: In a separate tube, pre-
pare the DNA plasmid rescue mix in a total volume of 50 μL of
Opti-MEM I media. We recommend using 0.8 μg of pCAGGS-
NP, 1.0 μg of pCAGGS-L, 0.8 μg of mPol-I S, and 1.4 μg of
mPol-I L (total 4 μg of plasmid DNA) for 6-well plates
(1.0–1.2 × 106 cells/well).
3. Preparation of the LPF2000/DNA mixture: Combine Opti-
MEM I-LPF2000 and Opti-MEM I-DNA plasmid by pipet-
ting 250 μL of Opti-MEM I-LPF2000 (step 1) into the Opti-
MEM I-DNA plasmid mixture (step 2) and incubate for
approximately 20–30 min at room temperature. During this
incubation period, prepare the cells for transient transfection in
suspension (see Note 9).
4. Preparation of cells: Before manipulating the BHK-21 cells,
bring the 1X PBS, DMEM 10% FBS, 1% PS media, and
trypsin-EDTA mixture to 37 °C for approximately 10 min
(see Note 10). Wash the cells, twice, with 5 mL of 1X PBS.
Trypsinize the cells using 1 mL trypsin-EDTA. Cell detach-
ment is usually accomplished by incubating the cells in a humi-
dified 37 °C, 5% CO2 chamber, for approximately 5 min.
124 Luis Martı́nez-Sobrido et al.

Gently tap the cell culture T75 flask to confirm all the BHK-21
cells have been detached. After BHK-21 cells are completely
detached from the T75 flasks, carefully resuspend the cells in
9 mL of DMEM 10% FBS, 1% PS. Place the total media/cell
mixture in a 15 mL centrifuge tube and centrifuge the cells for
5 min at 1000 × g. Remove the media and resuspend the
BHK-21 cells in 10 mL of fresh DMEM 10% FBS, 1%
PS. Count the cells using a hemocytometer or cell counter
and adjust the cell concentration to approximately
1.0–1.2 × 106 cells/mL.
5. LPF2000/DNA incubation with BHK-21 cells: After approxi-
mately 20–30 min of incubation, pipette into each LPF2000/
DNA tube (step 3) 1 mL containing approximately
1.0–1.2 × 106 cells. Incubate the LPF2000/DNA/BHK-21
cell mixture for approximately 5 min at room temperature.
6. LPF2000/DNA/BHK-21 cell plating: Transfer the
LPF2000/DNA/BHK-21 cell mixture (step 5) into individual
wells of a 6-well cell culture plate. Gently tap the plate to
distribute the cells uniformly and incubate the cells in a 5%
CO2 humidified 37 °C incubator for approximately 6–12 h.
7. Transfection media change: After approximately 6–12 h of
incubation, replace the cell culture supernatant with 2 mL of
infection media and return the transfected BHK-21 cells to the
5% CO2 humidified 37 °C incubator and incubate for an addi-
tional 48–72 h [43].
8. Cell passage: After 48–72 h of incubation, transfected BHK-21
cells should reach approximately 100% confluence. Remove the
cell culture supernatant, wash the cells twice with 1X PBS, and
trypsinize them by adding 500 μL of trypsin-EDTA/well.
Return the 6-well plates to the 5% CO2 humidified 37 °C
incubator and let them incubate for approximately 5 min.
Gently tap the plates to complete BHK-21 cell detachment
from the plate. Carefully resuspend the BHK-21 cells with
1 mL of DMEM 10% FBS, 1% PS, and transfer to a 1.5 mL
microcentrifuge tube. Centrifuge the cells for 5 min at
5000 × g, at 4 °C in a microcentrifuge. Remove the cell culture
supernatant and resuspend the BHK-21 cells in 1 mL of infec-
tion media and transfer to a T75 cell culture flask. Bring up the
volume in the cell culture T75 flask to 10 mL with infection
media. Gently shake the T75 flask to allow uniform distribu-
tion of the BHK-21 cells and incubate the cells at 37 °C, 5%
CO2, for an additional 72–96 h (see Note 11).
9. LASV recovery from cell culture supernatants: After 72–96 h of
incubation, collect the cell culture supernatants from the T75
cell culture flask (step 8) into a 15 mL centrifuge tube.
 LASV Reverse Genetics 125

Centrifuge at 2500 × g, at 4 °C for 5 min. Collect the cell

culture supernatants containing the rLASV and discard the cell
pellet.
10. Storage of virus: Aliquot the recovered rLASV in cryotubes and
store them at -80 °C (see Note 12). Aliquots can be stored at
-80 °C until confirmation of the presence of rLASV.

3.3 Confirmation of Mammarenaviruses, including LASV, do not display the classic

Successful rLASV cytopathic effect (CPE) observed with other negative-stranded
Rescue RNA viruses [1]. Thus, successful rescue of rLASV must be eval-
uated by immunofluorescence using LASV-specific antibodies
[14, 15, 31, 33–36, 39] (see Notes 13 and 14).
1. Preparation of Vero cells for LASV titration: prepare single cell
suspension of Vero cells at 2 × 105 cells/mL. Seed the cells in
96-well plates (100 μL/well) and gently tap the plate so that a
uniform cell monolayer of 80–90% confluence (~4 × 104 cells/
well) is reached the next day. Incubate the plates at 37 °C in the
5% CO2 incubator.
2. On the day of LASV titration, serially diluted (tenfold dilu-
tions) cell culture supernatants recovered from transfected
BHK-21 cells (or virus stocks) in Opti-MEM I.
3. Remove the media and wash Vero cells twice with 50 μL of 1X
PBS. Infect the cells with 50 μL of the serially diluted cell
culture supernatants. Allow virus adsorption at 37 °C, 5%
CO2, for 60–90 min, rocking the plates every 15 min to
allow uniform virus infection of the Vero cell monolayers.
4. After 60–90 min of viral absorption, remove the cell culture
supernatants and add 100 μL of infectious media. Allow the
cells to incubate for 16–18 h (see Note 15).
5. After 16–18 h of viral infection, remove the cell culture super-
natants, and fix the infected cells with 4% formaldehyde diluted
in 1X PBS for 15 min at room temperature, before permeabi-
lizing with 0.1% Triton X-100 in 1X PBS for 15 min at room
temperature (see Note 16).
6. Remove the permeabilization solution and wash the cells, three
times, with 50 μL of 1X PBS.
7. Block the cells with 2.5% BSA in 1X PBS for 1 h at room
temperature (see Note 17).
8. During cell blocking, prepare the primary antibody. Antibodies
specific to LASV antigens should be diluted in blocking solu-
tion (2.5% BSA), and centrifuge for 15 min at 3500 × g before
use (see Note 18).
9. Remove the blocking solution and incubate the cells with
50 μL of the primary LASV antibody. Incubate at 37 °C for 1 h.
126 Luis Martı́nez-Sobrido et al.

10. After 1 h of incubation with the primary LASV antibody, wash

the cells three times with 50 μL of 1X PBS. Incubate with
50 μL of the fluorescein-conjugated secondary antibody
diluted (following manufacturer recommendations) in block-
ing solution (2.5% BSA) at 37 °C for 30 min (see Note 19).
11. Following the 30 min of incubation, remove the secondary
antibody and wash the cells three times with 50 μL of 1X PBS.
12. At this moment, rLASV rescue (or viral titers) can be deter-
mined under a fluorescent microscope. Viral titration is calcu-
lated by counting the fluorescent focus-forming units (FFU)
and the respective dilutions.

3.4 Amplification of Successful rescue of rLASV depends on multiple factors, including,

Rescued rLASV among others, the proper maintenance of BHK-21 cells, the quality
of the plasmid preparations used for LASV rescues, and the trans-
fection efficiency in BHK-21 cells. Because of these reasons, we
recommend performing LASV rescues in triplicate to increase the
likelihood of a successful rescue. If successful, it is possible that
rLASV titers in cell culture supernatants from the initial rescue are
low and, therefore, the virus will need to be amplified to generate a
stock. To that end, we recommend infecting fresh Vero cells at low
multiplicity of infection (MOI) (0.01) and allow rLASV amplifica-
tion for 48–72 h before collecting the new cell culture supernatants
for viral titration, as previously described.

4 Notes

1. Because of its size, we recommend growing the mPol-I L

plasmid at 30°C for 24 h.
2. We have found that the orientation of the insertion dramati-
cally influenced the genetic stability in bacteria of the mPol-I
plasmid coding for the full-length L RNA. We recommend the
construct containing the cDNA for the L RNA in a genome
orientation with respect to the mPol-I promoter since the
plasmid containing the L RNA in the opposite antigenomic
orientation was significantly more prone to large deletions. It is
worth noting that mPol-I L plasmid with either genomic or
antigenomic orientation was successfully used to rescue rLASV
with similar levels of efficiency [43].
3. Plasmid concentrations can be determined using spectropho-
tometry at 260 nm, with DNA purity being estimated using the
260:280 nm ratio (it is optimal to reach a 1.8–2.0 ratio for
optimal LASV rescues.
 LASV Reverse Genetics 127

4. All LASV pCAGGS and mPol-I plasmids can be generated

using normal cloning techniques and sequenced using standard
protocols.
5. Other Pol-II-driven protein expression plasmids instead of
pCAGGS can be used for the expression of LASV NP and L
for viral rescue.
6. In this protocol, we describe LASV reverse genetics approaches
using the Pol-II/mPol-I system [43]. Alternatively, expression
of vRNA for the S and L genome segments can be achieved
with the use of T7-driven expression plasmids [29, 44]. In
contrast to mPol-I, the T7 RNA polymerase promoter works
similarly in different mammalian cells, but its activity requires
the incorporation into the transfection mix of an additional
Pol-II-driven expression plasmid (e.g., pCAGGS) for the
expression of the T7 RNA polymerase [29] (Fig. 4). The Pol-
II-driven T7 expressing plasmid should be added to the trans-
fection mixture for rLASV rescue. Alternatively, a cell line
stably expressing the T7 RNA polymerase can be used to
avoid the transfection of an additional plasmid. We recommend
the use of BSR-T7 cells, a BHK-21 cell line constitutively
expressing the T7 RNA polymerase [44].
7. Alternatively, DMEM 2% FBS, 1% PS, can be used during and
after LASV infections.
8. To increase the likelihood of successful rLASV rescue, we rec-
ommend performing transfections in triplicate. Therefore, pre-
pare enough Opti-MEM I-LPF2000 based on the number of
LASV rescues planned.
9. Alternatively, BHK-21 cells can be transiently transfected in
monolayer. However, we have observed better transfection
efficiencies for viral rescues when BHK-21 cells are transfected
in suspension.
10. LASV rescues can be performed in any rodent cell line because
of the use of the mPol-I promoter [43]. We recommend the
use of BHK-21 cells since they are easy to maintain, have high
transfection efficiencies, and support LASV growth [34, 35,
38, 39]. Alternatively, LASV rescues can be performed in any
cell type with the use of the T7-driven RNA polymerase plas-
mids [44]. In this case, add a T7 RNA polymerase expression
plasmid into the transfection mix. Alternatively, use cells con-
stitutively expressing the T7 RNA polymerase (e.g., BSR-T7
cells) [44].
11. Alternatively, cell culture supernatants collected from BHK-21
cells at 72 h post-transfection can be used to infect fresh
monolayers of Vero cells to produce stocks with increased
virus titers [43]. Usually, cell culture supernatants collected
128 Luis Martı́nez-Sobrido et al.

from Vero cells at 72 h post-infection consistently had titers of

approximately 106 focus-forming units (FFU)/mL.
12. We recommend making small volume viral aliquots to avoid
multiple thaw cycles, which may reduce LASV titers.
13. We recommend the use of immunofluorescence assays
(16–18 h) over plaque assays (5–6 days) to determine LASV
titers [14, 15, 31, 33–36, 39].
14. We recommend performing LASV titrations in triplicates,
using 96-well plates of Vero cells.
15. LASV infections over 18 h may lead to secondary infections
and, therefore, result in overestimation of viral titers.
16. Alternatively, fix and permeabilize the cells with 4% formalde-
hyde, 0.1% Triton X-100 diluted in 1X PBS for 15 min at room
temperature. After fixation, the plate can be moved out from
BSL4 to BSL2 according to the institutional biosafety
guidelines.
17. Alternatively, Vero cells can be blocked with 2.5% BSA over-
night at 4 °C.
18. An antibody specific to LASV NP is recommended since it is
the most abundantly viral protein produced during mammar-
enavirus infections and will assist in easy detection of LASV
[1]. Optimal conditions for the use of antibodies to detect
LASV antigen should be determined previously.
19. We normally use a secondary FITC-conjugated α-mouse anti-
body from Dako diluted 1:100 in 2.5% BSA [14, 15, 31, 33–
36, 39].

Acknowledgments

We thank past and present members in L.M-S and J.C.T labora-

tories of the development of mammarenavirus reverse genetics
techniques and plasmids, including LASV. Mammarenavirus
research in L.M-S laboratory was funded by the National Institutes
of Health (NIH) R03AI099681, R21AI119775, R21AI128097-
01, R21AI121550, R21AI1135284, RO1AI142985, and
R43AI119775-01 grants and by the Department of Defense
(DoD) W81XWH1810071 and W81XWH1910496 grants.
Research in J.C.T. laboratory is supported by grants
RO1AI047140, RO1AI077719, RO1AI079665, and
RO1AI142985.
 LASV Reverse Genetics 129

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 Chapter 9

Bacterial Artificial Chromosome Reverse Genetics

Approaches for SARS-CoV-2
Kevin Chiem, Aitor Nogales, Fernando Almazán, Chengjin Ye,
and Luis Martı́nez-Sobrido

Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new member of the Coronaviridae
family responsible for the coronavirus disease 19 (COVID-19) pandemic. To date, SARS-CoV-2 has been
accountable for over 624 million infection cases and more than 6.5 million human deaths. The develop-
ment and implementation of SARS-CoV-2 reverse genetics approaches have allowed researchers to geneti-
cally engineer infectious recombinant (r)SARS-CoV-2 to answer important questions in the biology of
SARS-CoV-2 infection. Reverse genetics techniques have also facilitated the generation of rSARS-CoV-
2 expressing reporter genes to expedite the identification of compounds with antiviral activity in vivo and
in vitro. Likewise, reverse genetics has been used to generate attenuated forms of the virus for their
potential implementation as live-attenuated vaccines (LAV) for the prevention of SARS-CoV-2 infection.
Here we describe the experimental procedures for the generation of rSARS-CoV-2 using a well-established
and robust bacterial artificial chromosome (BAC)-based reverse genetics system. The protocol allows to
produce wild-type and mutant rSARS-CoV-2 that can be used to understand the contribution of viral
proteins and/or amino acid residues in viral replication and transcription, pathogenesis and transmission,
and interaction with cellular host factors.

Key words Coronavirus, SARS-CoV-2, COVID-19, Bacterial artificial chromosome, Reverse genet-
ics, Recombinant virus, Infectious clone

1 Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is

the etiological agent of the coronavirus disease 2019 (COVID-19)
pandemic, a respiratory illness first identified in December 2019 in
Wuhan, China [1, 2]. SARS-CoV-2 is similar to other corona-
viruses that are responsible of causing seasonal mild respiratory
illness (e.g., 229E, OC43, HKU1, or NL63) and two previously
identified coronaviruses that caused fatal zoonotic disease in
humans: severe acute respiratory syndrome coronavirus (SARS-
CoV) in 2002 [3, 4] and Middle East respiratory syndrome

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_9,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

133
134 Kevin Chiem et al.

coronavirus (MERS-CoV) in 2012 [5, 6]. While SARS-CoV and

MERS-CoV have a higher fatality rate than that of SARS-CoV-
2 [7–10], the ability of SARS-CoV-2 to transmit more efficiently
and covertly cause asymptomatic infections has resulted in more
than 6.5 million human deaths and over 624 million human infec-
tion cases worldwide. Moreover, since late 2020/early 2021, the
emergence of SARS-CoV-2 variants of concern (VoC) and variants
of interest (VoI) has significantly impacted the state of the COVID-
19 pandemic that still is threatening global public health [11–
15]. Although prophylactic (vaccines) [16–20] and therapeutic
(antiviral drugs) [21–24] options are currently available to control
SARS-CoV-2 infection, there is still an urgent need for the efficient
control of SARS-CoV-2 infections in humans.
SARS-CoV-2 is a positive-sense, single-stranded, enveloped,
RNA virus with a genome of ~30 kb that belongs to the Corona-
viridae family within the order Nidovirales [25–28] (Fig. 1a).
Approximately, two-thirds of the 5′ viral RNA genome contains
two open reading frames (ORFs), ORF1a and ORF1b, the latter
being translated via a - 1 ribosomal frameshift [25–28] (Fig. 1a).
Both ORFs are translated into two co-amino terminal proteins,
pp1a and pp1ab, which are subsequently cleaved by viral and cellu-
lar proteases to yield a total of 16 mature nonstructural proteins
(nsps) that are involved in different steps in the viral replication
cycle (Fig. 1b). The 3′ one-third of the genome includes the genes
encoding for the structural and genus-specific proteins (S, 3a, 3b,
E, M, 6, 7a, 7b, 8, N, and 10), which are expressed from a nested
set of subgenomic mRNAs [25–28]. Much like other coronavirus,
SARS-CoV-2 virion contains the structural membrane (M), enve-
lope (E), and spike (S) proteins (Fig. 1c) in the viral envelope,
which surrounds the viral RNA genome associated with the nucle-
ocapsid (N) protein (Fig. 1c). These structural proteins are
involved in viral entry, replication and transcription, assembly, and
budding (Fig. 1b).
Reverse genetics have allowed the generation of recombinant
viruses, including those with modified genomes, to better under-
stand aspects of the virus life cycle, including viral entry, replication
and transcription, assembly, and budding [29–32]. Furthermore,
reverse genetics have also facilitated a greater grasp on the function
of viral proteins and/or the contribution of specific amino acids in
viral proteins in replication, pathogenicity, and transmission [33–
37]. Researchers may also use reverse genetics to generate recom-
binant viruses expressing reporter genes to easily identify the pres-
ence of the virus in cultured cells and/or in validated animal models
of infection [38–49]. More importantly, reverse genetics have
allowed investigators with the feasibility to generate attenuated
forms of viruses for their potential implementation as live-attenu-
ated vaccines (LAV) [50–62]. To study this newly emerging coro-
navirus, different laboratories have been able to develop several
 BAC-Based SARS-CoV-2 Reverse Genetics 135

A)
NCR NCR
Ribosomal
5’ SAR2 nsp2 nsp3 nsp4 SAR3 6 7 8 9 10 11 S 3a E M 6 7a 7b 8 N 10 3’
frameshift
ORF1a nsp Endo-
nsp12 Helicase SAR4 SAR5
RNAase
ORF1b
B)
Gene Locus Protein Product Start End Proposed functions
5’ UTR 5’ UTR None 1 177 N/A
ORF1a SAR2 Leader protein 178 717 Induce host mRNA cleavage
ORF1a nsp2 nsp2 718 2,631 Binds to Prohibitin-1/2
ORF1a nsp3 nsp3 2,632 8,466 Release NSPs 1, 2, 3 (Papain like proteinase)
ORF1a nsp4 nsp4 8,467 9,966 Membrane rearrangement
ORF1a SAR3 3C-like proteinase 9,967 10,884 Cleaves at 11 sites of NSP polyprotein (3C-like proteinase)
ORF1a nsp6 nsp6 10,885 11,754 Generates autophagosomes
ORF1a nsp7 nsp7 11,755 12,003 Dimerizes with NSP8
ORF1a nsp8 nsp8 12,004 12,597 Stimulates NSP12
ORF1a nsp9 nsp9 12,598 12,936 Binds to helicase
ORF1a nsp10 nsp10 12,937 13,353 Stimulates NSP16
ORF1b nsp11 nsp11 13,354 13,392 Unknown
ORF1b nsp12 RdRp 13,352 16,148 Copies viral RNA methylation (RNA polymerase)
ORF1b helicase helicase 16,149 17,951 Unwinds duplex RNA (Helicase)
5ʹ-cap RNA (3ʹ to 5ʹ exonuclease, guanine N7-
ORF1b SAR4 3’-5’ exonuclease 17,952 19,532
methyltransferase)
Degrade RNA to evade host defense
ORF1b endoRNAse endoribonuclease 19,533 20,570
(endoRNAse/endoribonuclease)
2’-O-ribose 5ʹ-cap RNA methylation (2ʹ-O-ribose-methyltransferase—
ORF1b SAR5 20,571 21,464
methyltransferase potential antiviral drug target)
S S Spike (S) 21,475 25,296 Glycoprotein
ORF3a 3a 3a 25,305 26,132 Not fully defined, has Ion channel activity
E E Envelope (E) 26,157 26,384 Envelope protein
M M Membrane (M) 26,435 27,103 Membrane protein
ORF6 6 6 27,114 27,299 Interferon antagonist
ORF7a 7a 7a 27,306 27,671 Immunomodulator and interferon antagonist
ORF7b 7b 7b 27,668 27,799 Apoptosis activation
ORF8 8 8 27,806 28,171 Immunomodulator, down-regulator of MHC-1
N N Nucleocapsid (N) 28,186 29,445 Nucleocapsid protein
ORF10 10 10 29,470 29,586 Interferon antagonist
3’ UTR 3’ UTR None 29,587 29,930 N/A

C)
Spike
(S)

Membrane
(M)

Envelope
(E)

Viral RNA
genome

Nucleocapsid
(N)

Fig. 1 SARS-CoV-2 genomic organization and virion structure. (a) Genome organization. At the end of the
SARS-CoV-2 genome ( 29,930 nucleotides) are located the 5′ and 3′ non-coding regions (NCR). The 5′ end
136 Kevin Chiem et al.

reverse genetic approaches to generate recombinant (r)SARS-CoV-

2 [63, 64]. However, some of these approaches rely on the produc-
tion of viral (v)RNA in vitro using a bacteriophage T7 promoter, a
process that is not as efficient and robust as generating recombinant
viruses by transfecting DNA into susceptible cells and, therefore, is
currently restricted to a few selective laboratories. To aid other
researchers in overcoming this major limitation, we have developed
a bacterial artificial chromosome (BAC)-based reverse genetics sys-
tem to generate rSARS-CoV-2 [30, 37, 46, 65–67]. Compared to
other methods, our BAC-based reverse genetics does not rely on
in vitro transcription of the vRNA using a T7 promoter and
subsequent RNA electroporation and simply uses transfection of
the infectious BAC clone using Lipofectamine 2000 into suscepti-
ble cells that is less technically challenging than electroporating cells
with RNA produced in vitro. This approach has also been shown to
overcome difficulties associated with the cloning and propagation
of the viral genome as cDNA clones in bacteria [68–70].
In this chapter, we describe the technical experimental steps
required for efficient recovery of rSARS-CoV-2 USA-WA1/2020
(WA-1) strain by transfection of a BAC in Vero E6 cells [30, 66, 67,
71] and provide different steps to detect the rescued rSARS-CoV-
2 based on the presence of cytopathic effect (CPE), immunofluo-
rescent assay (IFA), and plaque assay. The implementation of this
BAC-based reverse genetic approach to generate rSARS-CoV-
2 represents an excellent tool to ask important questions in the
biology of SARS-CoV-2, including viral infection, pathogenesis,
and transmission, by genetic manipulation of the viral genome.
Likewise, these reverse genetics can be used to generate attenuated
forms of the virus for their implementation as LAV for the prophy-
lactic treatment of SARS-CoV-2 or for the generation of rSARS-
CoV-2 expressing reporter genes to facilitate the identification and
characterization of therapeutic antivirals against SARS-CoV-2.
Moreover, the protocol described in this chapter can be
implemented to generate infectious BAC clones of recently identi-
fied SARS-CoV-2 VoC or VoI to generate similar recombinant

Fig. 1 (continued) of the genome contains two overlapping ORFs (1a and 1b), which are translated in two
co-amino terminal polyproteins named pp1a and pp1ab. Both polyproteins are cleaved into 16 mature nsps:
leader protein (SAR2), nsp2, nsp4, nsp4, 3C-like proteinase (SAR3), nsp6, nsp7, nsp8, nsp9, nsp10, nsp11,
RNA-dependent RNA polymerase (RdRp, nsp12), helicase, 3′-to-5′ exonuclease (SAR4), endoRNase, and
2′-O-ribose methyltransferase (SAR5). The structural genes encoding the viral S (green), E (orange), M (red),
and N (blue) structural proteins and the genes encoding the accessory proteins 3a, 6, 7a, 7b, 8, and 10, are
located downstream the ORF1b. (b) SARS-CoV-2 genes, proteins, and their functions: SARS-CoV-2 genes and
their respective encoded proteins, including their location in the viral genome and functions, are indicated. (c)
Schematic representation of SARS-CoV-2 virion: SARS-CoV-2 virion is decorated by the spike (S, green),
membrane protein (M, red), and envelope (E, orange) proteins. Inside the virion is the viral nucleocapsid (N,
blue) protein that encapsulates the positive sense, single-stranded RNA viral genome
 BAC-Based SARS-CoV-2 Reverse Genetics 137

viruses in order to more effectively combat the still ongoing

COVID-19 pandemic.

2 Materials

2.1 Biosafety SARS-CoV-2 is a biosafety level 3 (BSL3) pathogen, and therefore

Recommendations all experiments outlined in this book chapter describing the gener-
ation and characterization of rSARS-CoV-2 using a BAC-based
reverse genetics approach should be performed in appropriated
BSL3 laboratory facilities according to proper institutional regula-
tions. Laboratory researchers working with rSARS-CoV-2 should
be property trained before entering and conducting experiments at
BSL3 laboratories. Experiments not involving infectious natural
isolate or rSARS-CoV-2, such as cell culture procedures, are per-
mitted to be conducted at BSL2. Likewise, the development of viral
plaque assays and immunofluorescences can be conducted at BSL2
after proper inactivation of SARS-CoV-2 at BSL3 following estab-
lished institutional inactivation procedures. All materials used for
the recovery of rSARS-CoV-2 must be properly sterilized before
disposal, following approved institutional biosafety
recommendations.

2.2 SARS-CoV-2 SARS-CoV-2 WA-1 strain (NR-52281) was obtained from the
Biodefense and Emerging Infections Research Resources Reposi-
tory (BEI Resources), a National Institute of Allergy and Infectious
Diseases repository managed by the American Type Culture Col-
lection (ATCC). We selected SARS-CoV-2 WA-1 strain to develop
the reverse genetics approaches based on the use of the plasmid
pBeloBAC11 since it was isolated from a patient with respiratory
illness in Seattle, Washington, in 2020 and because the complete
sequence was available (No. MN985325) at the National Center
for Biotechnology Information (NCBI).

2.3 BAC and Plasmid The plasmid pBeloBAC11 (NEB) used for the generation of the
Extraction SARS-CoV-2 BAC-based reverse genetics has been previously
described [26, 53, 68, 72–74] and used to generate the pBelo-
BAC11-SARS-CoV-2 [30, 48, 66, 67]. The Qiagen Large-
Construct Kit was used for the preparation of ultrapure BACs (see
Note 1).

2.4 E. coli DH10B E. coli DH10B electrocompetent cells (Thermo Fisher Scientific)
Cells are used for efficient propagation of SARS-CoV-2 BAC (see Note
2). E. coli DH10B electrocompetent cells are stored at -80 °C to
avoid thaw-freeze cycles until use.
138 Kevin Chiem et al.

2.5 Culture Media for Reagents in this section could be purchased from different
DH10B Cells suppliers.
1. Luria Broth (LB) media: To prepare 1 L of LB media, mix 10 g
of Bacto Tryptone (BD), 5 g Bacto yeast extract (BD), and 10 g
NaCl (T Baker) in 900 mL of ddH2O. Adjust the volume to
1 L with ddH2O. Autoclave to sterilize and store at 4 °C.
2. Chloramphenicol (Thermo Fisher Scientific): Prepare a stock
of chloramphenicol at 12.5 mg/mL in 100% ethanol. Aliquot
and store at - 20 °C.
3. LB media with chloramphenicol: Dilute the working stock of
chloramphenicol to a final concentration of 12.5 μg/mL (1:
1000) in LB. LB media with chloramphenicol can be stored at
4 °C for up to 30 days.
4. LB agar plates with chloramphenicol: Prepare 1 L of LB media,
and add 15 g of Bacto agar (BD). Autoclave to sterilize, equili-
brate at 55 °C for 30 min, and add chloramphenicol to a final
concentration of 12.5 μg/mL. Dispense 25 mL of media to
petri dishes, and store at 4 °C.
5. SOC media: To prepare 1 L of SOC media, mix 20 g of Bacto
Tryptone (BD), 5 g Bacto yeast extract (BD), 0.5 g NaCl
(T Baker), and 2.5 mL of 1 M KCl in 900 mL of ddH2O,
and adjust the volume of the solution to 1 L with
ddH2O. Sterilize by autoclaving, and before use, add 10 mL
of 1 M MgCl2, 10 mL of 1 M MgSO4, and 20 mL of 1 M
glucose. SOC media can be stored at 4 °C.

2.6 Cell Line for the Vero E6 (African green monkey kidney epithelial cells) are available
Rescue of rSARS-CoV- from the ATCC (CRL-1586). Vero E6 cells are used in all experi-
2 ments outlined in the protocol to generate and characterize rSARS-
CoV-2 because of their acceptable transfection efficiency and their
ability to efficiently support SARS-CoV-2 replication. Vero E6 cells
are cultured and maintained in growth medium (described below in
Subheading 2.7) at 37 °C in a 5% CO2 atmosphere cell culture
humidified incubator (see Notes 3 and 4).

2.7 Cell Culture Reagents in this section could be purchased from other suppliers.
Media and Reagents
1. Cell growth medium: Dulbecco’s Modified Eagle’s Medium
for Rescue and
(DMEM, Gibco) supplemented with 5–10% fetal bovine serum
Propagation of rSARS- (FBS, Avantor Seradigm), 100 mg/mL penicillin/streptomy-
CoV-2 cin, and 2 mM L-glutamine (PSG, Sigma-Aldrich). Store at 4 °
C.
2. Virus growth medium: DMEM supplemented with 2% FBS,
100 mg/mL PSG (Sigma-Aldrich). Store at 4 °C.
3. Trypsin-EDTA solution (Sigma-Aldrich): 0.25% (w/v) trypsin,
0.02% (w/v) EDTA.
 BAC-Based SARS-CoV-2 Reverse Genetics 139

4. Phosphate-buffered saline (PBS): To prepare 1 L of PBS, mix

8 g of NaCl, 0.2 g of KCl, 1.15 g of Na2HPO4, and 0.2 g of
KH2PO4 in 900 mL of ddH2O. Adjust pH to 7.4, add ddH2O
up to 1 L, sterilize by autoclaving, and store at room
temperature.
5. 2X DMEM/F-12: Dissolve a packet of DMEM/F-12 (Gibco)
into 450 mL of cell culture grade water (Corning), along with
10 mL of PSG (Sigma-Aldrich), 6 mL of 35% BSA, 10 mL of
1 M HEPES buffer solution (Gibco), and 24 mL of 5% (w/v)
NaHCO3 (Sigma). Sterilize by filtration, and store at 4 °C.
6. 2% (w/v) agar (Oxoid): Dissolve 2 g of agar in 100 mL of
ddH2O (see Note 5). Sterilize by autoclaving, and store at
room temperature.
7. DMEM/F-12/agar: Mix 25 mL of 2X DMEM/F-12 media,
0.5 mL of 1% (w/v) DEAE-Dextran (MP Biomedicals), 1 mL
of NaHCO3, and 8.5 mL of cell culture grade water (Corning).
Before use, add 15 mL of pre-warmed 2% agar for a final
concentration of 0.5% (see Note 6).
8. Lipofectamine 2000 (LPF2000) transfection reagent
(Invitrogen).
9. Opti-MEM Reduced Serum Medium for BAC transfection
(Invitrogen).

2.8 Reagents for Reagents described in this section could be purchased for other
Plaque Assay and suppliers.
Immunostaining
1. Fixing solution: 10% (v/v) neutral buffered formalin solution
(Sigma-Aldrich) in PBS.
2. Permeabilization solution: 0.5% (v/v) Triton X-100 (Sigma-
Aldrich) in PBS.
3. Blocking solution: 2.5% (w/v) bovine serum albumin (BSA,
Sigma-Aldrich) in PBS.
4. Primary mouse anti-SARS nucleocapsid (N) protein 1C7C7
cross-reactive monoclonal antibody (mAb, Millipore Sigma)
(see Notes 7 and 8).
5. Vectastain ABC Kit, peroxidase mouse IgG (Vector Labora-
tories Inc.).
6. DAB Substrate Kit, Peroxidase (HRP), with Nickel (Vector
Laboratory Inc.).

2.9 Reagents for Reagents described in this section could be purchased for other
Immunofluorescence suppliers.
1. Fixing solution: 10% (v/v) neutral buffered formalin solution
(Sigma-Aldrich) in PBS.
140 Kevin Chiem et al.

2. Permeabilization solution: 0.5% (v/v) Triton X-100 (Sigma-

Aldrich) in PBS.
3. Blocking solution: 2.5% BSA (Sigma-Aldrich) in PBS.
4. Primary mouse anti-SARS nucleocapsid (N) protein 1C7C7
cross-reactive mAb (Millipore Sigma) (see Note 7).
5. Secondary rabbit polyclonal anti-mouse IgG-FITC conjugate
antibody (Dako).
6. 4′,6′-Diamidino-2-2phenylindole (DAPI, Research Organics).

2.10 Laboratory Equipment, reagents, and supplies in this section could be pur-
Equipment, Reagents, chased for other suppliers different to those recommended.
and Supplies 1. Benchtop refrigerated microcentrifuge (Thermo Fisher
Scientific).
2. High-speed refrigerated centrifuge (Thermo Fisher Scientific).
3. Water bath (VWR).
4. Power supply (Bio-Rad).
5. NanoDrop (Thermo Fisher Scientific) or similar
spectrophotometer.
6. MicroPulser electroporator (Bio-Rad).
7. Electroporation cuvettes, 0.2 cm gap (Bio-Rad).
8. Laboratory 5% CO2 cell culture humidified microbiological
incubator at 37 °C for maintenance of cells, virus rescue, and
amplification (PHC Corporation).
9. Class II biosafety cabinet (Baker).
10. Evos light microscope (Thermo Fisher Scientific).
11. Evos fluorescent microscope (Thermo Fisher Scientific).
12. Microwave oven to melt 2% Oxoid agar (Panasonic).
13. -80 °C freezer (PHC Corporation).
14. Autoclave.
15. MaxQ Large Incubated Benchtop Orbital Shaker (Thermo
Fisher Scientific).
16. Polystyrene T75 cell culture flask (Corning).
17. Cell culture plates (Greiner Bio-One).
18. Microcentrifuge tubes, 1.5 mL (VWR).
19. Nalgene® System 100 Cryogenic Vials (Thermo Fisher
Scientific).
20. Polypropylene sterile conical 15 mL and 50 mL conical tubes
(Greiner Bio-One).
21. Serological 5 mL, 10 mL, and 25 mL pipettes (Greiner
Bio-One).
 BAC-Based SARS-CoV-2 Reverse Genetics 141

22. Universal 20 μL, 200 μL, and 1000 μL filtered pipette tips
(VWR).
23. Bacteria orbital shaker (Thermo Fisher Scientific).

3 Methods

3.1 Preparation of In this chapter we describe the experimental procedure to generate

the Infectious BAC rSARS-CoV-2 WA-1 strain using reverse genetics techniques based
Clone for the Rescue of on the use of a BAC [30, 37, 66, 67, 71]. The generation of the
rSARS-CoV-2 BAC containing the full-length genome of SARS-CoV-2 (pBelo-
BAC11-SARS-CoV-2) has been previously described [30, 66, 67,
71]. Briefly, five DNA fragments covering the entire 29,930 bp viral
genome of SARS-CoV-2 WA-1 strain (NCBI, No. MN985325)
were chemically synthesized de novo (Bio Basic) and sequentially
assembled into the pBeloBAC11 plasmid (NEB) using unique
restriction enzymes and standard molecular biology techniques.
The full-length viral genome was cloned under the control of the
cytomegalovirus (CMV) promoter and flanked at the 3′ end by the
hepatitis delta virus (HDV) ribozyme (Rz) and the bovine growth
hormone (bGH) termination and polyadenylation sequences
(Fig. 2).
For efficient rescue of rSARS-CoV-2 using the BAC-based
reverse genetics approach, we recommend preparing highly pure
BAC with no bacteria DNA contamination. Different commercially
available kits can be used, but we recommend the use of the QIA-
GEN Large-Construct Kit since it includes an ATP-dependent

CMV ORF1a ORF1b S 3a E M 6 7a 7b 8 N 10 Rz bGH

ori2 Cmr
repE

pBeloBAC11
sopC

SARS-CoV-2
sopB sopA

Fig. 2 Generation of the pBeloBAC11-SARS-CoV-2. The full-length SARS-CoV-2 genome is cloned into the
pBeloBAC11 plasmid as a cDNA between the cytomegalovirus (CMV) polymerase II-driven promoter (gray
arrow) at the 5’ end and the hepatitis delta ribozyme (Rz) and bovine growth hormone (bGH) polyadenylation
signals (gray boxes) at the 3’ end. The pBeloBAC11 plasmid can accommodate large cDNA fragments as 1–2
copies into E. coli. The pBeloBAC11 regulatory genes (sopA, sopB, sopC, and repE), the F-factor replication
origin (Ori2), and the chloramphenicol resistance gene (Cmr) are indicated. Viral proteins are those previously
described in Fig. 1
142 Kevin Chiem et al.

exonuclease digestion step that removes bacteria DNA contamina-

tion, allowing high-purity BAC preparation, like that obtained
using a cesium chloride method (see Note 1). Prepare highly pure
BAC clone according to the following procedure:
1. Transform 100 μL of DH10B E. coli competent cells with
10–100 ng of pBeloBAC11-SARS-CoV-2 by electroporation
(2.5 kV, 600 Ω, and 10 μF) in a Gene Pulser® cuvette 0.2 cm
gap (see Note 9).
2. Transfer bacteria into a 15 mL culture tube containing 1 mL of
SOC media and incubated at 37 °C for 1 h in an orbital shaker
at 250 rpm.
3. Spread 100–200 μL of the bacteria onto pre-warmed LB agar
plates containing 12.5 μg/mL of chloramphenicol. Place the
LB agar plates inversely in an incubator at 37 °C for 16–24 h.
4. Transfer a single bacterial colony transformed with pBelo-
BAC11-SARS-CoV-2 into 3–5 mL of LB media supplemented
with 12.5 μg/mL of chloramphenicol and incubate at 37 °C
for 8 h in a bacteria orbital shaker with vigorous (250 rpm)
shaking.
5. Inoculate 1 mL of bacterial culture into a 2 L flask containing
500 mL of LB supplemented with 12.5 μg/mL of chloram-
phenicol, and grow the bacteria in a 37 °C shaking incubator at
200–250 rpm for 16–24 h, until an OD600 of 0.8–1.0 (see
Notes 10 and 11).
6. Collect the bacteria cells by centrifugation at 6000 × g for
15 min at 4 °C.
7. Purify the pBeloBAC11-SARS-CoV-2 using the QIAGEN
Large-Construct Kit following the manufacturer’s instructions
(see Note 1). Elute the BAC clone in 250–500 μL of ddH20
(see Note 12).
8. Quantify the concentration of the purified BAC using a nano-
drop (see Note 13). This purification method usually yields
20–30 μg of purified pBeloBAC11-SARS-CoV-2 (see Note
14).
9. If other purification kit not containing the DNase step is used,
treat the purified BAC with an ATP-dependent DNase kit to
remove the contaminated bacterial genome fragments.
10. Store the pBeloBAC11-SARS-CoV-2 at - 20 °C for short-
term storage or at - 80 °C for long-term storage.

3.2 Rescue of For the rescue of rSARS-CoV-2, we recommend three independent

rSARS-CoV-2 transfections in Vero E6 cells for each recombinant virus to increase
the probability of successful rescue. If multiple rescues are
attempted, scale up the following steps accordingly. We also
 BAC-Based SARS-CoV-2 Reverse Genetics 143

Plasmid Split Virus

transfection cells detection

pBeloBAC11
SARS-CoV-2
Media
change

Fig. 3 Rescue of rSARS-CoV-2 from the BAC infectious clone. Vero E6 cells in six-well plates are transfected in
suspension with 4 μg of pBeloBAC11-SARS-CoV-2 and LPF2000 as described in the method section. Two
days after transfection, cells are collected and scaled up into T75 flasks. After 48 h post-transfection, when
the CPE is clear, the cell culture supernatants are collected, and the presence of the virus is confirmed by the
CPE, IFA, and plaque assay

recommend transfecting the empty pBeloBAC11 plasmid as an

internal control. Infectious rSARS-CoV-2 can be successfully
recovered by transfecting Vero E6 cells with the purified BAC-
SARS-CoV-2 described in Subheading 3.1. A schematic represen-
tation of the experimental procedures and steps for the rescue of
rSARS-CoV-2 is shown in Fig. 3.
1. Plate Vero E6 cells in six-well plates (5 × 105 cells/well, tripli-
cates) in cell culture media the day before transfection (see
Notes 15–17).
2. Prepare the Opti-MEM-LPF2000 mixture: Mix 250 μL of
Opti-MEM media with 8–10 μL of LPF2000 per transfection.
Gently invert to mix, and incubate at room temperature for
5–10 min.
3. Prepare the plasmid transfection mixture: Add 4 μg of pBelo-
BAC11-SARS-CoV-2 per transfection into 250 μL of Opti-
MEM, and mix by inverting the tubes.
4. Prepare the Opti-MEM-LPF2000-DNA plasmid mixture: Add
the 250 μL Opti-MEM-LPF2000 mixture (step 2) into the
250 μL plasmid transfection mixture (step 3), and incubate the
mixture for 20–30 min at room temperature. Meanwhile, wash
the Vero E6 cell monolayer twice with 4 mL of growth medium
without antibiotics, and leave the cells in 1 mL of the same
medium (see Note 18).
5. Slowly add the 500 μL Opti-MEM-LPF2000-DNA plasmid
mixture (step 4) into the Vero E6 cells. Gently rock the plate,
and place in the 5% CO2 incubator at 37 °C for 6 h (see Note
19).
6. Approximately 6 h after transfection, remove the transfection
media (step 5), and add 2–4 mL of virus growth media.
7. Place the six-well plates back in the 37 °C incubator with 5%
CO2.
144 Kevin Chiem et al.

8. At 72 h post-transfection, scale up the transfected Vero E6 cells

to a T75 flask. To that end, wash the cells with PBS twice, and
then add 1 mL of trypsin-EDTA. When the cells are detached,
resuspend the cells in 10 mL of virus growth media, and
transfer to a 15 mL conical tube. Centrifuge the cells at
1500 rpm for 10 min. Remove the media, and resuspend the
cells in 12 mL of fresh virus growth media. Transfer the cells
into a T75 flask.
9. Prepare two additional T75 flasks with confluent monolayers of
Vero E6 cells (~5 × 106 cells/T75) mock-infected and infected
with parental SARS-CoV-2 at a multiplicity of infection (MOI)
of 0.01 plaque-forming units (PFU) as controls.
10. Incubate the T75 flasks in a 37 °C incubator with 5% CO2 for
2–3 days. Check the cells daily for the presence of cytopathic
effect (CPE) to assess the recovery of rescued rSARS-CoV-
2 (Fig. 4a) (see Note 20).

A) B)
Mock SARS-CoV-2 rSARS-CoV-2 Mock SARS-CoV-2 rSARS-CoV-2
FITC
DAPI

C) Mock SARS-CoV-2 rSARS-CoV-2

Merge

Fig. 4 Characterization of rescued rSARS-CoV-2. At 48–72 h after scaling up transfected Vero E6 cells into T75
flask, cell culture supernatants are collected and used to infect fresh monolayers of Vero E6 cells to confirm
the presence of rSARS-CoV-2 by CPE, IFA, and plaque assay. (a) CPE: Representative images of Vero E6 cells
mock-infected or treated with the cell culture supernatant from transfected Vero E6 cells are shown. As
internal control, Vero E6 cells were also infected with SARS-CoV-2 natural isolate. Scale bars, 400 μm. (b) IFA:
Presence of rSARS-CoV-2 is also evaluated by infecting fresh Vero E6 cells with cell culture supernatants
collected at 48–72 h after scaling up transfected Vero E6 cells into T75 flask and assessing the presence of
SARS-CoV-2 using the mAb 1C7C7 against the viral N protein. Vero E6 cells mock-infected or infected with
SARS-CoV-2 are included as internal controls. DAPI is used to stain the nucleus. Scale bars, 100 μm. (c)
Plaque assay: Confluent monolayers of Vero E6 cells in six-well plates were infected with tenfold serial
dilutions of the cell culture supernatants collected at 48–72 h after scaling up transfected Vero E6 cells into
T75 flask. After 1 h adsorption, cells are overlaid with agar, and 72 h after infection, the presence of the virus
was evaluated with the mAb 1C7C7 against the viral N protein by immunostaining. Mock-infected and Vero E6
cells infected with SARS-CoV-2 were used as controls to validate the plaque assay
 BAC-Based SARS-CoV-2 Reverse Genetics 145

11. If CPE is observed, transfer the cell culture supernatant con-

taining rSARS-CoV-2 to a 50 mL conical tube, and centrifuge
at 400 × g for 10 min at 4 °C to pellet cell debris.
12. Collect the cell culture supernatants, and aliquot them into
cryogenic tubes (0.5 mL/tube) for storage at - 80 °C.

3.3 Validation of To confirm the successful rescue of rSARS-CoV-2, we recommend

Viral Rescue and infecting fresh Vero E6 cells with the cell culture supernatants
Characterization of containing rSARS-CoV-2 generated in the previous section. The
rSARS-CoV-2 presence of rSARS-CoV-2 can be determined by IFA by detection
of SARS-CoV-2 N protein using the cross-reactive mouse anti-
SARS N protein mAb 1C7C7 (Fig. 4b) (see Note 7). We recom-
mend including mock-infected cells and Vero E6 cells infected with
the natural SARS-CoV-2 as negative and positive controls,
respectively.
1. Seed Vero E6 cells in 12-well plates (0.5 × 105 cells/well,
triplicates) the day before infection.
2. Thaw an aliquot of rSARS-CoV-2 and natural SARS-CoV-
2 isolate on ice. Prepare undiluted and 1:10 and 1:100 dilu-
tions of the viral stock in virus growth media.
3. Wash Vero E6 cells three times with virus growth media, and
infect them with 500 μL of undiluted or diluted rSARS-CoV-
2 or SARS-CoV-2 natural isolate in triplicates.
4. Incubate the plates in a 37 °C 5% CO2 incubator for 1 h.
5. After viral absorption, remove the viral inoculum, wash once
with virus growth media, and add 1 mL/well of fresh virus
growth media. Return the plates to the 37 °C 5% CO2 incuba-
tor, and incubate for 24 h.
6. Remove the cell culture supernatants from the infected Vero E6
cells, and submerge the plates in 10% neutral buffered formalin
for 16–24 h to fix the cells and inactivate the virus, as required
by institutional safety guidelines (see Note 21).
7. After inactivation, plates may be removed to the BSL2, based
on institutional safety guidelines.
8. Gently rinse the plates with tap water to remove remaining
excess of 10% neutral buffered formalin.
9. Wash the plates three times with PBS.
10. Add 500 μL/well of permeabilization solution, and incubate
for 10 min at room temperature.
11. Remove the permeabilization solution, and wash the cells three
times with PBS.
12. Add 500 μL/well of blocking solution, and incubate for 1 h at
room temperature (see Note 22).
146 Kevin Chiem et al.

13. Remove the blocking solution, and incubate the cells with
500 μL/well of a 1 μg/mL solution of the anti-SARS N
protein mAb 1C7C7 (1 μg/mL) in a 37 °C incubator for 1 h
(see Note 7).
14. Remove the primary antibody solution, and wash the cells
three times with PBS.
15. Add 500 μL/well of a 1:200 dilution of an anti-mouse IgG-
FITC-conjugated antibody and 1 mg/mL of DAPI in blocking
solution. Place the plates in a 37 °C incubator for 1 h.
16. Remove the secondary antibody/DAPI solution, and wash the
cells three times with PBS. Leave 1 mL of PBS in each well.
Samples can be stored at 4 °C protected from light with
aluminum foil.
17. Observe the samples under a fluorescence microscope. Detec-
tion of SARS-CoV-2 N protein is seen in green (FITC), while
the cell nucleus is seen in blue (DAPI). Only Vero E6 cells
exposed to cell culture supernatants containing rSARS-CoV-
2 or infected with the SARS-CoV-2 natural isolate should give
a positive FITC signal. Mock-infected cells should only be
stained with DAPI (Fig. 4B) (see Note 23).

3.4 Plaque Assay for To determine the titer of rSARS-CoV-2 from our rescue approach
Viral Titration and and to assess the plaque phenotype of the recombinant virus, we
Visualization of Plaque recommend conducting a plaque assay in Vero E6 cells (Fig. 4c).
Sizes Mock-infected cells and cells infected with the SARS-CoV-2 WA-1
natural isolate should be included as internal negative and positive
controls, respectively, in the plaque assay. An agar overlay is placed
on top of the Vero E6 cells infected with tenfold serial dilutions of
the rSARS-CoV-2 to restrict the spread of the virus to only neigh-
boring cells. Thus, each viral plaque is produced by a single virus.
1. Seed six-well plates of Vero E6 cells (0.5 × 106 cells/well) in
cell culture media the day before the plaque assay. Vero E6 cells
should be ~80–90% confluency (~1 × 106 cells/well) at the
time of viral infection. We recommend preparing enough
six-well plates to titrate each sample in triplicates.
2. Thaw an aliquot of the rSARS-CoV-2 and SARS-CoV-2 natural
isolate, and make tenfold serial dilutions in virus growth media.
3. Wash Vero E6 cells three times with virus growth media, and
infect each of the wells with 1 mL of the tenfold viral serial
dilutions. We recommend using 10-2–10-7 serial dilutions of
the virus stocks.
4. Incubate the plates at 37 °C in a 5% CO2 incubator for 1 h.
Gently rock the plates every 15 min.
 BAC-Based SARS-CoV-2 Reverse Genetics 147

5. After viral adsorption, remove the inoculum, add 3 mL of

pre-warmed DMEM F12/agar media, and let the agar solidify
at room temperature.
6. Once the agar has solidified, put the plates in a 37 °C incubator
with 5% CO2 for 3 days in an inverted position (see Note 24).
7. After 3 days incubation, fix and inactivate the cells by
completely submerging the plates in 10% neutral buffered for-
malin for 16–24 h, following institutional safety guidelines (see
Note 21).
8. After inactivation, plates may be removed to the BSL2, based
on institutional safety guidelines.
9. Gently rinse the plates with tap water to remove the excess of
10% neutral buffered formalin.
10. Carefully remove the agar, and wash the plates three times with
PBS.
11. Remove the PBS, and incubate the plates with permeabiliza-
tion solution for 10 min at room temperature.
12. After permeabilizing the cells, wash the plate with PBS three
times.
13. Add 1 mL of blocking solution, and incubate for 1 h at room
temperature (see Note 22).
14. Remove the blocking solution, and add 1 mL/well of 1 μg/
mL of the cross-reactive SARS-CoV N protein 1C7C7 mAb in
blocking solution, and incubate the plates at 37 °C for 1 h (see
Notes 7 and 8).
15. Remove the primary antibody solution, and wash the cells
three times with PBS.
16. Add 1 mL/well of a biotinylated anti-mouse secondary rabbit
antibody (Vector Laboratories) diluted in blocking solution.
Incubate the plates at 37 °C for 30 min.
17. Remove the secondary antibody, and wash the cells three times
with PBS.
18. Add 1 mL/well of Vectastain ABC reagent, and incubate the
plates at 37 °C for 30 min.
19. Wash with PBS three times, and visualize the viral plaques
using a DAB peroxidase kit for 3–5 min following the manu-
facturer’s instructions.
20. Calculate the viral titer by counting the number of visible
plaques in each well, average the triplicates, and then multiply
by the dilution factor, and divide by the volume used during
infection.
21. A representative result of a viral plaque assay is shown in
Fig. 4c.
148 Kevin Chiem et al.

3.5 Amplification of 1. Seed Vero E6 cells in T75 flasks (~5 × 106 cells/T75) in cell
rSARS-CoV-2 and growth media to reach ~80–90% confluent the day before
Generation of Virus infection.
Stocks 2. Wash the cells twice with virus growth media, and infect them
with a MOI of ~0.01–0.001 PFU in 5 mL of virus growth
media.
3. After viral adsorption, remove the virus inoculum, and add
15 mL of virus growth media. Place the T75 flask in a 37 °C
incubator with 5% CO2, and incubate the cells until CPE is
observed.
4. When CPE is ~25% (~48–72 h post-infection), collect the cell
culture supernatant into a 15 mL tube, and centrifuge at
2000 × g for 10 min at 4 °C to remove cell debris.
5. Collect the cell culture supernatant containing the virus in a
new 15 mL tube, and discard the cell pellet.
6. Aliquot the cell culture supernatant in labeled cryotubes, and
store it at - 80 °C (see Notes 25 and 26).
7. Thaw one cryotube aliquot, and assess the viral titer by plaque
assay as described previously in Subheading 3.4 (see Note 27).

4 Notes

1. BAC preparation is key for efficient rescue of rSARS-CoV-2.

We recommend high-purity BAC preparations with minimal
bacterial DNA contamination (e.g., Qiagen Large-Construct
Kit) for successful rescue of rSARS-CoV-2. Other commer-
cially available kits suitable for high-quality BAC purification
can also be used. If viral rescue is not achieved, we recommend
generating a new preparation of BAC clone.
2. DH10B are a recombination-defective E. coli strain designed to
avoid unwanted rearrangements, recommended for propaga-
tion of BACs containing large DNA fragments.
3. Proper maintenance of Vero E6 cells is key for successful rescue
of rSARS-CoV-2. We recommend keeping track of the cell
passage number and using low-passage cells (up to 30–35
passages). It is also important to maintain healthy Vero E6
cells without the presence of potential contaminants (e.g.,
mycoplasma). If viral rescue is not achieved, we recommend
using a new batch of fresh Vero E6 cells with a low passage
number.
4. Alternative cell lines besides Vero E6 cells can be used for
rescuing rSARS-CoV-2. We have successfully rescued rSARS-
CoV-2 using similar experimental procedures to those
described in this book chapter using Vero E6 cells
 BAC-Based SARS-CoV-2 Reverse Genetics 149

constitutively expressing human angiotensin converting

enzyme 2 (hACE2) and transmembrane serine protease
2 (TMPRSS2) (Vero E6-TMPRSS2-T2A-ACE2; BEI
NR-54970) as well as HEK293T constitutively expressing
hACE2 (HEK293T-hACE2; BEI NR-52511).
5. Alternative overlay media such as Avicel or methylcellulose can
also be used. However, the time for proper plaque formation
may vary using a different overlay media.
6. For the plaque assays, the DMEM/F-12 agar must be overlaid
over the cells at ~39 °C. Maintain the DMEM/F-12 mixture in
a 39 °C water bath before adding to wells.
7. Other primary mAb or polyclonal antibody (pAb) against
SARS-CoV-2 can be used. Optimal concentration of a different
mAb or pAb should be determined prior.
8. We recommend visualizing the presence of rescued rSARS-
CoV-2 plaques using a mAb or pAb against a viral protein.
Alternatively, the presence of viral plaques could be visualized
and quantified with dyes such as crystal violet staining.
9. Most electroporators are provided with specific programs for
different cell types. Choose the program recommended by the
provider for efficient electroporation of bacteria cells.
10. Do not overgrow the bacteria culture to avoid DNA degrada-
tion and potential unwanted rearrangements.
11. A large quantity of highly purified BAC is required and can be
generated with the provided protocol. If needed, use more
than 500 mL to obtain sufficient amount and quality of high-
purity BAC for viral rescue.
12. Avoid vortexing or shaking vigorously the BAC during purifi-
cation, as this may cause DNA shearing due to its large size.
13. It is recommended to generate and verify the purity of the
SARS-CoV-2 BAC preparation using a nanodrop to assess the
260:280 nm ratio and gel electrophoresis to guarantee success-
ful and optimal viral rescues.
14. The pBeloBAC11 is a low-copy number plasmid, and only one
to two copies are maintained per DH10B E. coli cell.
15. We recommend attempting to rescue rSARS-CoV-2 in tripli-
cates to ensure successful viral rescue.
16. Transfection of Vero E6 cells in suspension could increase the
transfection efficiency.
17. The density of Vero E6 cells on the day of the transfection
should be 80–90% confluent.
18. Addition of antibiotics may decrease transfection efficiency and
the rescue of rSARS-CoV-2.
150 Kevin Chiem et al.

19. For SARS-CoV-2 WA-1 strain, the optimal temperature for

viral rescue and growth is 37 °C. However, successful rescue
of rSARS-CoV-2 mutants or different strains may require dif-
ferent temperatures.
20. CPE can be determined by the presence of rounded and
birefringent cells detached from the flask and floating in the
cell culture supernatant. CPE is an indication of successful viral
rescue. However, full validation by IFA and/or plaque assay is
required.
21. Fixation with 10% neutral buffered formalin for 16 h will
inactivate SARS-CoV-2 and will permit safe removal of the
plates from the BSL3 laboratory to continue the characteriza-
tion of the virus at BSL2. Biocontainment procedures to
remove samples out of BSL3 must still be strictly followed.
22. A different blocking solution can be used to block the cells.
Cells can also be incubated with the blocking solution over-
night at 4 °C.
23. Immunofluorescence can be observed immediately under a
fluorescent microscope of visualized later. We recommend
storing the samples at 4 °C and keeping them protected from
the light for long-term storage.
24. The viral plaque size is dependent on the duration in which the
Vero E6 cells are incubated after viral infection. We do not
recommend stopping the plaque assay earlier than 48 h post-
infection or later than 96 h post-infection.
25. We recommend storing the rSARS-CoV-2 in small-volume
aliquots to prevent multiple freeze-thaw cycle that will result
in reduced viral titer.
26. We recommend sequencing the rescued rSARS-CoV-2 using
next-generation sequencing technique to identify possible
mutations introduced in the viral genome during amplification
of the virus in Vero E6 cells.
27. Successful viral rescue in Vero E6 cells can yield ~105–106
PFU/mL of rSARS-CoV-2.

Acknowledgments

Research on SARS-CoV-2 in L.M-S laboratory was partially sup-

ported by grants W81XWH2110103 and W81XWH2110095
from the Department of Defense (DoD) Peer Reviewed Medical
Research Program (PRMRP); 1R43AI165089-01,
1R01AI161363-01, and 1R01AI161175-01A1 from the National
Institutes of Health (NIH); the Center for Research on Influenza
Pathogenesis and Transmission (CRIPT), one of the National
 BAC-Based SARS-CoV-2 Reverse Genetics 151

Institute of Allergy and Infectious Diseases (NIAID)-funded Cen-

ters of Excellence for Influenza Research and Response (CEIRR;
contract # 75N93021C00014); the Antiviral Countermeasures
Development Center (AC/DC) (1U19AI171403-01), the Center
for Antiviral Medicines & Pandemic Preparedness (CAMPP)
(1U19AI171443-01), and the QCRG Pandemic Response Pro-
gram (1U19AI171110-01), three of the National Institutes of
Health (NIH)-funded Antiviral Drug Discovery Centers for Patho-
gens of Pandemic Concern; the San Antonio Partnership for Preci-
sion Therapeutics; and the San Antonio Medical Foundation.
Research in L.M-S was also partially supported by NIH
R01AI145332, R01AI142985, and R01AI141607 grants and by
DoD W81XWH1910496 PRMRP grant.

Materials Availability The pBeloBAC11-SARS-CoV-2 BAC

clone described in this book chapter for the generation of rSARS-
CoV-2 is available at this website: https://www.txbiomed.org/
services-2/reverse-genetics-plasmids. Other BAC clones as well as
their respective rSARS-CoV-2 are also available at the same website.

Conflict of Interest C.Y, F.A, and L. M.-S are co-inventors on a

patent application directed to BAC-based reverse genetics
approaches to generate rSARS-CoV-2.

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 Chapter 10

Construction of Infectious Clones for Human Enteroviruses

Thinesshwary Yogarajah and Justin Jang Hann Chu

Abstract
The infectious clone has been constructed for years via various mechanisms using reverse genetics of viral
RNA into cDNA. The mechanism of construction has evolved to DNA-launch plasmids which simplify
infectious clone manipulation and expression in mammalian cells. Infectious clones have enormously
allowed manipulation of the enterovirus genome to discover antivirals, viral replication mechanisms, and
functions of essential viral proteins. Here we will be discussing methods for the production of DNA-launch
human enterovirus infectious clones and viral genome engineered with a fluorescent reporter gene.

Key words Enterovirus, DNA-launch plasmids, Infectious clone, Reverse genetics, Fluorescence
reporter gene

1 Introduction

Enteroviruses (EVs) are members of the genus Enterovirus and

belong to the family of Picornaviridae [1]. EVs can be categorized
into 12 species inclusive of EV A-L and RV A-C [2]. The serological
and phylogenetic analysis of the human EV is classified into four
different groups accordingly: HEV-A to HEV-D [3]. All entero-
viruses are non-enveloped virions of about 30 nm diameter with a
capsid made up of 60 protomers where each protomer consists of
4 polypeptides (VP1, VP2, VP3, and VP4) [4]. The capsid encloses
a naked RNA genome consisting of a single-stranded, positive-
sense RNA of approximately 7.5 kilo bases. The enterovirus
genome consists of a basic viral protein, VPg, covalently linked to
the 5′ end of the 5′UTR, followed by the open reading frame
(ORF) and 3′UTR with a poly(A) tail [5]. The 5′UTR region is
highly conserved among human enteroviruses and consists of a
clover leaf-like structure called IRES, which is involved in viral
RNA replication and translation. The 5′UTR has an important
role in the internal initiation of translation [4].

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_10,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

155
156 Thinesshwary Yogarajah and Justin Jang Hann Chu

The construction of an infectious clone is also one of the

indispensable tools for experimental virology. Human EV infec-
tious clones have been developed to be used for various research
in understanding viral protein functions, viral replication mechan-
isms, viral vaccine productions, and therapeutics [6–9]. Thus far,
cDNA of the viral genome is placed downstream of bacteria RNA
transcription promoter (SP6 or T7) in an infectious clone construct
[10]. Howbeit, such design required tedious post-preparation such
as in vitro transcription before expression in mammalian cells. In
this study, a more direct and rapid approach is illustrated by using a
cytomegalovirus (CMV) eukaryotic promoter instead of a bacteria
promoter [11]. In this manner, the viral genome could be intro-
duced into host cells eliminating the need for in vitro transcription
prior to transfection as viral transcripts function as messenger RNA
to produce viral proteins. In our infectious clone design, the viral
gene is also engineered with a fluorescent reporter gene which
allows tracing of in vitro interaction and viral replication in the
host, making it a convenient tool for in vitro studies [12–14].
In all production of infectious clones, there are always various
options that need to be considered such as the size and type of
plasmids, type of bacterial competent cells (E. coli), and PCR
polymerase. These hurdles are also accommodated with the insta-
bility of viral cDNA due to developed mutation, deletions, and
rearrangements of the viral genome. These usually occur due to
the toxicity of viral cDNA in plasmids during propagation in E. coli.
This chapter presents a step-by-step guide of modern molecular
cloning procedures and exquisite insights to overcome the impedi-
ments mentioned above. It will also provide fundamental design
and procedures needed to establish a restriction endonuclease
digestion cloning experiment. This strategy can be used for the
generation of any human enterovirus and other RNA virus infec-
tious clones.
We will be walking through the production of a DNA-launch
infectious cDNA clone for enterovirus D68 (EV-D68) using high-
copy number plasmid vector pcDNA3.1(+) and E. coli strain Stellar
competent cells and STBL3 competent cells as bacterial hosts. The
construction and modification of the EV-D68 infectious cDNA
clone are summarized in Fig. 1. The breakdown of these methods
will be strategically explained in the following steps: (1) virus cul-
ture for viral RNA purification and cDNA synthesis via reverse
transcription, (2) double-stranded (ds) cDNA fragment produc-
tion via PCR amplification of reverse-transcribed single-stranded
cDNA into three fragments comprising the complete viral genome
and an addition of self-cleaving ribozyme, (3) cloning of the ds
cDNA fragments into high-copy number plasmid vector followed
by transformation into a bacterial host for propagation, (4) trans-
fection of the DNA-launch EV-D68 infectious clone into
 Construction of Infectious Clones for Human Enteroviruses 157

Fig. 1 Approach for the construction of EV-D68 complete genome DNA-launch infectious cDNA clone. Virus is
cultured in RD cells, and virus culture supernatant is used for RNA extraction. Viral RNA is reverse transcribed
into cDNA. Three overlapping fragments were produced via PCR labeled Fragment 1, Fragment 2, and
Fragment 4. Fragment 4 was produced via fusion PCR with additional hepatitis delta virus sequence encoding
for a self-cleaving ribozyme (to be henceforth referred to as “HDV”). Each fragment was cloned into high-copy
number plasmid vector sequentially via unique restriction sites. The complete genome clone is transfected
into permissive mammalian cells for EV-D68 replication to rescue clone-derived virus. The fluorescence
reporter gene is then inserted into complete genome clone for obtaining the EV-D68 fluorescence reporter
gene expression virus

mammalian cells to generate the clone-derived enterovirus D68

virus, (5) propagation of the generated enterovirus D68 to deter-
mine genetic and phenotypic characterization in comparison to the
parental virus, (6) designing insertion of fluorescence reporter gene
and generation of fluorescence reporter gene via PCR, (7) and
cloning of fluorescence reporter gene and transfection of the
DNA-launch EV-D68 infectious clone with fluorescence reporter
gene for characterization.
This molecular cloning procedures and methods have been
approved by the Genetic Modification Advisory Committee
(GMAC), Singapore (Protocol ID: 23512). It is crucial to under-
stand and follow each genetic modification advisory committee
guideline at residential institution/country before the initiation of
genetic modification experiments.
158 Thinesshwary Yogarajah and Justin Jang Hann Chu

2 Materials

2.1 Viral RNA 1. Virus culture supernatant containing EV-D68.

Extraction 2. NanoDrop 2000 (Thermo Scientific) (see Note 1).
3. QIAamp Viral RNA Mini Kit (see Note 1).

2.2 Reverse 1. Reverse transcriptase: 200 U Maxima H Minus Reverse Tran-

Transcription scriptase (see Note 1), 5× RT buffer, 40 U ribonuclease inhibi-
tor, 0.5 mM dNTPs, 10 mM DTT, and 30 pmol
oligonucleotide primer.
2. Nuclease-free water (Ambion).

2.3 PCR PCR reaction: Q5 Hot Start High-Fidelity DNA 2X Master Mix
Amplification (see Note 2). 10 pmol oligonucleotide primer, nuclease-free water.

2.4 Agarose Gel 1. 0.8% TAE-agarose gel: 0.8 g agarose, 100 mL 1× TAE buffer
Electrophoresis and consists of 1 mM EDTA, 40 mM tris acetate.
Gel Extraction 0.5% TAE-agarose gel: 0.5 g agarose, 100 mL 1× TAE
buffer consists of 1 mM EDTA, 40 mM tris acetate.
2. 100 bp DNA ladder and 1 kb plus DNA ladder.
3. Ethidium bromide (EtBr) for DNA visualization.
4. Agarose gel documentation system with trans-UV lamp and a
blue light transilluminator.
5. QIAquick Gel Extraction Kit (see Note 1).

2.5 DNA Cloning 1. pcDNA3.1(+) Invitrogen Cloning Kit. pEGFP Clontech.

2. Stellar Competent Cells (Takara Bio) and STBL3 cells
(Thermo Scientific).
3. LB broth with ampicillin: 1% tryptone (w/v), 0.5% yeast
extract (w/v), 1% NaCl (w/v), 100 μg/mL ampicillin.
4. LB agar with ampicillin: 1% tryptone (w/v), 0.5% yeast extract
(w/v), 1% NaCl (w/v), 0.7% agar (w/v), 100 μg/mL
ampicillin.
5. Restriction endonuclease digestion: 1× buffer, 10 U of NheI-
HF, NotI-HF, EcoRI-HF, AgeI-HF (New England BioLabs)
(see Note 3).
6. DNA ligation: 1× T4 DNA ligase buffer, 1 U T4 DNA ligase.
7. SOC medium: 2% tryptone (w/v), 1% yeast extract (w/v),
20 mM glucose, 2.5 mM KCl, 8.5 mM NaCl, 10 mM MgCl2.
8. Plasmid extraction kits: QIAprep Spin Miniprep Kit (QIA-
GEN), QIAGEN Plasmid Plus Midi Kit (QIAGEN) (see
Note 1).
9. QIAquick PCR purification kit (see Note 1).
 Construction of Infectious Clones for Human Enteroviruses 159

2.6 Recovery and 1. Transfection reagents: jetPRIME buffer, jetPRIME reagent

Generation of (see Note 1).
Infectious Clone- 2. Cell culture media: Dulbecco’s Modified Eagle Medium
Derived EV-D68 Virus (DMEM) high glucose supplemented with 10% FBS or
and Fluorescence 2% FBS.
Reporter Gene 3. Cell lines: Human embryonic kidney 293T (HEK293T) cells,
Expression EV-D68 human rhabdomyosarcoma (RD) cells.
Virus
4. 1× Phosphate-buffered saline (PBS).
5. Six-well tissue culture plate (cell-culture treated).
6. Tissue culture flasks (T75, cell-culture treated).
7. Olympus IX81 fluorescence microscope.

3 Methods

3.1 EV-D68 Viral RNA 1. Propagate virus in suitable cell culture until the cytopathic
Extraction and Viral effect is observed. Following that harvest virus culture super-
cDNA Synthesis natant from EV-D68-infected cells (see Note 4). Viral RNA is
extracted using QIAamp Viral RNA Mini Kit according to the
manufacturer’s instructions. Viral RNA is eluted in 50 μL of
AVE buffer. Quantitate RNA concentration in ng/μL using
NanoDrop 2000.
2. A first strand cDNA is synthesized covering the complete
genome of EV-D68 with Maxima H minus Reverse Transcrip-
tase (see Note 1). Assemble a reverse transcription reaction with
a total volume of 20 μL in a 0.2 mL tube. Mix appropriate
volume of viral RNA (1 μg) and 3 μL (30 pmol) of the oligo-dT
anchored primers (see Note 5), and heat for 5 min at 25 °C.
Then keep on ice for 5 min. Then add 200 U Maxima H Minus
Reverse Transcriptase, 5× RT buffer, 40 U ribonuclease inhibi-
tor, 0.5 mM dNTPs, 10 mM DTT, and makeup balance vol-
ume with nuclease-free water. Incubate the mixture at 55 °C
for 30 min, followed by incubation at 85 °C for 5 min for the
inactivation of the enzyme (see Note 5). Store the cDNA
product at -20 °C until required.

3.2 Viral cDNA 1. Amplify cDNA fragments using EV-D68-specific primers into
Fragment three fragments with different lengths. Each primer set is used
Amplification with PCR in PCR with Q5 Hot Start High-Fidelity DNA 2X Master Mix.
For the amplification of Fragment 1 from cDNA, NheI, a
unique restriction site, is inserted upstream of the 5′UTR
viral sequence, and NotI, also a unique restriction site, is
inserted downstream of the 2C viral sequence to ease cloning
into pcDNA3.1(+) plasmid vector (Fig. 2).
160 Thinesshwary Yogarajah and Justin Jang Hann Chu

Fig. 2 Primer sets and schematic representation of Fragment 1 and Fragment

2 from EV-D68 genome

2. Carry out PCR reactions in a 0.2 mL tube with a reaction

volume of 25 μL following Q5 Hot Start High-Fidelity DNA
2X Master Mix protocol. In brief, the PCR reaction uses 1× Q5
Hot Start High-Fidelity Master Mix which amounts to 12.5 μL
and 0.5 μM each forward and reverse primer and 2 μL of cDNA
(from Subheading 3.1, step 2). Carry out Fragment 1 amplifi-
cation with cDNA from Subheading 3.1, step 2. Assemble a
PCR reaction with an initial denaturation at 98 °C 30 s, fol-
lowed by 40 cycles of 98 °C 10 s, 64–68 °C 30 s, and 72 °C
30 s/kb, and a final incubation at 72 °C for 2–5 min (see
Note 5).
3. For the amplification of Fragment 2 from cDNA, the PCR
forward primer is designed at the already present AgeI, a
unique restriction site in the 2C region of the EV-D68 viral
sequence. The reverse primer on the other hand is inserted with
a unique restriction site downstream of the 3B viral sequence,
NotI (Fig. 2). PCR of the fragment is carried out according to
the mentioned Subheading 3.2, step 2.
4. For the amplification of Fragment 3 from cDNA, the PCR
forward primer is designed at the already present EcoRI, a
 Construction of Infectious Clones for Human Enteroviruses 161

unique restriction site in the 3B region of the EV-D68 viral

sequence. The reverse primer is incorporated with poly-A
sequence (see Note 6) and part of HDV sequence (see Note
6) to ease fusion PCR (Fig. 3). PCR of the fragment is carried
out with primer set 1 (Fig. 3) according to the mentioned
Subheading 3.2, step 2. For the amplification of HDV frag-
ment, primer set 2 (Fig. 3) is used where PCR forward primer is
incorporated with poly-A sequence ahead of HDV sequence
and reverse primer is inserted with a unique restriction site,
NotI, downstream of the HDV sequence to ease cloning into
pcDNA3.1(+) plasmid vector. PCR of the fragment is carried
out with primer set 2 (Fig. 3) according to the mentioned
Subheading 3.2, step 2.
5. Carry out agarose gel electrophoresis to determine the
expected sizes of resulting PCR products from cDNA amplifi-
cation. Run 10 μL of each PCR product through 0.8%
TAE-agarose gel at approximately 120 volts in 1× TAE buffer
(see Note 7). DNA on agarose gel documentation system is
visualized using trans-UV lamp. Once the correct band size is
identified, mix 25 μL of PCR product with dye loading buffer
before running through 0.8% TAE-agarose gel at approxi-
mately 120 volts in 1× TAE buffer. DNA is viewed on a blue
light transilluminator. The expected band size is excise from the
gel followed by gel extraction using QIAquick Gel Extraction
Kit (see Note 8).
6. Fusion PCR will be carried out to produce Fragment 4 which is
the combination of both Fragment 3 and HDV fragment.
Fusion PCR involves two-step PCR. The first step will be to
carry out PCR reaction using 1× Q5 Hot Start High-Fidelity
Master Mix which amounts to 12.5 μL and 2 μL of gel excised
and purified Fragment 3 and 2 μL of gel excised and purified
HDV fragment (from Subheading 3.2, step 5) and the addi-
tion of nuclease-free water to a total volume of 25 μL. Carry
out the fusion PCR step 1 amplification with an initial denatur-
ation at 98 °C 30 s, followed by 15 cycles of 98 °C 10 s,
64–68 °C 30 s, and 72 °C 30 s/kb, and then a final incubation
at 72 °C for 2 min. Followed by fusion PCR step 2 amplification
with 1× Q5 Hot Start High-Fidelity Master Mix which
amounts to 12.5 μL and 0.5 μM each forward and reverse
primer of set 3 (Fig. 3) and the addition of nuclease-free
water to a total volume of 25 μL. Carry out the fusion amplifi-
cation with an initial denaturation at 98 °C 30 s, followed by
40 cycles of 98 °C 10 s, 64–68 °C 30 s, and 72 °C 30 s/kb, and
then a final incubation at 72 °C for 2 min. Carry out agarose gel
electrophoresis, and excise PCR products of the expected sizes.
Gel purify Fragment 4 using QIAquick Gel Extraction Kit.
162 Thinesshwary Yogarajah and Justin Jang Hann Chu

Fig. 3 Fusion PCR primer sets and schematic representation of Fragment

4 production
 Construction of Infectious Clones for Human Enteroviruses 163

Fig. 4 The construction of EV-D68 complete genome DNA-launch infectious cDNA clone using three
fragments. Three overlapping fragments starting from Fragment 1 followed by Fragment 2 and then Fragment
4 were sequentially inserted via specific RE digestion into pcDNA3.1(+) plasmid vector

3.3 Viral cDNAs 1. All purified fragments and pcDNA3.1(+) plasmid vector are
Cloning into a Plasmid quantitated using NanoDrop 2000. Double digestion by two
Vector restriction endonuclease, PCR purification kit, and ligated and
transformed into plasmid sequentially (Fig. 4). For the assem-
bly of Fragment 1, restriction endonucleases Nhel and NotI are
used for double digestion. Restriction endonuclease digestion
was set up according to New England BioLabs manufacturer’s
protocol. Restriction endonucleases are used to generate phos-
phorylated ends of the purified cDNA fragments and plasmid
vector for subsequent sticky-end cloning (see Note 8).
164 Thinesshwary Yogarajah and Justin Jang Hann Chu

2. A typical restriction digest reaction is carried out with 1 μg of

purified cDNA, 5 μL of 10× NEBuffer (see Note 9), 10 units/λ
DNA of NheI and NotI (see Note 9), and nuclease-free water
to make a total volume of 50 μL. The mixture is incubated at
37 °C for 1 h, 65 °C for 20 min.
3. Take 8 μL of the double-digested cDNA Fragment 1 and the
linearized plasmid DNA, and separate them by gel electropho-
resis on 0.8% TAE-agarose gel to confirm the presence of right-
sized bands and the complete linearization of the plasmid.
Purify the remainder of the sample using QIAquick PCR puri-
fication kit. Evaluate the quality and quantity of purified DNA
by NanoDrop 2000.
4. Assemble a ligation reaction with a volume of 20 μL in a 0.2 mL
tube. Universally utilize insert-to-vector molar ratios of 1:1 or
3:1 (see Note 10). Initially mix 50 ng of vector with an accurate
amount of insert fragment, 1 U of T4 DNA ligase and 1× T4
DNA ligase buffer and makeup balance volume with nuclease-
free water. Carry out the ligation reaction overnight at 16 °C.
The next day use 2–5 μL of the DNA ligation mix for transfor-
mation. Assembled ligation reaction should not be freeze and
thawed before transformation.
5. Thaw frozen Stellar competent cells on ice for 10 min till
completely thawed. Place DNA ligation mix on the ice to
equilibrate to a similar temperature as competent cells. Carry
out transformation in 100 μL of Stellar competent cells by
adding 2–5 μL of the DNA ligation mix with the dropwise
method. Then gently dab the tube to mix (do not mix by
pipetting). Incubate mix on ice for 30 min. Then heat-shock
the tube in a 42 °C water bath for 45 s without shaking.
Immediately transfer the tube on ice, incubate for 2 min,
then add 200 μL of pre-warmed to 37 °C SOC media, and
mix gently. Incubate the tube in a shaker incubator pre-set to
37 °C with continuous shaking at 200× g for 1 h for recovery.
Following recovery, warm LB agar containing ampicillin
100 μg/mL plates, and spread the transformation mixture
(50 μL) before incubation at 30 °C for 18–24 h (see Note 11).
6. Individual E. coli colonies harboring transformed plasmid vec-
tor are picked and propagated in LB broth comprise of ampi-
cillin 100 μg/mL in a shaker incubator at 30 °C for 18–24 h at
200× g (see Note 12). Isolate the plasmid DNA using the
QIAprep Spin Miniprep Kit, and characterize the plasmid
DNA using a unique restriction endonuclease digestion present
only in the insert of the plasmid DNA. Concurrently sequence
the region of insert of the cDNA clones to ensure that no
mutations have been incorporated during the reverse transcrip-
tion, PCR, and cloning steps.
 Construction of Infectious Clones for Human Enteroviruses 165

7. Assemble Fragment 2 into the plasmid containing Fragment

1. Initially carry out a double digestion with the unique restric-
tion site that is present in Fragment 2: NotI inserted into
primer set 2 from Fig. 2 and AgeI that is naturally existing in
the region of 2C of EV-D68. Double digestion is carried out as
mentioned in Subheading 3.3, step 2, and purification, liga-
tion, and transformation are carried out as mentioned in Sub-
heading 3.3, steps 3–5. Individual E. coli colonies harboring
transformed plasmid vector are picked, propagated, and
isolated using the QIAprep Spin Miniprep Kit before screening
of plasmid DNA using unique restriction endonuclease analy-
sis, which was incorporated, AgeI. Sequencing of Fragment
2 in the isolated plasmid is carried out to determine the pres-
ence of fragment of interest.
8. Assembly of Fragment 4 into plasmid containing Fragments
1 and 2. First perform a double digestion with a site that is
naturally existing in the region of 3B of EV-D68, EcoRI, and
the unique restriction site that is integrated into the HDV PCR
primers, NotI. Double digestion and purification are carried
out as mentioned in Subheading 3.3, step 2 and 3. Ligation
and transformation are modified for this fragment due to the
high toxicity of the EV-D68 3D region propagation in E. coli
bacterial cells. Assemble a ligation reaction with a volume of
20 μL in a 0.2 mL tube. Initially mix 50 ng of vector DNA with
a pertinent amount of insert, 1× T4 DNA ligase buffer, and 1 U
of T4 DNA ligase. Carry out the ligation reaction overnight at
16 °C. For transformation, utilize about 2–5 μL from the DNA
ligation mix.
9. Due to the high toxicity of EV-D68 3D region, competent cells
were switched to STBL3 cells (see Note 13). Initially, thaw
frozen competent cells on ice for 10 min till completely
thawed. Place DNA ligation mix on the ice to equilibrate to a
similar temperature as competent cells. Carry out transforma-
tion in 100 μL of STBL3 competent cells by adding 2–5 μL of
the DNA ligation mix, dab the tube gently to mix, and incubate
on ice for 30 min. Then warm water bath at 42 °C and heat-
shock STBL3 competent cells containing DNA ligation mix for
45 s without shaking, immediately transfer the tube to ice, and
hold for 2 min. Add 200 μL of pre-warmed to 37 °C SOC
media, and mix gently. Incubate the tube in a shaker incubator
pre-set to 30 °C with continuous shaking at 200× g for 1 h for
recovery. Following recovery, warm LB agar containing ampi-
cillin 100 μg/mL plates, and spread the transformation mix-
ture (50 μL) before incubation at 30 °C for 18–24 h (see
Note 11).
166 Thinesshwary Yogarajah and Justin Jang Hann Chu

10. Individual E. coli colonies harboring transformed plasmid vec-

tor are picked and propagated in LB broth comprised of ampi-
cillin 100 μg/mL in a shaker incubator at 25 °C for 24–36 h at
200× g (see Note 13). The clones are propagated and isolated
using the QIAGEN Plasmid Plus Midi Kit and characterized
using restriction endonuclease analysis unique to Fragment
4, EcoRI. Sequencing of the complete genome cDNA clones
(pcDNA3.1(+) EV-D68) is carried out to confirm that no
mutations have been incorporated during the reverse transcrip-
tion, PCR, and cloning steps.

3.4 Plasmid 1. Grow HEK293T cells in DMEM supplemented with 10% FBS,
Transfection to and then seed cells into cell-culture treated 6-well tissue culture
Recover Infectious plate at a seeding density of 600,000 cells per well to achieve
Clone-Derived EV-D68 60–70% confluency at the time of transfection. Incubate plate
Virus for 18–24 h before transfection (see Note 14).
2. Replace DMEM supplemented with 10% FBS with 2.5 mL of
fresh DMEM supplemented with 2% FBS about 30 min prior
to transfection. Keep plate in 37 °C until use.
3. Transfect purified plasmid DNA into cells using jetPRIME
reagent. Dilute 5 μL jetPRIME reagent in 200 μL of jetPRIME
buffer, then add 2–3 μg of plasmid DNA, and mix gently.
Incubate mixture for 10 min at room temperature. Add com-
plex by dropwise method into cells, and mix side by side before
incubation at 33 °C incubator for 4–7 days (see Note 15). Then
freeze the transfected six-well tissue culture tray in a -80 °C
freezer.

3.5 Propagation and 1. Seed RD cells in a T75 flask at a seeding density of 12 million
Characterization of cells/flask or 80% confluency. Incubate the cells 18–24 h before
Clone-Derived EV-D68 infection with clone-derived EV-D68 (see Note 14).
2. Thaw the transfected six-well tissue culture tray mentioned in
Subheading 3.4, step 3 in a 37 °C incubator until completely
thawed. Harvest the supernatant from transfected wells into a
cryovial. Infect 1 mL of the virus culture supernatant from
cryovials into the seeded T75 flask, and incubate at 33 °C for
60 min. Following that remove unbound virus by washing with
warm 1× PBS. Add 10 mL of DMEM with 2% FBS, and
incubate at 33 °C with 5% CO2 until cytopathic effect (CPE)
is visible. Virus harvest can be carried out upon observation of
80–90% CPE. Unless there is no visible CPE, upon consider-
ation of cell health in flask, harvest virus after infection upon
4–6 days (see Note 16).
3. Phenotypically characterize the clone-derived EV-D68 virus by
comparing plaque morphology on RD cells by performing
plaque assay, and then compare growth kinetics to the parental
virus.
 Construction of Infectious Clones for Human Enteroviruses 167

4. For plaque assay, RD cells were seeded on a 24-well plate at a

density of 2.8 × 104 cells/mL and incubated overnight at
37 °C with 5% CO2. Viral supernatants were subjected to
dilution from 10-1 to 10-6 in DMEM with 2% FBS, and the
cells were infected with 100 μL of diluted supernatant for 1 h at
33 °C with 5% CO2. The cells were then washed twice with 1×
PBS and overlaid with 1 mL of DMEM with 2% FBS and 0.5%
agarose. The plates were incubated for 4 days at 33 °C with 5%
CO2 for plaque formation. Cells were fixed and stained with 4%
paraformaldehyde and 1% crystal violet. Then, the size of
plaque formation was measured between clone-derived
EV-D68 virus and parental virus, and the number of viral
plaques was counted manually to determine the viral titer in
plaque-forming units (PFU)/mL.
5. For growth kinetics, RD cells were seeded on a 24-well plate
at a density of 2.8 × 104 cells/mL and incubated overnight at
37 °C with 5% CO2. Cells were infected with virus at MOI of
1 and incubated for 1 h at 33 °C with 5% CO2. The cells were
then washed twice with 1× PBS and added with 1mL of
DMEM with 2% FBS. Cells were harvested at intervals of 2 h
up to 12 h, and plaque assays were used to determine viral
titers.
6. Secondly, genetically characterize the clone-derived EV-D68
virus by sequencing of the viral complete genome. Use 280 μL
of clone-derived EV-D68 virus culture supernatant for viral
RNA extraction as described in Subheading 3.1, and carry
out reverse transcription with 1 μg of viral RNA and 30 pmol
of the oligo-dT anchored primers. Perform PCR as described in
Subheading 3.2 for the complete genome using primer sets
from Fragment 1 to Fragment 4. Use BigDye terminator tech-
nology to sequence all fragments by primer walking. Compare
the sequence of cloned-derived EV-D68 virus to the parental
viral sequence.

3.6 Introduction of a 1. To insert a fluorescence reporter gene, PCR is carried out using
Fluorescence Reporter a pEGFP plasmid containing EGFP gene. The insertion of
Gene into the Complete EGFP gene in between 5′UTR and VP4 gene is accommo-
Genome cDNA Clone dated by producing three fragments of PCR and stitching them
together using fusion PCR. Each primer set is used in PCR
with Q5 Hot Start High-Fidelity DNA 2X Master Mix. For the
amplification of Fragment 1 from pcDNA3.1(+) EV-D68, a
unique restriction site, NheI is inserted upstream of the 5′UTR
of viral sequence, and 15 base of EGFP sequence is inserted
in the reverse primer in set 1 to ease fusion PCR (Fig. 5).
Fragment 2 is amplified from pEGFP plasmid using a 15 base
of 5′UTR sequence addition in the forward primer and an
addition of a unique 2A cleavage sequence in the reverse primer
168 Thinesshwary Yogarajah and Justin Jang Hann Chu

Fig. 5 Fusion PCR primer sets and schematic representation of Fragment

5 production. Three overlapping fragments—Fragment 1, Fragment 2, and
Fragment 3—were stitched using fusion PCR for the formation of Fragment 5
 Construction of Infectious Clones for Human Enteroviruses 169

in set 2 (Fig. 5). Fragment 3 is amplified from pcDNA3.1(+)

EV-D68, a 15 base of 2A cleavage site, and VP4 is included in
forward primer, and BbvCI, a unique restriction site, is inserted
downstream if reverse primer.
2. Fusion PCR is carried out to produce Fragment 5 which is the
combination of Fragments 1, 2, and 3 from Subheading 3.6,
step 1. Fusion PCR involves a two-step PCR. The first step will
be to perform PCR reaction using 1× Q5 Hot Start High-
Fidelity Master Mix which amounts to 12.5 μL and 2 μL of
gel excised and purified Fragments 1, 2, and 3 premixed at a
ratio of 1:1:1 and the addition of nuclease-free water to a total
volume of 25 μL. Carry out the fusion PCR step 1 amplification
with an initial denaturation at 98 °C 30 s, followed by 15 cycles
of 98 °C 10 s, 64–68 °C 30 s, and 72 °C 30 s/kb, and then a
final incubation at 72 °C for 2 min. Then carry out fusion PCR
step 2 amplification with 1× Q5 Hot Start High-Fidelity Mas-
ter Mix which amounts to 12.5 μL and 0.5 μM each forward
and reverse primer of set 3 (Fig. 5) and the addition of
nuclease-free water to a total volume of 25 μL. Perform the
fusion amplification with an initial denaturation at 98 °C 30 s,
followed by 40 cycles of 98 °C 10 s, 64–68 °C 30 s, and 72 °C
30 s/kb, and then a final incubation at 72 °C for 2 min.
Perform agarose gel electrophoresis and excise PCR products
of the expected sizes. Gel purify Fragment 5 using QIAquick
Gel Extraction Kit.
3. For the insertion of Fragment 5 into pcDNA3.1(+) EV-D68,
restriction endonucleases Nhel and BbvCI are used for double
digestion of Fragment 5 and pcDNA3.1(+) EV-D68. Restric-
tion endonuclease digestion was set up according to New
England BioLabs manufacturer’s protocol. Ligation, transfor-
mation, and recovery of the infectious clone were carried out as
mentioned in Subheadings 3.3, steps 9 and 10, and 3.4. For
the recovery of pcDNA3.1(+)-EGFP EV-D68 infectious clone,
transfected HEK293T cells were viewed under Olympus IX81
fluorescence microscope to determine the production of EGFP
during virus replication (Fig. 6).
4. For growth kinetics, RD cells were seeded on a 24-well plate
at a density of 2.8 × 104 cells/mL and incubated overnight at
37 °C with 5% CO2. Cells were infected with EGFP EV-D68
virus and wild-type EV-D68 virus at MOI of 1 and incubated
for 1 h at 33 °C with 5% CO2. The cells were then washed twice
with 1× PBS and added with 1 mL of DMEM with 2% FBS.
Cells were harvested at intervals of 2 h up to 12 and at 6 h of
interval thereafter up to 24 h, and plaque assays were used to
determine viral titers for comparison.
5. Secondly, genetically characterize the clone-derived EGFP
EV-D68 virus by sequencing of the viral complete genome.
170 Thinesshwary Yogarajah and Justin Jang Hann Chu

Fig. 6 The construction and propagation of EGFP EV-D68 DNA-launch infectious cDNA clone. Fragment 5 was
inserted via specific RE digestion into pcDNA3.1(+) EV-D68 plasmid vector. Clone-derived vector was
transfected into HEK293T cells, and EGFP expression was viewed using Olympus IX81 fluorescence
microscope

Use 280 μL of clone-derived EV-D68 virus culture supernatant

for viral RNA extraction as described in Subheading 3.1 and
carry out reverse transcription with 1 μg of viral RNA and
30 pmol of the oligo-dT anchored primers. Perform PCR as
described in Subheading 3.2 for the complete genome using
primer sets from Fragment 5. Sequence purified PCR product
by primer walking using BigDye terminator technology. Com-
pare the sequence of cloned-derived EGFP EV-D68 virus to
the parental viral sequence and EGFP sequence.
 Construction of Infectious Clones for Human Enteroviruses 171

4 Notes

1. Usage of specific kits and reagents mentioned in our material

can be substituted with comparably same kits and reagents.
2. The use of Hot Start Q5 allows room temperature reaction
setup. This is due to hot start DNA polymerase with 3′ → 5′
exonuclease activity and an addition of an aptamer-based inhib-
itor. The use of Q5 without Hot Start would preferably require
a reaction setup on ice.
3. The NEB restriction endonucleases used are high-fidelity
(HF) options to ensure reduced star activity and rapid diges-
tion. However, non-high-fidelity and other brand restriction
endonucleases that function the same can be used.
4. The viral titer from virus culture of EV-D68 in RD cells must
be a minimum of 107 PFU/mL to yield a high quantity of viral
RNA. For QIAamp Viral RNA Mini Kit, 280 μL (twice the
volume recommended by the manufacturer’s protocol) of virus
supernatant can be used for each viral RNA extraction in a
single mini column.
5. In the preparation of cDNA, the oligo-dT anchored primers
can be replaced with random hexamer primers or EV-D68 3′
UTR-specific primers. When using EV-D68 3′UTR-specific
primers, ensure to optimize the incubation temperature to
match primer annealing temperature. When using Hot Start
Q5 polymerase, the online tool (NEB Tm Calculator) is
recommended to be used to determine the optimal annealing
temperature for the primer set used.
6. The poly-A residues which are incorporated in the reverse
primer should be >25 nucleotides for enteroviruses to ensure
proper cleavage during translation and transcription in trans-
fected mammalian cells. The reverse primers also include
13 nucleotides specific to the 3′ terminus of the 3′ fragment
for amplification of the viral genome. The HDV sequence does
not possess any upstream sequence composition prerequisite
which enables direct insertion of HDV immediately after the
EV-D68 poly-A sequence. Since HDV allows for the genera-
tion of homogeneous RNA 3′ ends, it ensures precision and
consistency in the length of the generated RNA transcript and
does not exceed the maximum packaging limit of the virus
[15, 16].
7. Gel electrophoresis voltage can range from 90 to 135 volts
depending on band separation duration. Usage of TAE buffer
is recommended as compared to TBE buffer since TAE buffer
provides a high resolution for short DNA fragments.
172 Thinesshwary Yogarajah and Justin Jang Hann Chu

8. To enhance cloning efficiency and avoid damage to DNA by

UV light while excising the expected DNA band, the PCR
products and plasmid vector in agarose gel are exposed only
to visible light.
9. With the use of NEB restriction endonuclease, it is important
to take note of the NEB buffer recommendation for each RE
used. For digestion with 2 RE in the same reaction, both REs
should be compatible in NEB buffer with a higher digestion
activity and at the recommended optimal temperature. Sequen-
tial digestion should be carried out for incompatible RE. For
the first digestion reaction, 3–5 μg of cDNA and DNA is used
to ensure that the concentration of PCR fragments and plasmid
DNA, respectively, is sufficiently high following the second
restriction endonuclease digestion.
10. Carry out pcDNA 3.1(+) control ligation containing only a
plasmid vector without inserts in an effort to approximate
background colonies.
11. For transformation mixture plating onto LB agar for plasmid
with Fragments 1 and 2, the optimal temperature for STELLA
competent cells is used for high-copy plasmid propagation.
However, with transformation mixture plating onto LB agar
of Fragment 3 containing an unstable EV-D68 3D region,
STBL3 competent cells are used, and a lower temperature is
used to ensure slow propagation of plasmid in bacterial cells.
12. Grow a small-scale preparation of the high-copy number
pcDNA3.1(+) in 10 mL of LB broth containing 100 μg/mL
of ampicillin. This small-scale preparation is used solely for
sequencing and restriction endonuclease digestion to ensure
the plasmid contains the correct insert. For further analysis of
the insertion of Fragment 2 and Fragment 3, a medium-scale
preparation of the plasmid was used. The plasmid was grown
for 6–8 h in LB broth containing 100 μg/mL ampicillin, and
then 200 μL was transferred into 200 mL of LB broth contain-
ing 100 μg/mL ampicillin for growth of 18–24 h before plas-
mid purification.
13. The change of competent cells from Stellar competent cells and
STBL3 competent cells is due to the latter being specifically
designed for cloning unstable inserts and conferring structural
and maintenance stability [17]. After insertion of Fragment
3, pcDNA3.1(+) was grown on 800 mL of LB broth contain-
ing 100 μg/mL ampicillin and was grown for 24–36 h before
plasmid purification. To obtain a high yield of plasmid DNA,
retransform plasmid DNA, and pick freshly grown colony to
grow bacteria culture.
 Construction of Infectious Clones for Human Enteroviruses 173

14. For the transfection of pcDNA3.1(+) EV-D68 and pcDNA3.1

(+)-EGFP EV-D68, we used HEK293T cells since these cells
have a high transfectability as it contains the SV40 T-antigen.
However, propagation and characterization of EV-D68 and
EGFP EV-D68 derived from clones were carried out in RD
cells since RD cells are the gold standard for EV-D68 propaga-
tion based on the Centers for Disease Control and Prevention
(CDC) and the American Type Culture Collection (ATCC).
RD cells are well permissive to EV-D68 infection.
15. Transfected cells should be observed daily for cytopathic effect.
There are possible times to confuse with cytopathic effect and
cellular toxicity. The expression of EV-D68 clone-derived plas-
mid (>10 kb) takes 3–4 days. If a high cellular toxicity is
observed, reduce the quantity of plasmid DNA to 1 μg, or
exchange the media containing plasmid DNA and jetPRIME
reagent 24 h post-transfection.
16. It is crucial to carry out phenotypic characterization of the
clone-derived EV-D68 virus and parental EV-D68 virus to
compare for similarity. This is to ensure that the clone-derived
virus characteristic is the same as the parental strain and that the
addition of EGFP after 5′UTR does not affect virus replication.
It is important to use clone-derived virus from the first or the
second passage for phenotypic characterization.

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1128/mSphere.00104-16
 Chapter 11

Reverse Genetics of Hepatitis C Virus Using an RNA

Polymerase I-Mediated Transcription
Ryosuke Suzuki and Tetsuro Suzuki

Abstract
The reverse genetics system commonly used for the production of hepatitis C virus (HCV), which is a major
causative agent of liver diseases, involves introduction of the viral genomic RNA synthesized in vitro into
human hepatoma cells by electroporation. As an alternative methodology, we describe a cell culture system
based on transfection with an expression plasmid containing a full-length HCV cDNA clone flanked by
RNA polymerase I promoter and terminator sequences to generate infectious virus particles from trans-
fected cells.

Key words Plasmid-based reverse genetics, Hepatitis C virus, RNA polymerase I, Promoter, Termi-
nator, Infectious cDNA clone

1 Introduction

Hepatitis C virus (HCV) is a hepatotropic enveloped virus with a

9.6-kb, single-stranded RNA genome of positive polarity that
belongs to the Hepacivirus genus within the Flaviviridae family.
Chronic HCV infection is one of the leading causes of chronic liver
disease, including cirrhosis and hepatocellular carcinoma
[1, 2]. The viral RNA genome codes for one long polyprotein
that is co- and posttranslationally processed into the mature gene
products. The RNA cis-elements that control HCV RNA genome
translation largely reside in highly structured 5′- and 3′-
-untranslated regions (UTRs) of the genome. Unlike most cellular
mRNAs, the 5′ end of HCV genome does not have a cap nucleotide
attached that governs cap-dependent translation initiation. Instead,
HCV translation is mediated by an internal ribosomal entry site
located primarily in the 5′-UTR [3, 4].
In the reverse genetics system commonly used for basic HCV
research and evaluation of antiviral drugs, the viral genomic RNA is
generally synthesized in vitro from plasmids containing full-length

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_11,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

175
176 Ryosuke Suzuki and Tetsuro Suzuki

HCV cDNA, which is then electroporated into human hepatoma-

derived cell lines. Virus genome replication in cells has been ana-
lyzed, and infection experiments have been conducted using viruses
produced in this culture system [3]. To find an alternative approach
to develop a reverse genetics system for HCV, we have turned to the
RNA polymerase I (Pol I)-mediated expression system, which has
been utilized in the virus production from cloned cDNAs of influ-
enza [5–9] and other RNA viruses [10–16]. The Pol I systems
consist of a viral cDNA or a reporter gene flanked by the Pol I
promoter and terminator to produce transcripts with correct 5′-
and 3′-ends that are free of modifications such as cap structures and
poly(A) tails.
We have demonstrated that the HCV full-length RNA pro-
duced using this system is non-spliced with precise sequences at
both ends and is replicated in the cytoplasm of transfected cells to
produce infectious particles [17]. This methodology can be applied
not only to transient HCV production systems but also to the
generation of stable cell lines that constitutively produce HCV
and to trans-encapsidation systems by transient transfection of
two plasmids [17, 18] (see Note 1).

2 Materials

2.1 HCV This protocol is described for the rescue of HCV. Work with HCV
should follow biosafety laboratory rules and regulations appropri-
ate for this type of work. All material used for the rescue of HCV
should be sterilized before disposal.
Reverse genetic manipulation of HCV infectious cDNA clones
can be successfully performed using HCV strain that can propagate
in vitro. This method was described based on the experience using
HCV JFH-1 strain [17, 19].

2.2 Cloning Vector The plasmid containing the human RNA polymerase I promoter
(Pol I-P) and the mouse terminator (Pol I-T) sequences (e.g.,
pHH21 [5]) is used for plasmid-based HCV reverse genetics sys-
tems. The constructed plasmid allowed researchers to generate
wild-type and mutant viruses by transfection of plasmid without
RNA transcription. Viral genomic RNA is transcribed in the
nucleus of transfected cells, which is exported to the cytoplasm to
initiate RNA replication, following viral assembly and secretion
(Fig. 1).

2.3 Plasmid 1. E. coli DH5α competent cells.

Preparation 2. LB medium.
3. LB agar plates with ampicillin (100 μg/mL).
 RNA Polymerase I-Mediated HCV Reverse Genetics 177

pHH21 carrying full-length HCV cDNA

E1 p7 NS4A NS5A
Pol I P Pol I T
C E2 NS2 NS3 NS4B NS5B

Transfection Release

Assembly

RNA
Transcription replication

nucleus

Translation
ER

Fig. 1 Overview of the reverse genetics of hepatitis C virus based on the Pol I-
mediated transcription
Huh-7 or Huh7.5.1 cells are transfected with plasmid containing the full-length
HCV cDNA under control of RNA polymerase I promoter (Pol I-P). A genomic viral
RNA generated in the nucleus is transported to the cytoplasm, leading to
expression of the viral proteins in a cap-independent manner. The HCV genome
is synthesized by the replication complex, which consists of viral and host cell-
derived proteins, in tight association with structurally rearranged vesicle-like
cytoplasmic membranes. The genomic RNA synthesized and the viral structural
proteins are assembled into the viral particle. Matured infectious virus is
released from cells

4. Restriction endonucleases, T4 DNA ligase, high-fidelity DNA

polymerase (see Note 2), and reverse transcriptase. These
enzymes can be purchased from various commercial sources.
5. PCR primers (see Notes 3 and 4).
6. Molecular Biology Kits for RNA extraction and plasmid
extraction.
7. RNase-free water.

2.4 Cell Line and 1. The human hepatoma cell line, Huh-7, or its derivative cell
Reagents lines such as Huh7.5.1 [20].
2. Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with nonessential amino acids, 100 U of penicillin/ml, 100 mg
of streptomycin/ml, and 10% fetal bovine serum (FBS).
3. OPTI-MEM I reduced serum medium (Invitrogen).
4. Phosphate-buffered saline (PBS).
178 Ryosuke Suzuki and Tetsuro Suzuki

5. Trypsin-EDTA solution: 0.25% (w/v) trypsin, 0.02%

(w/v) EDTA.
6. DMEM with 100 U of penicillin/ml, 100 mg of streptomy-
cin/ml, 10% fetal bovine serum (FBS), and 0.8% carboxy-
methyl cellulose.
7. TransIT-LT1 transfection reagent (Mirus).
8. Acetone.
9. Methanol.
10. Antibodies specific for HCV protein.
11. Fluorescein-labeled secondary antibodies.
12. Laboratory equipment: fluorescent microscope, Centrifuge,
CO2 incubator.

3 Methods

3.1 Isolation of Viral Virus RNA is isolated from virus stock or virus-infected cells by
RNA standard procedures using silica-based (e.g., RNeasy Mini Kit,
Qiagen) or organic-based (e.g., TRIzol reagent, Invitrogen)
reagents following procedures recommended by the manufacturer.
Extracted RNA is eluted in RNase-free water.

3.2 PCR 1. The first-strand HCV cDNA is synthesized from extracted

Amplification of Full- RNA using the appropriate reverse transcriptase kit (e.g.,
Length HCV Genome SuperScript IV First-Strand Synthesis System, Invitrogen)
according to the manufacturer’s instructions. A random hex-
amer is used to prime first-strand cDNA synthesis.
2. Amplification of multiple PCR fragments covering the entire
HCV genome is carried out according to the manufacturer’s
instructions with the high-fidelity polymerase (e.g., Q5 High-
Fidelity DNA Polymerase, NEB) using the specific primer (see
Note 2).
3. PCR products with the expected size by agarose gel electro-
phoresis are purified using the gel extraction kit (e.g., Monarch
DNA Gel Extraction Kit, NEB) according to the manufac-
turer’s instruction.
4. The purified fragments are cloned into PCR cloning vector
(e.g., Zero Blunt TOPO PCR Cloning Kits, Thermo Fisher),
and sequenced to ensure that no unexpected mutations have
occurred.

3.3 Assembly of the 1. Digest the PCR products from cloning vector with
Full-Length cDNA corresponding restriction endonuclease.
Clone 2. Digest the vector containing the human Pol I-P and the mouse
Pol I-T with corresponding restriction endonuclease (e.g.,
BsmBI for pHH21 vector) (Fig. 2).
 RNA Polymerase I-Mediated HCV Reverse Genetics 179

Amp

pHH21

Pol I P Pol I T

HCV cDNA
Pol I P BsmBI Pol I T BsmBI
-GGGTTATT GGAGACGGTACCGTCTCCT CCCCCCCA- CGTCTCNTATTACCTGCCC----GATCATGTCCCNGAGACG
-CCCAATAA CCTCTGCCATGGCAGAGGA GGGGGGGT- GCAGAGNATAATGGACGGG----CTAGTACAGGGNCTCTGC

BsmBI BsmBI

BsmBI digestion BsmBI digestion

-GGGT T CCCCCCCA- TATTACCTGCCC----GATCATG

-CCCAATAA GGGGT- TGGACGGG----CTAGTACAGGG

HCV cDNA
Pol I P Pol I T
-GGGTTATT ACCTGCCC----GATCATGT CCCCCCCA-
-CCCAATAA TGGACGGG----CTAGTACA GGGGGGGT-

Fig. 2 Cloning of full-length HCV cDNA into the pHH21 plasmid

pHH21 contains the human RNA polymerase I promoter (Pol-I P) and the mouse RNA polymerase I terminator
(Pol I-T) sequences to direct the synthesis of the genomic HCV RNA. The fragments covering the entire viral
cDNA, two of which contain the UTR sequence and BsmBI restriction site, are amplified by RT-PCR. BsmBI-
digested PCR products are ligated into the BsmBI-digested pHH21 containing the same nucleotide overhangs.
(Modified from Neumann et al. [5])

3. Ligate the corresponding fragments using T4 DNA ligase

according to the manufacturer’s instruction.
4. Introduce the ligated DNA into the E. coli competent cells
(e.g., DH5α).
5. Plate the cells onto LB agar plate with 100 μg/mL ampicillin.
6. Incubate the plates overnight at 37 °C.
7. Select individual bacterial colonies for screening, and verify the
inserts by restriction enzyme and DNA sequencing.
8. The amplification and isolation of plasmid DNA containing
full-length cDNA clone are performed using standard proce-
dures. Amplified plasmid can be used directly for transfection.
Linearization by restriction enzyme is not required.
180 Ryosuke Suzuki and Tetsuro Suzuki

3.4 Generation of 1. Transfection of plasmid into Huh-7 or Huh7.5.1 cells:

Infectious HCV from (i) Prepare 80–90% confluent Huh-7 or Huh7.5.1 cell
Plasmid monolayers in 100 mm dishes a day before transfection,
and culture in a CO2 incubator at 37 °C.
(ii) Prepare transfection mixtures at RT:
(a) Add 500 μL Opti-MEM into a 1.5 mL
centrifuge tube.
(b) Add 5 μg of plasmid DNA into Opti-MEM, and
then tap the tube gently to mix.
(c) Add 20 μL TransIT-LT1 to the diluted DNA mix-
ture, and then tap the tube gently to mix.
(d) Incubate the Opti-MEM/Plasmid/TransIT-LT1
mixture at room temperature for 15 min.
(iii) Add the TransIT-LT1 Reagent/DNA complexes drop-
wise to different areas of the dish.
(iv) Gently rock the dish to distribute the TransIT-LT1
Reagent/DNA complexes.
(v) Incubate for 24 h at 37 °C.
(vi) Replace transfection medium with 8 mL of fresh DMEM
with nonessential amino acids, 100 U of penicillin/ml,
100 mg of streptomycin/ml, and 10% FBS.
(vii) Continue incubating the dishes at 37 °C for 2–5 days.
2. Preparation of virus stock:
(i) Collect virus-containing supernatant from transfected
cells from 3 to 6 days post-transfection. Add warmed
fresh media for subsequent collection of the supernatant.
(ii) Spin down the supernatant for 5 min at 4 °C to remove
cell debris.
(iii) Filter through 0.22 μm membrane.
(iv) Store the virus-containing supernatant sample at -80 °C
until further use.

3.5 Determination of 1. Prepare 80–90% confluent Huh-7 or Huh7.5.1 cell monolayers

the Infectious Titers of in 96-well plates a day before infection, and culture in a CO2
HCV incubator at 37 °C.
2. Remove media from each well of cell culture.
3. Inoculate 100 μL of serially diluted virus sample.
4. Incubate the plate at 37 °C for 6 h to allow virus infection.
5. Replace with DMEM containing 10% FBS and 0.8% carboxy-
methyl cellulose.
 RNA Polymerase I-Mediated HCV Reverse Genetics 181

6. Incubate the plate at 37 °C for 72 h. Incubation period

depends on the HCV strains.
7. The monolayers were fixed in cold acetone/methanol (1:1),
for 30 min at -20 °C, followed by removing acetone/metha-
nol and drying the plate.
8. Block with a nonfat milk solution (Block Ace; DS Pharma
Biomedical) for 30 min at room temperature. Then, wash cell
monolayers with PBS once.
9. Incubate with the HCV-specific antibodies (e.g., anti-NS5A
antibody) for 1 h at 37 °C.
10. Wash cell monolayers with PBS three times.
11. Incubate with fluorescein-labeled secondary antibodies (e.g.,
Alexa Fluor 488-conjugated anti-rabbit secondary antibody
(Invitrogen)) for 1 h at 37 °C.
12. Wash cell monolayers with PBS three times.
13. Stained foci were counted using fluorescence microscope, sub-
jecting to calculation of the titers of focus-forming units
(FFU)/ml.
14. Quantification of HCV RNA copy numbers (see Note 5) or
HCV core protein levels (see Note 6) are also useful for the
evaluation of virus samples.

4 Notes

1. DNA-based HCV subgenomic replicon is also generated by

deleting most of core-NS2 region from the full-length plasmid
[17]. Trans-complemented HCV, which is infectious but sup-
ports only single-round infection and is unable to spread, can
be generated by transient transfection of two plasmids: HCV
core-NS2 expression plasmid and Pol I-mediated subgenomic
replicon plasmid [17, 18].
2. It is important to use high-fidelity enzymes for RT-PCR ampli-
fication. This will reduce the inadvertent introduction of nucle-
otide changes during RT-PCR amplification.
3. The entire sequence of the HCV strain including the 5′- and
3′-ends of the viral genome should be obtained before starting
cloning.
4. To design the primers for PCR amplification, appropriate
restriction enzyme site and terminal overhangs for vector
should be considered. cDNAs covering 5′- or 3′-UTR were
amplified by PCR using primers containing BsmBI sites for
cloning in pHH21 vector (Fig. 2).
182 Ryosuke Suzuki and Tetsuro Suzuki

5. For the detection of HCV RNA in the supernatant of trans-

fected cells, DNase treatment is essential to minimize the con-
tamination of the plasmid DNA. Side-by-side experiment using
a control plasmid carrying the mutated HCV genome that
abolishes RNA-dependent RNA polymerase activity is
recommended.
6. For the detection of HCV in the supernatant of transfected
cells, quantification of HCV core protein by using an enzyme
immunoassay is also useful.

Acknowledgments

We thank Dr. Mami Matsuda for her constructive suggestions. This

work was supported by the Japan Agency for Medical Research and
Development (AMED), under Grant Numbers JP21fk0210053,
JP22fk0210086, JP22fk0210090, JP22fk0210066,
JP22fk0210110, and JP22fk0210109, and by MEXT KAKENHI
Grant Number JP20K08852.

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 Chapter 12

Reverse Genetics of Zika Virus Using a Bacterial Artificial

Chromosome
Aitor Nogales, Luis Martı́nez-Sobrido, and Fernando Almazán

Abstract
Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has become a global threat to
human health. Although ZIKV has been known to circulate for decades causing mild febrile illness, the
more recent ZIKV outbreaks in the Americas and the Caribbean have been associated with severe neuro-
logical disorders and congenital abnormalities. The development of ZIKV reverse genetics approaches have
allowed researchers to address key questions on the biology of ZIKV by genetically engineering infectious
recombinant (r)ZIKV. This has resulted in a better understanding of the biology of ZIKV infections,
including viral pathogenesis, molecular mechanisms of viral replication and transcription, or the interaction
of viral and host factors, among others aspects. In addition, reverse genetics systems have facilitated the
identification of anti-ZIKV compounds and the development of new prophylactic approaches to combat
ZIKV infections. Different reverse genetics strategies have been implemented for the recovery of rZIKV. All
these reverse genetics systems have faced and overcome multiple challenges, including the viral genome
size, the toxicity of viral sequences in bacteria, etc. In this chapter we describe the generation of a ZIKV full-
length complementary (c)DNA infectious clone based on the use of a bacterial artificial chromosome
(BAC) and the experimental procedures for the successful recovery of rZIKV. Importantly, the protocol
described in this chapter provides a powerful method for the generation of infectious clones of other
flaviviruses with genomes that have stability problems during bacterial propagation.

Key words Zika virus, Reverse genetics, Recombinant virus, Bacterial artificial chromosome, cDNA
clone, Infectious clone

1 Introduction

The recent coronavirus disease 2019 (COVID-19) pandemic

caused by severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) reminds us of how vulnerable the world’s
human population is to zoonotic viruses (https://COVID-19.
who.int/). Zika virus (ZIKV) is an enveloped positive-sense single-
stranded RNA arbovirus with an icosahedral-like structure that
belongs to the genus Flavivirus within the Flaviviridae family

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_12,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

185
186 Aitor Nogales et al.

A)
Envelope (E) protein

Membrane (M) protein

Capsid (C) protein

Genomic RNA

B)
0 2 4 6 8 10 11 kb

5’ UTR 3’ UTR
Cap C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5

Structural Proteins Non-structural Proteins

Fig. 1 ZIKV structure and genome organization. (a) Virion structure. The viral particle is constituted by a
nucleocapsid core composed by the C protein associated to the genomic RNA, surrounded by a lipid bilayer
that contains the structural proteins M and E arranged with icosahedral symmetry on the surface. (b) Genetic
structure of the ZIKV genome. The viral genome is a positive single-stranded RNA molecule that contains a cap
structure at the 5′-end and a single open reading frame (ORF) flanked by 5′- and 3′ untranslated regions
(UTR). The coding regions from the structural (C, prM/M, and E) and nonstructural (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) proteins are illustrated by colored boxes. A scale in kb is included at the top as
reference of the genomic position of the coding regions

(Fig. 1a) [1, 2]. ZIKV genome is approximately 10.8 kb and

encodes a large polyprotein of about 3423 amino acids that is
processed by viral and cellular proteases into 3 structural proteins
(capsid [C], pre-membrane/membrane [prM/M], and envelope
[E]) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) (Fig. 1b) [1]. The clinical presentation of
ZIKV infection is mostly asymptomatic or associated to mild febrile
illness with most of the cases not requiring health care [3–5]. How-
ever, ZIKV is also associated with neurological disorder such as
Guillain-Barré syndrome in adults and severe fetal abnormalities
and microcephaly in newborn infants [6–10].
ZIKV was first isolated from a rhesus macaque in the Zika forest
of Uganda in 1947 [11] and was declared by the Word Health
Organization (WHO) as a global public health emergency in
February 2016 [12, 13], after an epidemic in French Polynesia
 BAC-Based ZIKV Reverse Genetics 187

and an increase in the number of clinical cases in Brazil [4, 5, 14–

16]. Currently, ZIKV has been reported in more than 80 countries
with autochthonous transmission of the virus in the Americas, the
Caribbean, Asia, and Africa [14–16]. However, the number of
reported ZIKV cases has diminished significantly since the Latin
American peak of infections in 2016. This fact could indicate that
ZIKV cases are probably underreported or that surveillance efforts
have been reduced. Nevertheless, the development of ZIKV vac-
cines are still an emergency need because of the pregnancy compli-
cations associated with infections in ZIKV endemic areas where the
virus is circulating or the mosquito vector is present [17–19]. In
addition, there is also an unmet need to identify antivirals to treat
ZIKV infections [20, 21].
The development and implementation of ZIKV reverse genet-
ics techniques has been a powerful tool for the direct manipulation
of the viral genome to generate recombinant (r)ZIKV and to
answer important questions in the biology of ZIKV [22–28]. In
addition, reverse genetics approaches have allowed the develop-
ment of candidate vaccines [24, 29–31] and the identification of
antivirals [20, 21, 32, 33] to prevent or treat ZIKV infections,
respectively. Multiple ZIKV reverse genetics strategies have been
implemented [23, 25–29, 34–42], and all of them have different
advantages and disadvantages that need to be taken into consider-
ation before deciding which will be used to generate rZIKV
(revised in [23]).
In this chapter, we describe a detailed protocol for the engi-
neering of a full-length complementary (c)DNA infectious clone of
ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) [10, 29]
assembled in the low-copy-number bacterial artificial chromosome
(BAC) pBeloBAC11 [43]. The use of a BAC reduces the toxicity
associated to the viral genome, which makes its propagation diffi-
cult in bacteria using standard high-copy-number plasmids [44–
47]. The cDNA of ZIKV-RGN strain is assembled under the
control of the human cytomegalovirus (CMV) immediate-early
promoter, to allow the expression of the viral (v)RNA in the
nucleus of transfected cells by cellular RNA polymerase II, and
flanked at the 3′-end by the hepatitis delta virus (HDV) ribozyme
(Rz) followed by the sequences of the bovine growth hormone
(BGH) termination and polyadenylation signals (Fig. 2). This
approach allows the generation of RNAs bearing authentic 5′-
and 3′-ends of the viral genome. In addition, a detailed protocol
to rescue and titer the recovered rZIKV in mammalian Vero cells is
provided. Importantly, Vero cells are approved by the Food and
Drug Administration (FDA) for the development of human vac-
cines [48], which represent an important advantage for the use of
reverse genetics to generate vaccines.
188 Aitor Nogales et al.

Pml I Afe I BstB I

Asc I (3,347) (5,969) (9,127) Mlu I
ZIKV 1 ZIKV 3
CMV C prM E NS1 NS4A NS4B NS5
5’UTR ZIKV 2 ZIKV 4
NS2A NS2B NS3 NS5 Rz BGH
3’UTR

ApaL I 5’ UTR 3’ UTR BamH I

CMV C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 Rz BGH

GTTGA…ACCGT AGTTG GTCTT GGGTC…AGGGA

pBAC-ZIKV

Cmr
parC

Transcription start (18,308 bp) Ribozyme cleavage site

parB parA repE OriS

Fig. 2 Strategy to assemble a ZIKV-RGN BAC cDNA clone. After the selection of appropriated restriction sites in
the viral genome (genomic positions indicated in parenthesis), four overlapping fragments (ZIKV 1 to ZIKV 4)
covering the entire viral genome and flanked by the selected restriction sites were chemically synthesized and
sequentially cloned into the pBeloBAC11-Afe I plasmid to generate the pBAC-ZIKV infectious clone. Fragment
ZIKV 1 contains the human cytomegalovirus (CMV) promoter to allow the expression of the viral RNA into
transfected cells. Fragment ZIKV 4 contains the last nucleotides of the viral genome fused to the hepatitis delta
virus (HDV) ribozyme (Rz) and the bovine growth hormone (BGH) termination and polyadenylation sequences to
produce viral RNAs with authentic 3′-ends. Both, the fusion of CMV-5′ end and 3′ end-Rz are shown, where
the transcription start and the Rz cleavage site are indicated. Acronyms for coding region and regulatory
element are as described in Fig. 1. The BAC regulatory genes (parA, parB, parC, and repE), the F-factor
replication origin (OriS), and the chloramphenicol resistance gene (Cmr) are also indicated

2 Materials

2.1 Biosafety This chapter describes the assembly of ZIKV-RGN strain [10, 29]
Recommendation (accession number KU527068) in the pBeloBAC11 BAC (see Note
1). The rescue and manipulation of the recovered rZIKV should be
performed in a biosafety level 2 (BSL2), BSL2+, or BSL3 labora-
tory according to the country and institutional regulations and
required permits regarding ZIKV handling and storage. All mate-
rial used for the recovery of rZIKV must be sterilized before dis-
posal, following the appropriated institutional biosafety
indications.

2.2 Plasmids and 1. Plasmid pBeloBAC11: This is a single-copy plasmid derived

Bacterial Strains for from the Escherichia coli (E. coli) F-factor that encode the
the Generation of an genes parA, parB, and parC to ensure the accurate partitioning
Infectious ZIVK of plasmids to daughter cells. Moreover, pBeloBAC11 also
Infectious cDNA Clone contains the gene repE and the element oriS involved in initia-
tion and orientation of DNA replication, the chloramphenicol
 BAC-Based ZIKV Reverse Genetics 189

resistance gene (Cmr), the lacZ gene for color-based identifica-

tion of recombinants by α-complementation, and a multiclon-
ing site containing restriction sites for ApaL I, Sfo I, BamH I,
and Hind III. For the assembly of ZIKV-RGN full-length
infectious cDNA clone, we used the modified pBeloBAC11
plasmid pBeloBAC11-AfeI, which lacks the Afe I restriction
site [29] (see Note 2).
2. Electrocompetent DH10B cells: E. coli DH10B electrocompe-
tent cells could be acquired from different suppliers (Electro-
MAX DH10B Cells from Thermo Fisher is recommended) or
prepared as previously described [49]. E. coli DH10B cells [F-
mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1
endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL nupG]
were designed for efficient propagation of BACs containing
large DNA fragments (see Note 3).

2.3 Culture Media for 1. Luria broth (LB) liquid media: 1% (w/v) tryptone, 0.5% (w/v)
E. coli yeast extract, 1% (w/v) NaCl, pH 7.0. It can be purchased from
suppliers or prepared in the laboratory. To prepare LB liquid
media, mix 10 g of Bacto Tryptone, 5 g Bacto Yeast Extract,
and 10 g NaCl in 900 mL of ddH2O. Adjust the pH to 7.0 with
5 N NaOH, and adjust the volume of the solution to 1 L with
ddH2O. Sterilize by autoclaving on liquid cycle, and store at 4 °
C.
2. LB liquid media with chloramphenicol: Add chloramphenicol
to a final concentration of 12.5 μg/mL from a stock solution of
34 mg/mL. LB liquid media with chloramphenicol can be
stored at 4 °C for 1 month.
3. LB agar plates with chloramphenicol: Prepare 500 mL LB
liquid media, and, just before autoclaving, add 15 g/L of
Bacto Agar. After autoclaving on liquid cycle, equilibrate at
55 °C for 30 min, and add chloramphenicol to a final concen-
tration of 12.5 μg/mL. Dispense 20–25 mL of media to
90 mm petri dishes, and store LB agar plates at 4 °C.
4. SOC liquid media: 2% (w/v) tryptone, 0.5% (w/v) yeast
extract, 0.05% (w/v) NaCl, 2.5 mM KCl, 10 mM MgCl2,
10 mM MgSO4, 20 mM glucose, pH 7.0. SOC liquid media
can be purchased from suppliers or prepared in the laboratory.
To prepare SOC media, mix 20 g of Bacto Tryptone, 5 g Bacto
Yeast Extract, 0.5 g NaCl, and 2.5 mL of 1 M KCl (final
concentration 2.5 mM) in 900 mL of ddH2O. Adjust the pH
to 7.0 with 5 N NaOH, and adjust the volume of the solution
to 1 L with ddH2O. Autoclave and, before use, add 10 mL of
1 M MgCl2, 10 mL of 1 M MgSO4, and 20 mL of 1 M glucose.
Store SOC liquid media at 4 °C.
190 Aitor Nogales et al.

5. Chloramphenicol stock (34 mg/mL): Dissolve solid chloram-

phenicol in ethanol to a final concentration of 34 mg/mL, and
store the solution in a light-tight container at -20 °C.
6. LB freezing buffer: 40% (v/v) glycerol in LB medium. Sterilize
by passing it through a 0.45 μm disposable filter, and store at
4 °C.

2.4 Enzymes, The enzymes, reagents, and kits described in this section could be
Reagents, and Kits for purchased from other suppliers different to those recommended
the Assembly and here.
Manipulation of BAC 1. Restriction endonucleases (New England Biolabs, Inc.): Afe I,
Clones ApaL I, Asc I, BamH I, BstB I, Mlu I, and Pml I (see Note 4).
2. Shrimp Alkaline Phosphatase (SAP, Promega).
3. T4 DNA ligase (Promega).
4. Expand high-fidelity DNA polymerase (Roche).
5. PCR Nucleotide Mix (dNTP).
6. UltraPure distilled water (ddH2O).
7. SeaKem LE Agarose (Lonza) (see Note 5).
8. QIAEX II Gel Extraction Kit (Qiagen).
9. QIAGEN Plasmid Midi Kit (Qiagen).
10. QIAGEN Large-Construct Kit (Qiagen) (see Note 6).

2.5 Cell Culture Other suppliers different to those recommended here could also be
Media and Reagents used.
for Rescue and
1. Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco).
Propagation of rZIKV
2. Fetal bovine serum (FBS).
3. Growth medium: DMEM supplemented with 5% FBS,
2 mM L-glutamine (Sigma-Aldrich), 1% nonessential amino
acids (Sigma-Aldrich), and 100 mg/mL penicillin/streptomy-
cin (Sigma-Aldrich). Store at 4 °C.
4. Virus growth medium: DMEM supplemented with 2% FBS,
2 mM L-glutamine (Sigma-Aldrich), 1% nonessential amino
acids (Sigma-Aldrich), and 100 mg/mL penicillin/streptomy-
cin (Sigma-Aldrich). Store at 4 °C.
5. Trypsin-EDTA solution (Sigma-Aldrich): 0.25% (w/v) trypsin,
0.02% (w/v) EDTA.
6. Phosphate-buffered saline (PBS): PBS (138 mM NaCl, 3 mM
KCl, 8.1 mM of Na2HPO4, 1.5 mM KH2PO4, pH 7.4) can be
purchased from suppliers or prepared in the laboratory. To
prepare PBS, mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na2HPO4, and 0.24 g of KH2PO4 in 900 mL of
ddH2O. Adjust pH to 7.4, add ddH2O up to 1 L, sterilize by
autoclaving, and store at room temperature.
 BAC-Based ZIKV Reverse Genetics 191

7. Lipofectamine 2000 (LPF2000, Invitrogen).

8. Opti-MEM I Reduced Serum Medium (Invitrogen).

2.6 Cell Line for the Vero cells (African green monkey kidney epithelial cells) are avail-
Rescue of rZIKV able from the American Type Culture Collection, ATCC, CCL-81
(see Note 7). Vero cells have been shown to have a high transfection
efficiency, and they are the best cell line for producing high ZIKV
titers. Vero cells are growth and maintained in growth medium at
37 °C in a 5% CO2 atmosphere cell culture humidified incubator.

2.7 Reagents for the The reagent described in this section could be purchased from
Titration and Detection other suppliers different to those recommended here.
of ZIKV

2.7.1 Plaque Assay and 1. Overlay solution: Virus growth medium containing 1% DEAE-
Immunostaining dextran (Sigma-Aldrich) and 0.6% agar noble (Difco, Thermo
Fisher) (see Note 8).
2. 37% formaldehyde (Sigma-Aldrich).
3. Crystal violet solution: 0.5% crystal violet (Sigma-Aldrich),
20% methanol (Merck) (see Note 9).
4. Immunostaining blocking solution: 10% FBS in PBS.
5. Primary monoclonal antibody (mAb) pan-flavivirus E protein
4G2 (BEI Resources NR-50327) (see Note 10).
6. Biotinylated anti-mouse secondary antibody (Vector Labora-
tories, Inc.).
7. Vectastain ABC Kit (Vector Laboratories, Inc.).
8. DAB Substrate Kit, Peroxidase (HRP), with Nickel (Vector
Laboratory, Inc.).

2.7.2 Immuno- 1. Fixing solution: 4% paraformaldehyde (PFA) in PBS.

fluorescence 2. Permeabilization solution: 0.5% Triton X-100 (Sigma-Aldrich)
in PBS.
3. Blocking solution: 2.5% bovine serum albumin (BSA, Sigma-
Aldrich) in PBS.
4. Primary mAb pan-flavivirus E protein 4G2 (BEI Resources
NR-50327) (see Note 10).
5. Secondary anti-mouse IgG-FITC conjugate antibody (Dako).
6. DAPI (4′,6-diamidino-2-phenylindole).
7. ProLong Gold Antifade Reagent (Thermo Fisher).

2.8 Laboratory 1. Desktop refrigerated microcentrifuge with a rotor capable of

Equipment reaching up to 12,000 × g and microcentrifuge tubes.
2. High-speed refrigerated centrifuge.
192 Aitor Nogales et al.

3. Water bath or heat block.

4. Electrophoresis system and a power supply.
5. NanoDrop (or similar spectrophotometer).
6. MicroPulser electroporator and cuvettes fitted with electrodes
spaced 0.2 cm.
7. Microbiological incubator.
8. Thermocycler and PCR tubes.
9. Laboratory 5% CO2 cell culture humidified incubators at 37 °C
for maintenance of cells.
10. Class II biosafety cabinet.
11. Light microscope.
12. Fluorescent microscope.
13. Microwave oven.
14. Cell culture plasticware.
15. -80 °C freezer.
16. Autoclave.

3 Methods

3.1 Assembly of a The strategy for the assembly and generation of the ZIKV-RGN
ZIKV Infectious cDNA strain infectious clone using the BAC has been previously described
Clone in the BAC [10, 29] (Fig. 2).
1. The first step for the assembly of the full-length cDNA clone is
the choice of appropriate restriction endonuclease sites. For
that, select unique restriction sites in the viral genome that
are absent in the pBeloBAC11-AfeI (Fig. 2). In the case of
ZIKV-RGN, the unique restriction sites Pml I, Afe I, and
BstB I (genomic positions 3347, 5969, and 9127, respectively)
were selected to generate the infectious clone (Fig. 2) (see
Note 11).
2. Design and chemically synthesize four overlapping cDNA frag-
ments spanning the full-length viral genome (ZIKV 1 to ZIKV
4 fragments, Fig. 2) flanked by the appropriated regulatory
elements and restriction sites (see Note 12). The full-length
cDNA must be flanked by the CMV promoter at the 5′-end
(ZIKV 1) and by the HDV Rz and the BGH termination and
polyadenylation sequences at the 3′-end (ZIKV 4) (Fig. 2).
Fragment ZIKV 1 contains the CMV immediate-early pro-
moter precisely fused to the first 3350 nucleotides of the viral
genome (until restriction site Pml I) flanked at the 5′-end by
ApaL I (to clone into the pBeloBAC11-AfeI) and Asc I (absent
in the viral genome and in the vector) and at the 3′-end by the
 BAC-Based ZIKV Reverse Genetics 193

restriction sites selected for the assembly of the infectious clone

(Pml I, Afe I, and BstB I), followed by Mlu I (absent in the viral
genome and the vector) and BamH I (to clone into the pBe-
loBAC11-AfeI). Fragments ZIKV 2 and 3 cover the genomic
area from restriction sites Pml I to Afe I and Afe I to BstB I,
respectively (Fig. 2). Fragment ZIKV 4 contains the genomic
area from the restriction site BstB I to the end of the viral
genome, followed by the HDV Rz, the BGH termination and
polyadenylation sequences, and the restriction sites Mlu I
(absent in the viral genome and the vector) and BamH I
(Fig. 2). The full-length cDNA clone (pBAC-ZIKV) is assem-
bled by sequential cloning of the four overlapping cDNA frag-
ments into the pBeloBAC11-AfeI (Fig. 2).
3. Digest 2 μg of pBeloBAC11-AfeI plasmid and 1 μg of ZIKV
1 fragment with 20 units of ApaL I and BamH I for 2 h at 37 °
C (see Note 13).
4. Dephosphorylate the BAC vector by adding 2.5 μL (2.5 units)
of SAP to the digested BAC, and incubate at 37 °C for 30 min.
Heat-inactivate the SAP by incubation at 75 °C during 15 min.
5. Purify the digested ZIKV 1 fragment and the vector by agarose
gel electrophoresis using QIAEX II Gel Extraction Kit, which is
specially designed to purify DNA fragments from 40 bp to
50 kb (see Note 14).
6. Determine the concentration of the BAC vector and the insert
in the NanoDrop.
7. Perform the ligation reaction to obtain the plasmid pBAC-
ZIKV1. For that, mix 150 ng of purified BAC vector, an
amount of the purified insert (fragment ZIKV 1) equivalent
to a molar ratio of vector-to-insert of 1:3, 1.5 μL of 10x T4
DNA ligase buffer, 1 μL (3 units) of T4 DNA ligase, and
ddH2O to a final volume of 15 μL (see Note 15). As control
of the ligation reaction, set up in parallel a reaction without
insert. Incubate the ligation reaction for 16–20 h at 16° and
heat-inactivate the ligase by incubation at 65 °C for 15 min (see
Note 16).
8. Transform 50 μL of electrocompetent DH10B cells with 2 μL
of the ligation reaction by electroporation, using a MicroPulser
electroporator (25 μF capacitance, 2.5 kV, and 100 Ω resis-
tance) and cuvettes fitted with electrodes spaced 0.2 cm, fol-
lowing the manufacturer’s instructions (see Note 17).
9. Immediately after the electrical pulse, remove the cuvette, and
transfer the cells to a polypropylene tube with 1 mL of
pre-warmed SOC medium. Incubate the cells at 37 °C for 1 h
with gentle shaking (200–250 rpm).
194 Aitor Nogales et al.

10. Plate the cells onto LB agar plates containing 12.5 μg/mL
chloramphenicol, and incubate them at 37 °C for 16–24 h.
11. Pick about ten bacterial colonies, make a replica on a fresh LB
agar plate containing 12.5 μg/mL chloramphenicol, and test
whether they contain the correct insert by direct PCR analysis
using specific oligonucleotides and expand high-fidelity DNA
polymerase, following the manufacturer’s instruction (see
Note 18).
12. Pick a positive colony from the replica plate, and grow it in
100 mL of LB containing 12.5 μg/mL of chloramphenicol at
37 °C for 16 h with shaking (200–250 rpm). Isolate the BAC
plasmid using the QIAGEN Plasmid Midi Kit, following the
protocol for purification of large low-copy plasmids (see
Note 19).
13. Verify the genetic integrity of the isolated plasmid by restriction
analysis and sequencing using specific oligonucleotides.
14. After confirming the genetic identity of the plasmid pBAC-
ZIKV1, we recommend to prepare a glycerol stock of positive
bacteria. For that, mix 0.5 mL of LB freezing buffer with
0.5 mL of an overnight bacterial culture in a cryotube, freeze
in ethanol–dry ice, and store at -80 °C.
15. Starting from the generated plasmid pBAC-ZIKV1, sequen-
tially clone fragments ZIKV 2 to ZIKV 4 using the selected
restriction sites (Pml I /Afe I for ZIKV2, Afe I /BstB I for
ZIKV 3, and BstB I/Mlu I for ZIKV 4) to generate the full-
length infectious pBAC-ZIKV cDNA clone (Fig. 2), following
similar experimental approaches as described above for ZIKV
1 fragment.

3.2 Preparation of For the rescue of rZIKV using the infectious BAC cDNA clone, it is
Ultrapure pBAC-ZIKV recommended to prepare high-purity pBAC-ZIKV with no bacteria
Infectious Clone DNA contamination. Any commercial kit designed for BAC purifi-
cation is suitable, but we recommend using the QIAGEN Large-
Construct Kit. This kit includes an ATP-dependent exonuclease
digestion step that removes bacteria DNA contamination, allowing
the isolation of the BAC plasmid with a grade of purity similar to
that using cesium chloride method.
1. Inoculate a single colony of bacteria carrying the pBAC-ZIKV
infectious clone from a freshly streaked plate (LB agar plate
containing 12.5 μg/ml chloramphenicol) in 5 mL of LB con-
taining 12.5 μg/mL of chloramphenicol, and incubate at 37 °C
for 8 h with vigorous shaking (200–250 rpm).
2. Add 1 mL of the bacterial culture to 500 mL of LB with
12.5 μg/mL of chloramphenicol, and grow the bacteria at
37 °C with vigorous shaking (200–250 rpm) for 14–16 h
until an OD600 of 0.8–1 (late logarithmic growth phase) (see
Note 20).
 BAC-Based ZIKV Reverse Genetics 195

3. Harvest the bacterial cells by centrifugation at 6000 × g for

15 min at 4 °C, and purify the pBAC-ZIKV infectious cDNA
clone with the QIAGEN Large-Construct Kit, following the
specifications recommended by the manufacturer (see
Note 21).
4. Quantify the concentration of the purified BAC cDNA, adjust
the concentration to 0.2–0.4 μg/μL, and store the plasmid at
4 °C until its use (see Note 22). Following this approach, yields
of 25–30 μg of ultrapure BAC cDNA clone can be obtained.

3.3 Rescue of rZIKV Overall, rescue of rZIKV is carried out using experimental methods
similar to those previously described [23, 24, 29, 30, 50]. To
increase the likelihood of successfully recovering rZIKV, we recom-
mend three independent transfections. Infectious rZIKV is recov-
ered by the transfection of Vero cells with the generated pBAC-
ZIKV cDNA clone, using LPF2000 as transfection reagent. A
schematic representation of the protocol to generate rZIKV is
shown in Fig. 3. The following protocol is established for six-well
plates.
1. The day before transfection, plate 6 × 105 Vero cells/well on
six-well plates using growth medium without antibiotics to
raise 90% confluent cell monolayers on the day of transfection
(see Note 23). On the day of transfection, check the cells under
a light microscope to confirm the presence of a uniform cell
monolayer.
2. Just before transfection, equilibrate the Opti-MEM I medium
at room temperature, put the DNA and the LPF2000 reagent
on ice, and prepare sterile microfuge tubes.
3. Prepare Opti-MEM I-LPF2000 and plasmid transfection
mixtures.
4. Opti-MEM I-LPF2000 mixture: For each transfection sample,
mix 250 μL of Opti-MEM I medium and 12 μL of LPF2000
(gently vortex before use), and incubate for 5 min at room
temperature. Meanwhile, prepare the plasmid transfection
mixture.
5. Plasmid transfection mixture: Dilute 4 μg of pBAC-ZIKV plas-
mid in 250 μL of Opti-MEM I medium. Avoid prolonged
pipetting to prevent plasmid shearing.
6. Combine the Opti-MEM I-LPF2000 (step 4) with diluted
DNA (step 5), mix carefully, and incubate for 20–30 min at
room temperature.
7. During this incubation period, wash the Vero cell monolayers
twice with 4 mL of growth medium without antibiotics, and
leave the cells in 1 mL of the same medium (see Note 23).
196 Aitor Nogales et al.

CMV C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5 Rz BGH

pBAC-ZIKV

Cmr
parC

(18,308 bp)

parB parA repE OriS

TRANSFECTION

Vero cells

4-6 DAYS

rZIKV

VIRUS AMPLIFICATION

Fig. 3 Rescue of rZIKV from the pBAC cDNA clone. Vero cell monolayers at 90% confluence (six-well plate
format, triplicates) are transfected with 4 μg of the pBAC-ZIKV cDNA clone (Fig. 2) using LPF2000. When CPE
is evident (4–6 days post-transfection), cell cultured supernatants are collected and titrated in Vero cells, and
the presence of the rZIKV is confirmed using different experimental approaches. Finally, the rescued rZIKV is
amplified and titrated in Vero cells (Fig. 4) and stored at -80 °C

8. After the 20–30 min incubation, distribute the DNA/transfec-

tion reagent mixture (step 5) drop by drop onto the Vero cells,
mix them by rocking the plate gently, and incubate the cells in a
5% CO2 humidified incubator at 37 °C for 6–8 h.
9. Remove the transfection medium, add 2 mL of fresh growth
medium with antibiotics, incubate the cells in a 5% CO2 humi-
dified incubator at 37 °C, and check every day for the presence
of cytopathic effect (CPE) (see Note 24). As a reference for
healthy cells, mock-transfected control cells may be observed in
parallel.
 BAC-Based ZIKV Reverse Genetics 197

A) B) C)
MOCK CRYSTAL VIOLET MOCK

rZIKV IMMUNOSTAINING rZIKV

Fig. 4 In vitro characterization of the rescued rZIKV. (a) CPE. Confluent (~90%) monolayers of Vero cells
(six-well plate format, triplicates) were mock-transfected or transfected with pBAC-ZIKV, and CPE was
analyzed daily. Pictures show the presence of CPE at 4 days after transfection with pBAC-ZIKV (bottom) but
not in mock-transfected cells (top). Scale bars, 50 μm. (b) Plaque assays. Vero cells at 90% confluence
(six-well plate format, triplicates) were infected with tenfold serial dilutions of rZIKV, and at 3–4 days post-
infection, viral plaques were visualized by crystal violet staining (top) or by immunostaining using the
pan-flavivirus E protein mAb 4G2 (bottom). Representative images containing ~25–50 PFU are shown. (c)
Immunofluorescence assay (IFA). Vero cells at 90% confluence (24-well plate format, triplicates) were mock-
infected (top) or infected (bottom) with 0.5 PFU/cell of rescued rZIKV. At 18 h post-infection, the expression of
the viral E protein was assessed by fluorescent microscopy using the pan-flavivirus E protein mAb 4G2. Cell
nuclei were stained with DAPI. Scale bars and 20 μm

10. After four to 6 days of transfection, when CPE is around

50–75% (Fig. 4a), collect the cell culture supernatants contain-
ing rZIKV in 15 mL conical tubes, and centrifuge at 2000 × g
for 10 min at 4 °C to remove cells’ debris.
11. Aliquot the supernatant in cryotubes, and store at -80 °C. The
cell culture supernatant is used to confirm the presence of
rescued rZIKV and for the generation of virus stocks.

3.4 Titration of Plaque assay is a direct quantitative measurement of the number of

Recovered rZIKV by infectious viruses present in cell culture supernatants from trans-
Plaque Assay fected cells. A semisolid overlay is placed over a monolayer of Vero
cells to prevent the virus from infecting cells other than the neigh-
boring cells; therefore, each arising plaque will have originated
from a single virus. The presence of virus plaques could be visua-
lized and quantified by crystal violet staining (Fig. 4b, top) or by
immunostaining using the pan-flavivirus E protein mAb 4G2
(Fig. 4b, bottom).
198 Aitor Nogales et al.

3.4.1 Plaque Assay and 1. The day before infection, seed six-well plates with approxi-
Crystal Violet Staining mately 6 × 105 Vero cells/well in growth medium to raise
90% confluent cell monolayers by the time of titration. We
recommend conducting the titration of the recovered rZIKV
in triplicates.
2. Next day, make tenfold serial dilutions (10-1–10-6) of the
virus sample in virus growth medium without FBS. For that,
add 900 μL of virus growth medium without FBS in six micro-
tubes. Then, add 100 μL of virus sample in the first microtube
(dilution 10-1), and vortex the tube for 5 s. Pipet 100 μL of the
10-1 dilution, and transfer into the second tube (dilution 10-
2
). Repeat these steps for the other dilutions. Change the tip to
avoid any cross-contamination between samples.
3. Wash the cells twice with PBS, and starting with the most
diluted sample, add 800 μL of the virus dilution to each of
the wells. Place plates in a 5% CO2 humidified incubator at 37 °
C for a 90 min adsorption period, rocking the plates every
15 min to prevent the cells from drying.
4. Remove the viral inoculum, and overlay the cells with 3 mL of
virus growth medium containing 1% DEAE-dextran and 0.6%
agar noble. Let the plate(s) sit for 10 min at room temperature,
as the agar overlay solidifies, and incubate the plates at 37 °C in
the 5% CO2 incubator for 3–4 days (see Note 8).
5. When viral plaques are visible, fix the plates with a 2 mL of 4%
formaldehyde in PBS for 1 h at room temperature.
6. Carefully remove the overlay media.
7. Add 1 mL of 0.5% crystal violet solution to each well, and
incubate for 30 min at room temperature.
8. Discard the crystal violet solution, wash the cells gently with
ddH2O, and allow the plates to air-dry.
9. Count the plaques in the lowest two dilutions with visual
plaques, and calculate the viral titer in plaque-forming units
(PFU)/mL using the following formula: PFU/mL = number
of plaques x virus dilution x 1/volume of inoculum (mL).

3.4.2 Plaque Assay and To perform the immunostaining, we recommend the use of a
Immunostaining commercial kit as Vectastain ABC Kit (Vector Laboratories, Inc.),
following the manufacture’s indications. The following protocol is
designed for a six-well plate format.
1. Perform cell infections and plaque assay as indicated above in
Subheading 3.4.1 (steps 1–6).
2. After fixing and removal of the overlay agar, wash the cells twice
with PBS.
3. Permeabilize the cells by incubation with 1 mL/well of 0.5%
Triton X-100 in PBS for 15 min at room temperature.
 BAC-Based ZIKV Reverse Genetics 199

4. Wash the cells three times with PBS, and incubate them with
1 mL/well of blocking solution (10% FBS in PBS) for 1 h at
room temperature.
5. Remove the blocking solution, and incubate the cells with
1 mL/well of the pan-flavivirus E protein mAb 4G2 diluted
in blocking solution (1 μg/mL) for 1 h at 37 °C.
6. Wash the cells three times with PBS, add 1 mL of biotinylated
anti-mouse secondary antibody diluted (following the manu-
facturer’s recommendation) in blocking solution, and incubate
for 1 h at 37 °C.
7. Wash the cells three times with PBS to remove biotinylated Ab
solution thoroughly.
8. Prepare VECTASTAIN ABC Reagent by following manufac-
turer’s instructions. Add 50 μL of Reagent A (Avidin, ABC)
and 50 μL of Reagent B (Biotinylated HRP, ABC) to 5 mL of
PBS. Add 1 mL/well of VECTASTAIN ABC Reagent, and
incubate for 30 min at 37 °C.
9. Wash cells three times with PBS. Remove PBS, and dry the
plate.
10. Prepare developing solution by following manufacturer’s
instructions (DAB Substrate Kit with Nickel).
11. Add 1 mL of developing solution to each well and wait for
3–5 min to visualize viral plaques.
12. Stop the reaction by removing the developing solution, and
wash with PBS (see Note 25).
13. Take images of the different plates.

3.5 Confirmation of To confirm the identity of the rescued virus, Vero cells are infected
rZIKV Rescue by IFA with rZIKV, and the expression of ZIKV E protein is analyzed by
IFA using the pan-flavivirus E protein mAb 4G2 (Fig. 4c). Alterna-
tively, the virus identity can be confirmed directly by sequencing.
Using the IFA assay, the virus titers in the cell culture supernatants
can be also determined by immunofocus assay (fluorescent focus-
forming units (FFU)/ml) in 96-well plate format.
1. The day before performing the IFA, put sterile coverslips in
24-well plates, and seed 1 × 105 Vero cells/well in growth
medium to raise 90% confluent cell monolayers by the time of
infection. Place the plates at 37 °C in a 5% CO2 humidified
incubator.
2. The next day, wash the cells twice with PBS, and infect them in
triplicate with a multiplicity of infection (MOI) of 0.5 PFU/-
cell of the rescue rZIKV in virus growth medium without FBS
(final volume 250 μL/well). Incubate the plates at 37 °C for
90 min.
200 Aitor Nogales et al.

3. After viral adsorption, remove the viral inoculum, add 0.5 mL

of fresh virus growth medium, and incubate the cells in a 5%
CO2 humidified incubator at 37 °C for 18 h.
4. Remove the cell culture supernatants, and fix the cells with
150 μL/well of 4% paraformaldehyde in PBS for 20 min at
room temperature.
5. Wash the cells with PBS, and permeabilize them with 150 μL/
well of 0.5% Triton X-100 in PBS (permeabilization solution)
for 15 min at room temperature.
6. After permeabilization, wash the cells three times with PBS,
and incubate the cells with150 μL/well of blocking solution
(2.5% BSA in PBS) for 1 h at room temperature (see Note 26).
7. Remove the blocking solution, and incubate the cells with
150 μL/well of pan-flavivirus E protein mAb 4G2 diluted in
blocking solution (1 μg/mL) for 1 h at 37 °C.
8. Wash the cells three times with PBS, and incubate the cells with
150 μL/well of Alexa Fluor 488-conjugated anti-mouse sec-
ondary antibody diluted in blocking solution, as recommended
by the manufacturers, for 1 h at room temperature.
9. Wash the cells three times with PBS, and incubate them with
150 μL/well of DAPI (diluted 1:200 in PBS) for 15 min at
room temperature.
10. Remove the DAPI solution, wash the cells three times with
PBS, and mount the coverslips on ProLong Gold Antifade
Reagent (other antifade mounting medium could be used).
11. Analyze the samples under a fluorescence microscope (see Note
27).

3.6 Amplification of 1. The day before infection, seed 6 × 106 Vero cells in 100 mm
ZIKV and Production of plates in growth medium to raise 90% confluent cell mono-
Virus Stocks layers by the time of infection.
2. The next day, wash the cells twice with PBS, and infect them
with an MOI of 0.1 PFU/cell as described before (see Note
28).
3. When the CPE is around 75% (approximately 2–3 days post-
infection), collect the cell culture supernatant into a 15 ml
falcon tube, and centrifuge at 2000 × g for 10 min at 4 °C to
remove cells debris.
4. Collect the supernatant containing rZIKV into a new 15 mL
falcon tube, and discard the cell pellet.
5. Aliquot the supernatant in cryotubes, and store them at -80 °
C (see Note 29).
6. Determine the viral titer by plaque assay as described before
(Subheading 3.4).
 BAC-Based ZIKV Reverse Genetics 201

4 Notes

1. Although in this chapter we describe the reverse genetics

approach for ZIKV-RGN strain, this protocol can be used for
other ZIKV strains after the selection of appropriate restriction
sites.
2. Plasmid pBeloBAC11-AfeI was generated to use the restriction
site Afe I (genomic position 5970) in the virus genome assem-
bly process.
3. E. coli DH10B strain is a recombination-defective strain used
for the propagation of BACs to avoid unwanted
rearrangements.
4. These restriction enzymes can be different for the development
of reverse genetics approaches of different ZIKV strains.
5. Other agaroses can also be used for DNA gel electrophoresis.
6. BAC preparation is one of the key factors for efficient rZIKV
rescue. We recommend having ultrapure plasmid preparations
with reduced bacterial DNA contaminations for successful viral
rescue attempts.
7. It is important to keep track of the cell passage number, since it
can affect virus rescue efficiency and/or virus propagation. The
use of late-passage cells (above passage 30–35) is not
recommended.
8. It is possible to use other overlay media such as Avicel or
methylcellulose [32, 33, 51]. Importantly, the time for proper
plaque formation will vary depending on the overlay used and
the ZIKV strain.
9. Crystal violet is a powerful staining solution. Handle it
carefully.
10. Other primary antibodies against ZIKV, such us the mAb
against ZIKV E protein 1176–56 (BioFront Technologies),
can be used, and the optimal antibody concentration for the
assay should be determined.
11. In the case that no appropriate restriction sites are available in
the viral genome, generate new restriction sites appropriately
spaced in the viral genome by introducing silent nucleotide
mutations during the design of the ZIKV cDNA fragments.
Likewise, natural restriction sites could be eliminated to facili-
tate the assembly of the infectious clone. Importantly, these
modifications can be used as genetic markers to demonstrate
the recombinant nature of the recovered virus.
12. Otherwise, the fragments (ZIKV 1 to ZIKV 4) could be pro-
duced by reverse transcriptase and polymerase chain reaction
(RT-PCR) using specific oligonucleotides.
202 Aitor Nogales et al.

13. When two enzymes requiring different buffers are used to

digest the DNA, carry out the digestion sequentially with
both enzymes. Clean the DNA after the first digestion by
using the Qiagen QIAEX II Gel Extraction Kit following the
protocol for purifying DNA fragments from aqueous solutions.
14. Other commercial kits suitable for the purification of DNA
fragments larger than 10 kb can be also used.
15. Due to the low transfection efficiency of large-sized BACs, it is
essential to use a larger amount of vector, insert, and T4 DNA
ligase than when using conventional plasmids.
16. In the case of blunt-ended DNAs, use 225 ng of vector, the
corresponding amount of insert, and 6 units of T4 DNA ligase,
and incubate the ligation reaction for 20 h at 14 °C.
17. Most electroporation machines contain specific programs for
different cell types. In this case, choose the program recom-
mended to electroporated bacteria cells.
18. A mix of small and large colonies can suggest toxicity of the
cloned DNA fragment in the bacteria. If this is the case, select
small colonies, which may contain the correct DNA insert, and
grow the bacteria at 30 °C to minimize potential toxicity. In
addition, if one DNA fragment is toxic for the bacteria, we
recommend inserting this toxic DNA fragment into the BAC
in the last cloning step.
19. Other commercial kits designed for isolation of BACs can also
be used. Although the BAC DNA prepared by this method is
contaminated with up to 20% of bacterial genomic DNA, it is
suitable for further analysis, including restriction digestion,
sequencing, and cloning.
20. To avoid DNA degradation and unwanted rearrangements, do
not grow the culture up to the late stationary growth phase.
21. To facilitate the resuspension of the BAC DNA, it is recom-
mended to use a swinging bucket rotor for the last isopropanol
precipitation step. After washing with 70% ethanol, air-dry the
pellet for only 5 min (never use vacuum to dry the DNA pellet,
as overdrying the pellet will make the dissolution of the BAC
DNA difficult).
22. Due to the large size of the BAC cDNA clone, avoid vortexing
or pipetting to prevent plasmid shearing.
23. The addition of antibiotics during the transfection process may
decrease transfection efficiency and cause cell toxicity.
24. ZIKV CPE in Vero cells is characterized by the presence of
rounded and birefringent cells that detach and float in the cell
culture supernatant.
 BAC-Based ZIKV Reverse Genetics 203

25. It is important not to wait for too long once the plaques are
visible to prevent the entire cells from turning black.
26. Cells can be blocked using alternative blocking solutions (e.g.,
10% FBS in PBS) or overnight at 4 °C.
27. For long-term storage, store samples at 4 °C, protected from
light.
28. Adjust the volumes according with the plaque size.
29. We recommend making small-volume aliquots to prevent mul-
tiple freeze-thaw cycles, which may reduce virus titers.

Acknowledgments

We thank the Biodefense and Emerging Infectious Research

Resources Repository (BEI Resources) for providing the
pan-flavivirus E protein mAb 4G2 (NR-50327). This research was
partially funded by the Spanish Ministry of Economy and Compet-
itiveness (MINECO) (grant number BFU2016-79127-R) to F.A.;
by a “Ramon y Cajal” incorporation grant (RYC-2017) from the
Spanish Ministry of Science, Innovation and Universities to A.N.;
and by the R21AI130500 grant from the National Institute of
Allergy and Infectious Diseases (NIAID) at the National Institutes
of Health (NIH) to L.M-S. and F.A.

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 Chapter 13

A Stable Reverse Genetics System of Zika Virus Based

on a Self-Splicing Group II Intron
Zhong-Yu Liu, Xiao-Feng Li, and Cheng-Feng Qin

Abstract
Zika virus (ZIKV) is a mosquito-borne flavivirus of the Flaviviridae family first isolated from a sentinel
monkey in the Zika Forest, Uganda, in 1947. Since 2007, the virus has had a vast geographic expansion that
extended to the Americas in 2015, leading to a series of large outbreaks. Although mainly transmitted by
the bite of Aedes mosquitoes, human infection of ZIKV can also happen through unconventional routes
such as sexual intercourse and, more importantly, vertical transmission. The genome of ZIKV is a single-
stranded, positive-sense RNA molecule about 11 kb in length. The genome contains a single opening
reading frame (ORF) flanked by highly structured 5′ and 3′ untranslated regions. To understand the
mechanisms about ZIKV replication, transmission, and pathogenesis, reverse genetic tools are of great
importance. In this chapter, a novel system is described for the generation and manipulation of a ZIKV
infectious clone stabilized by a self-splicing group II intron, a mobile element with ribozyme activity. The
intron can be spliced in vitro, and thus full-length vRNA can be prepared allowing virus genome manipula-
tion required for further studies.

Key words Zika virus, Reverse genetics, Recombinant virus, Ribozyme, Self-splicing, Intron, Infec-
tious clone

1 Introduction

Zika virus (ZIKV) is a mosquito-borne flavivirus of the Flaviviridae

family. Since its isolation from a sentinel monkey in the Zika Forest,
Uganda, in 1947 [1], only a few human infections of ZIKV were
reported in Africa and Asia for several decades [2–5], and the
clinical outcomes were in general mild and self-limiting. However,
since 2007, the virus expanded rapidly in the pacific oceans and
reached the Americas in 2015, resulting in a series of large out-
breaks [6, 7]. Although mainly transmitted by the bite of Aedes
mosquitoes, human infection of ZIKV can also happen through
unconventional routes such as sexual intercourse [8]. Importantly,
vertical transmission of ZIKV was confirmed during the
2015–2016 outbreak in the Americas [9]. Accordingly, the clinical

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_13,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

207
208 Zhong-Yu Liu et al.

outcomes of ZIKV infections were found to be more complicated

with severe manifestations, such as Guillain-Barre syndrome in
infected adults [10, 11], spontaneous miscarriage in the pregnant
women, and congenital Zika syndrome in the newborns [12]. To
understand the mechanisms about ZIKV replication, transmission,
and pathogenesis, reverse genetic tools are of great importance.
The genome of ZIKV is a single-stranded, positive-sense RNA
molecule about 11 kb in length. The genome contains a single
opening reading frame (ORF) flanked by highly structured 5′ and
3′ untranslated regions. The viral ORF directs the translation of a
polyprotein precursor containing about 3400 residues, which is
processed into 3 structural (capsid C, premembrane/membrane
prM/M, and envelope E proteins) and 7 nonstructural proteins
(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The viral genome
is 5′-capped but lacks a 3′ poly(A) tail. The viral genome is started
by 5′-AG and ended by 3′-CU, which is a conserved feature in the
Flavivirus genus. A series of cis-acting RNA elements locate in the
5′- and 3′-UTRs, as well as in the C-coding region [13, 14]. The
integrity of these elements is crucial for viral RNA (vRNA) replica-
tion, translation, and/or nucleocapsid assembly.
Since 2016, a series of ZIKV reverse genetic tools have been
developed and widely used to clarify the function of evolutionary-
acquired substitutions in ZIKV proteins in virus transmission and
virulence [15–21]. Yet, the instability of full-length virus cDNA in
commonly used E. coli cloning strains hindered the generation and
application of ZIKV infectious clones. To overcome this drawback,
several alternative strategies to construct the reverse genetic systems
for ZIKV have been developed, such as the bacterium-free, in vitro
assembly of full-length viral cDNA [22] and the generation of
artificial intron-contained, “infectious DNA” clones [18, 19].
In this section, we will describe the generation and manipula-
tion of a ZIKV infectious clone stabilized by a self-splicing group II
intron, a mobile element with ribozyme activity [23]. The intron
can be spliced in vitro, and thus full-length vRNA can be prepared
as the classical infectious clones, which ensures the compatibility
with the requirements of different investigations.

2 Materials and Methods

2.1 Isolation of ZIKV 1. ZIKV strain GZ01 (GenBank: KU820898) was isolated from
RNA from Culture the serum samples of an imported case of Zika infection
Supernatants [24]. Baby hamster kidney cell line BHK-21 (ATCC
CCL-10) is cultured using DMEM supplemented with 5%
FBS, 25 mM HEPES, 100 U/mL penicillin, and 100 μg/mL
streptomycin.
2. DMEM medium, low glucose (Thermo Fisher Scientific).
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 209

3. Cell culture flasks, 25 cm2.

4. Sterile 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4).
5. Trypsin (0.25%, sterilized by filtration).
6. Fetal bovine serum (Biowest, Gibco, or other suppliers).
7. RNeasy Mini Kit (Qiagen) or E.Z.N.A. Total RNA Kit I
(Omega Bio-tek).
8. Absolute ethanol (analytical reagent grade, see Note 1).
9. Dithiothreitol (DTT, 2 M, stored at -20 °C, see Note 2).

2.2 RT-PCR for ZIKV 1. Superscript III reverse transcriptase (Thermo Fisher Scientific).
cDNA Fragments 2. Pyrobest high-fidelity DNA polymerase (Takara).
3. LA Taq DNA polymerase (Takara).
4. dNTP mixture (2.5 mM and 10 mM for each dNTP) (Takara).
5. Nuclease-free water.
6. Agarose (Biowest).
7. 50×TAE (dissolve 242 g Tris-free base in ultrapure water, then
add 57.1 mL HOAc and 100 mL 0.5 M EDTA, pH 8.0, adjust
the volume to 1 L).
8. 10× loading solution (Takara).
9. Ethidium bromide (5 mg/mL).
10. Wizard SV Gel and PCR Clean-Up System (Promega).

2.3 Molecular 1. The pACNR-E70, an infectious clone of Japanese encephalitis

Cloning and Assembly virus [25], served as the backbone for the assembly of the full-
of the pACNR-GZ01- length ZIKV cDNA clone.
Intron-IC 2. T4 DNA ligase.
3. pGEM-T Easy (Promega).
4. Nuclease-free water.
5. E. coli TOP10 chemically competent cells.
6. LB broth (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast
extract).
7. Ampicillin (100 mg/mL, sterilized by filtration).
8. LB agar plate with ampicillin (100 μg/mL).
9. IPTG (20 mg/mL, sterilized by filtration).
10. X-gal (40 mg/mL in N,N-dimethylformamide).
11. 2×GoTaq Green Master Mix (Promega).
12. Absolute ethanol (analytical reagent grade, see Note 1).
13. Wizard Plus SV Minipreps DNA Purification System
(Promega).
210 Zhong-Yu Liu et al.

14. 50× TAE (242 g Tris-free base, 57.1 mL HOAc, and 100 mL
0.5 M EDTA, pH 8.0 in 1 L of ultrapure water).
15. 10× loading buffer (Takara).
16. Wizard SV Gel and PCR Clean-Up System (Promega).
17. Restriction endonucleases NotI-HF, KpnI-HF, KasI, BstEII-
HF, XhoI, SacII, and HindIII-HF.

2.4 In Vitro 1. PureLink HiPure Plasmid Filter Maxiprep Kit (Thermo Fisher
Transcription Scientific).
2. Q5 high-fidelity DNA polymerase (NEB).
3. Wizard SV Gel and PCR Clean-Up System (Promega).
4. RiboMax SP6 large-scale RNA production system (Promega).
5. m7GpppG analogue (Promega).
6. Nuclease-free water.
7. RNeasy Mini Kit (Qiagen) or E.Z.N.A. Total RNA Kit I
(Omega Bio-tek).

2.5 In Vitro Splicing 1. 2× in vitro splicing buffer (80 mM Tris–HCl, pH 7.4, 2 M

NH4Cl, 20 mM MgCl2, and 0.04% SDS).
2. Nuclease-free water.
3. M-MLV Reverse transcriptase/RNase H- (Takara).
4. LA Taq DNA polymerase (Takara).
5. Absolute ethanol (analytical reagent grade, see Note 1).
6. RNeasy Mini Kit (Qiagen) or E.Z.N.A. Total RNA Kit I
(Omega Bio-tek).

2.6 RNA Transfection 1. 24-well plates.

2. Lipofectamine 2000 (Thermo Fisher Scientific).
3. Opti-MEM I (Thermo Fisher Scientific).

2.7 Titration 1. 12-well plates.

2. Low-melting agarose (2% in ultrapure water, autoclaved).
3. 2× DMEM (dissolve two packs of the DMEM in approximate
600 mL ultrapure water, then add 4.0 g NaHCO3
pre-dissolved in ultrapure water, and mix well. Adjust the
volume to 1 L, and sterilize by filtration through a 0.2 μm
filtration apparatus. Store at 4 °C before use).
4. 4% formaldehyde solution.
5. 1% crystal violet (dissolved in 20% ethanol).
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 211

2.8 vRNA Copy 1. 48-well plates.

Determination 2. Lipofectamine 2000 (Thermo Fisher Scientific).
3. Opti-MEM I (Thermo Fisher Scientific).
4. Nuclease-free water.
5. E.Z.N.A. Total RNA Kit I (Omega Bio-tek).
6. One-Step PrimeScript RT-PCR Kit/Perfect Real Time
(Takara).
7. LightCycler 20-μL Capillaries (Roche).
8. LightCycler 2.0 qRT-PCR system (Roche).

2.9 Immuno- 1. 24-well plates.

fluorescent Staining 2. Sterile square cover slips (10 mm × 10 mm).
3. 1× PBS.
4. Fixation solution (70% methanol, 30% acetone).
5. DAPI (1 mg/mL in N,N′-dimethylformamide).
6. Anti-ZIKV E protein monoclonal antibody (BioFront
Technologies).
7. Alexa Fluor 488-labeled goat anti-mouse IgG antibody.

3 Methods

3.1 Isolation of ZIKV 1. Seed BHK-21 cells into a 25 cm2 flask, and incubate in a 37 °C
RNA from Culture incubator with 5% CO2 until the cells reach approximately 90%
Supernatants confluency.
3.1.1 Propagation of ZIKV 2. Infect the BHK-21 cells using 0.1 MOI of ZIKV GZ01 strain,
in BHK-21 Cells and change the virus-containing medium with fresh DMEM
containing 2% FBS after a two-hour incubation. Culture the
infected cells at 37 °C with 5% CO2 until visible CPE is
apparent.
3. Freeze and thaw the infected cells together with the superna-
tant for one or two times, transfer the culture into a sterile
50 mL centrifuge tube, and centrifuge at 6000 g for 10 min at
4 °C. Discard the precipitation, aliquot the supernatant into
sterile screw cap cryotubes. The virus aliquot can be stored at -
80 °C for long-term storage. Steps 2 and 3 require a BSL-
2 facility.

3.1.2 ZIKV RNA Isolation The vRNA can be isolated from the virus-containing supernatants
from Culture Supernatants in Subheading 3.1.1. A brief procedure based on the RNeasy Mini
Kit is listed below.
212 Zhong-Yu Liu et al.

1. Mix 200 μL of viral supernatant, 200 μL Buffer RLT contain-

ing freshly added 40 mM DTT, and an equal volume of 100%
ethanol together. Mix well by pipetting, and do not centrifuge.
2. Transfer the mixed sample into the RNeasy mini column, and
centrifuge at 12,000 rpm for 1 min. Discard the flow-through.
3. Add 700 μL Buffer RW1, and centrifuge at 12,000 rpm for
1 min. Discard the flow-through.
4. Add 500 μL Buffer RPE containing 80% ethanol, and centri-
fuge at 12,000 rpm for 30 s. Discard the flow-through.
5. Repeat the above step.
6. Centrifuge the column at 12,000 rpm for 2 min, transfer the
RNeasy column to a RNase-free 1.5 mL Eppendorf tube, add
50 μL nuclease-free water to the bottom center of the column,
and do not let the tip of the pipette touch the silica membrane.
Centrifuge at 12,000 rpm for 1 min to elute the RNA. The
purified vRNA can be stored at -80 °C for at least 6 months
(see Note 3).

3.2 RT-PCR 1. Generation of cDNA from the isolated vRNA. Set up the
reaction as following:
3.2.1 Reverse
Transcription
Components Volume
Isolated vRNA 8 μL
dNTP mixture (10 mM each) 1 μL
Primer P-Z-R14B (20 μM, see Note 4) 1 μL
RNase-free H2O 2 μL
Total 12 μL

2. Incubate the reaction at 65 °C for 5 min, and then cool on ice

immediately. Centrifuge the reaction tube briefly, and add the
following components:

Components Volume
Denatured mixture in step 1 12 μL
5× first-strand buffer 4 μL
0.1 M DTT 2 μL
RNase inhibitor 1 μL
Superscript III reverse transcriptase 1 μL
Total 20 μL
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 213

Table 1
Primers for the amplification of ZIKV GZ01 fragments

Primers Sequences(5′-3′) Application

P-Z-NotI-SP6-F CTTCAGCGGCCGCATTTAGG S1 amplification
TGACACTATAGAGTTGTTGA
TCTGTGTGA
P-Z-KpnI-R CACCCCGGTACCGCATCTCG
TCTC
P-Z-KpnI-F GAGATGCGGTACCGGGGTG S2 amplification
TTTGTCTAT
P-Z-KasI-R TAAAGTTGGCGCCCATCTC
TGAAATG
P-Z-KasI-F AACTGACATTTCAGAGATG S3 amplification
P-Z-BstEII-R TCCTTTCAATGCGGTTACCAA
TGAT
P-Z-BstEII-F ATCATTGGTAACCGCA S4′ amplification
TTGAAAGG
P-Z-R14B GGCCAGCGTGGTGGAAACTC
P-Z-Tail AACGGGAAACGTCTTGCTC S4 amplification with P-Z-BstEII-
F (also includes a XhoI site and an
additional 16-bp downstream sequence)

3. Perform the RT reaction at 50 °C for 50 min, followed by the

inactivation of the reverse transcriptase at 85 °C for 5 min.
Store the prepared cDNA at -20 °C or -80 °C (the cDNA
can be stored up to a few weeks).

3.2.2 PCR Amplification 1. Using the above obtained cDNA as the template, amplify the
four DNA fragments (S1, S2, S3, S4′, see Note 5). Primer
sequences are listed in Table 1.
Set up the reactions as the following:

Components Volume
cDNA product 1 μL
Forward primer (20 μM) 1 μL
Reverse primer (20 μM) 1 μL
10× LA Taq Buffer 5 μL
dNTP mixture (2.5 mM each) 8 μL
LA Taq DNA polymerase (see Note 6) 0.5 μL
Nuclease-free water 33.5 μL
Total 50 μL
214 Zhong-Yu Liu et al.

Set the reaction parameters as below:

Steps Temperatures Durations Cycles

1 Pre-denaturation 95 °C 5 min ×1
2 Denaturation 95 °C 30 s ×1
3 Annealing 54 °C, decreased by 30 s ×1
0.5 °C per cycle
(see Note 7)
4 Elongation 72 °C 1 min/kb Jump to
step 2, ×6
5 Denaturation 95 °C 30 s ×1
6 Annealing 51 °C 30 s ×1
7 Elongation 72 °C 1 min/kb Jump to
step
5, ×25
8 Incubation 72 °C 5 min ×1
9 Hold 16 °C 10 min ×1

2. Pour 1% TAE/agarose gels containing 1 μg/mL of EtBr. Add

1/10 volume of 10× loading buffer into the PCR reactions,
and load them onto the agarose gels. Perform standard elec-
trophoresis in TAE buffer to separate the PCR products. Excise
the bands with expected sizes using a clean scalpel, and transfer
the gel slice into clean 1.5 mL collection tubes.
3. Purify the PCR products using the Wizard SV Gel and PCR
Clean-up System. Add an equal volume of membrane binding
solution into the tubes containing the gel slices, and incubate at
50 °C for approximately 10 min with mixing by inverting the
tubes for two to three times to dissolve the gel slices.
4. Transfer the samples into SV columns, and centrifuge at
12,000 rpm for 1 min. Discard the flow-through.
5. Wash the column with 750 μL membrane wash solution con-
taining 80% ethanol by centrifugation, and then perform the
washing step with 500 μL membrane wash solution containing
80% ethanol again.
6. Insert the columns back into the adapter tubes, and centrifuge
at 12,000 rpm for 2 min.
7. Transfer the SV columns to nuclease-free 1.5 mL centrifuge
tubes, and then add 50 μL nuclease-free water to each column
to elute the PCR products, and store at -20 °C before use.
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 215

3.3 Generation of the 1. Order the following oligonucleotides from a local supplier.
Infectious Clone P-ZSK-1: 5′-GGCCGCGCTAGATGCGGTACCTTTC
Plasmid AAGATGGGCGCCAATTACACTGCCGGCATATCATT
3.3.1 Construction of an
GGTAACCATTCCATTC-3′
Assemble Backbone P-ZSK-2: 5′-TCGAGAATGGAATGGTTACCAATGATAT
GCCGGCAGTGTAATTGGCGCCCATCTTGAAAGGT
ACCGCATCTAGCGC-3′
2. Dissolve the oligos into 20 μM solutions using nuclease-free
water (see Note 8). Mix 20 μL of the two oligos together, and
incubate the sample in boiling water for 5 min. Then left the
mixture in the water bath until it cools to below 30 °C (may
take a few hours). The oligos are now annealed to form a
dsDNA containing sticky ends of NotI and XhoI, respectively.
3. Digest the pACNR-E70 plasmid with NotI-HF/XhoI, separate
the digested plasmid DNA by agarose gel electrophoresis, and
retrieve the 2.3 kb length vector band by using a Wizard SV Gel
and PCR Clean-Up System. Ligate the annealed ZSK adaptor
dsDNA with the pACNR vector by using T4 DNA ligase at
4 °C for 16 h.
4. Transform the ligation reaction into E. coli TOP10 chemically
competent cells. Thaw the competent cell on ice. Add 15 μL of
the ligation reaction to 100 μL competent cell. Mix well, and
incubate on ice for 30 min.
5. Heat shock at 42 °C for 90 s. Then transfer the competent cell
to an ice bath immediately, and incubate for 2–3 min.
6. Add 500 μL LB broth, and rotate at 180–200 rpm under 37°C
for 50 min.
7. Transfer 200 μL of the transformed cells to a LB agar plate with
100 μg/mL ampicillin. Dispense evenly with a sterile spreader.
Incubate the agar plate inversely at 37 °C for 12–16 h when the
agar surface is dry.
8. Pick several clones, and incubate them into 3 mL of LB broth.
Culture in a shaker bath at 37 °C for 12–16 h. Isolate plasmid
from the clones. Then screen the recombinants by restriction
endonuclease analysis combined with Sanger sequencing to
obtain an aimed pACNR-GZ01-linker backbone plasmid.

3.3.2 Sub-cloning of ZIKV 1. Clone the PCR-prepared S2 fragment into the pACNR-GZ01-
cDNA Fragments linker by using KpnI-HF/KasI double digestion, which results
in the pACNR-GZ01-S2 recombinant plasmid. Follow the
basic cloning procedure in Subheading 3.3.1.
2. Clone the S1, S3, and S4′ fragments into pGEM-T Easy vector
by AT-cloning. Follow the basic procedure in Subheading
3.3.1, and add 20 μL of IPTG and X-gal before the spreading
of the bacterial suspension. Screen the resultant clones by blue-
216 Zhong-Yu Liu et al.

white screening, colony PCR, restriction endonuclease analysis,

and Sanger sequencing. 2× GoTaq Green Master Mix is ideally
suitable for colony PCR analysis. Other similar products can
also be used.
3. Order the following oligos from a local supplier.
P-S4ter-1: 5′-ATGAGTTTCCACCACGCTGGCCGCCAGG
CACAGATCGCCGAATAGCGGCGGCCGGTGTGGG
GAAATCCATGGTTTCTCGAGCAAGACGTTTCCCG
TT-3′
P-S4ter-2: 5′-AACGGGAAACGTCTTGCTCGAGAAACCA
TGGATTTCCCCACACCGGCCGCCGCTATTCGGC
GATCTGTGCCTGGCGGCCAGCGTGGTGGAAACT
CAT-3′

Anneal the oligos as described in Subheading 3.3.1 to obtain a

short dsDNA S4ter containing the last 77-nt of ZIKV genome.
Then fuse the S4ter with the cloned S4′ fragment by using over-
lapping PCR as described below.
Reaction setup:

Components Volume
Cloned S4′ fragment (PCR prepared, 20–50 ng) 1 μL
S4ter (annealed, 20 μM) 1 μL
P-Z-BstEII-F 1 μL
P-Z-Tail 1 μL
10× LA Taq Buffer 5 μL
dNTP mixture (2.5 mM each) 8 μL
LA Taq DNA polymerase 0.5 μL
Nuclease-free water 32.5 μL
Total 50 μL

Set reaction parameter as follows:

Steps Temperatures Durations Cycles

1 Pre- 95 °C 5 min ×1
denaturation
2 Denaturation 95 °C 30 s ×1
3 Annealing 54 °C, decreased 30 s ×1
by 0.5 °C per cycle
4 Elongation 72 °C 2 min 40 s Jump to step 2, ×6
5 Denaturation 95 °C 30 s ×1

(continued)
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 217

Steps Temperatures Durations Cycles

6 Annealing 51 °C 30 s ×1
7 Elongation 72 °C 2 min 40 s Jump to step 5, ×25
8 Incubation 72 °C 5 min ×1
9 Hold 16 °C 10 min ×1

Purify the PCR product by agarose gel electrophoresis and

recovery as described in Subheading 3.2.2. Perform the cloning
and screening as described in Subheading 3.3.2, step 2.

3.3.3 Design of the Order a full-gene synthesis service for the Pylaiella littoralis mito-
Modified P.li.LSUI2 Intron chondrial rRNA intron 2 (P.li.LSUI2) from a local supplier. Substi-
tute the exon-binding sequences (EBS) 1, 2, and 3 with the
complement sequence to the 2460–2464, 2467–2472, and 2473
positions in ZIKV GZ01 genome (Fig. 1). In addition, ZIKV
sequences were added to the upstream and downstream of the
self-splicing intron sequence to facilitate further molecular manip-
ulations. The secondary structure of the resultant P.li.LSUI2-
mZika is shown in Fig. 1.
Fuse the S1 fragment with the P.li.LSUI2-mZika sequence by
using overlapping PCR method as described in Subheading 3.3.2,
step 3. Clone the resultant S1-intron fragment into pGEM-T Easy,
and perform screening as described in Subheading 3.3.2.

3.3.4 Assembly of the 1. Stepwise assemble the S3-, S4-, and S1-intron fragments into
Full-Length pACNR-GZ01- the pACNR-GZ01-S2 plasmid by sub-cloning using the
Intron-IC corresponding restriction sites. Perform these sub-cloning
steps as described in Subheading 3.3.1. For the last round of
the assembly, shifting the culturing temperature from 37 °C to
30 °C may increase the positive rate of recombinants.
2. Identify the obtained pACNR-GZ01-Intron-IC by using
restriction endonuclease analysis (HindIII, SacII, and
NotI-HF/KpnI), and confirm the full-length genomic
sequence in the correct clone by Sanger sequencing.
3. Cryo-store the E. coli TOP10 strain containing the pACNR-
GZ01-Intron-IC. Add 200 μL of 50% sterile glycerol to 800 μL
of fresh overnight culture. Mix well, seal, and label the Eppen-
dorf tubes before storing them at -80 °C.

3.4 In Vitro 1. Prepare a 2 mL overnight culture for the E. coli TOP10/

Transcription pACNR-GZ01-Intron-IC.
2. Transfer 150 μL of the overnight culture into 300 mL of LB
broth containing 100 μg/mL ampicillin. Incubate the flask in a
30 °C shaking incubator, and culture overnight at
218 Zhong-Yu Liu et al.

Fig. 1 Structure model of the P.li.LSUI2-mZika intron. Magenta, the EBS1-IBS1 sequence; cyan, the EBS2 and
IBS2 sequence; red, the EBS3 and IBS3 sequence

200–250 rpm. The culturing time should not be longer than

16 h.
3. Purify the infectious clone plasmid by using PureLink HiPure
Plasmid Filter Maxiprep Kit following the manufacturer’s
instructions. Collect the culture pellet by centrifugation with
6000 rpm for 10 min, and resuspend with 10 mL of RNase
A-containing Buffer R3. Add 10 mL of Buffer L7, and invert
the tube for five times. Lyse for no longer than 5 min. Then
add 10 mL Buffer N3, and mix by inverting. Transfer the lysate
to an equilibrated HiPure Maxi column, and let the liquid pass
the column by gravity flow. Wash the inner filtration cartridge
with 10 mL of Buffer W8, then discard the filtration cartridge,
and wash the column with 50 mL of Buffer W8. Elute the DNA
with 15 mL of Buffer E4. Add 10.5 mL of isopropanol to the
eluted solution. Mix well by inverting for 20 times, and centri-
fuge at 12,000 g, 4 °C for 40 min. Discard the supernatant
carefully, and wash the pellet plasmid with 5 mL of 70% etha-
nol. Air-dry the precipitated plasmid DNA, and add
100–300 μL nuclease free water, 0.5× TE or 1× TE (supplied
A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 219

in the kit). Over-dehydration of DNA may render it hard to be

dissolved. Determine the concentration of the plasmid prepa-
ration by using a UV-Vis micro-volume spectrophotometer,
and check its quality by performing a regular TAE/agarose
electrophoresis. Store the plasmid at -20 °C. Usually
20–50 μg of plasmid can be obtained from 300 mL bacterial
culture.
4. Linearize the plasmid by digestion using XhoI endonuclease at
37 °C for 8 h.

Components Volume
Plasmid DNA 35 μL (approximately 5–15 μg)
10× NEB CutSmart Buffer 5 μL
XhoI 1 μL
Nuclease-free water 9 μL
Total 50 μL

5. Check the efficiency of the digestion by using standard TAE/

agarose gel electrophoresis. Then purify the linearized plasmid
using a Wizard SV Gel and PCR purification system. Briefly,
dilute the reaction with 150 μL 1× TE, then add 300 μL
membrane binding solution, and mix well. Transfer the sam-
ples into SV columns, and centrifuge at 12,000 rpm for 1 min.
Discard the flow-through. Perform the second round of col-
umn washing as described in Step X. Insert the columns back
into the adapter tube, and centrifuge at 12,000 rpm for 2 min.
Transfer the SV columns to a nuclease-free 1.5 mL centrifuge
tube, then add 35 μL nuclease-free water to the bottom center
of column, centrifuge at top speed for 1 min, transfer the elute
back to the column, and perform the centrifugation again.
Determine the concentration of the linearized plasmid DNA,
and store at -20 °C before use (see Note 9).
6. Set up the in vitro transcription reactions as following:

Volume

Control
Components Sample reaction
Template* 8 μL 2 μL
5× SP6 Buffer 6 μL 2 μL
NTP mixture (A:C:U:G=3:3:3:1, 100 mM in 6 μL 2 μL
total)
m7GpppG Cap analogue 1 μL 0.33 μL

(continued)
220 Zhong-Yu Liu et al.

Volume

Control
Components Sample reaction
SP6 Enzyme Mix 3 μL 1 μL
Nuclease-free water 6 μL 2.67 μL
Total 30 μL 10 μL

*The total amount of the template DNA should be at least 500 ng in a 30-L reaction. 1–
2 g of template DNA usually works well

Use the control template supplied in the RiboMax SP6 large-

scale RNA production system for the positive control reaction.
7. Incubate at 37 °C for 3–4 h, then add RQ1 RNase-Free DNase
I (1 U/μg template) to the sample reaction, and incubate at
37 °C for 15 min.
8. Perform a regular TAE/agarose gel electrophoresis to check
the efficiency of in vitro transcription. Ideally, a bright band
migrating similarly to a 2.5–5 kb dsDNA will be detected for
full-length ZIKV RNA.
9. The in vitro transcribed RNA can be purified using RNeasy
Mini Kit following the RNA cleanup protocol. In brief, dilute
the sample into 100 μL by using nuclease-free water, and
transfer the sample to a RNase-free 1.5 mL centrifuge tube.
Add 350 μL Buffer RLT and 250 μL absolute ethanol, mix well
by pipetting, and do not centrifuge. Transfer the 700 μL sam-
ple to a RNeasy mini column, and centrifuge at 12,000 rpm for
1 min. Discard the flow-through. Perform two times of column
washing using 500 μL of Buffer RPE containing 80% ethanol.
Centrifuge the column at 12,000 rpm for 2 min, transfer the
RNeasy column to a RNase-free 1.5 mL Eppendorf tube, add
50 μL nuclease-free water to the bottom center of the column,
and do not let the tip of the pipette touch the silica membrane.
Centrifuge at 12,000 rpm for 1 min to elute the vRNA. Deter-
mine the concentration by a UV-Vis spectrophotometer, and
check the integrity of the RNA preparation using TAE/agarose
gel electrophoresis. The purified RNA can be stored at -80 °C
for at least one year.

3.5 In Vitro Splicing 1. Incubate the 2× splicing buffer (80 mM Tris–HCl, pH 7.4,
2 M NH4Cl, 20 mM MgCl2, and 0.04% SDS) at 45 °C for
several minutes to dissolve any salt precipitations.
2. Mix with the RNA preparation with an equal volume of 2×
splicing buffer, and incubate at 45 °C for 1 h.
A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 221

3. Run a regular TAE/agarose gel or a formaldehyde denatured

agarose gel to check the success of in vitro splicing. The excised
intron band can be used as an indicator for successful splicing.
4. The splicing event can also be monitored by performing
RT-PCR using the following primer pair which spans the
intron region:
P-Z-F4: 5′-AGCATTTGAAGCCACTGTG-3′
P-Z-R4.1: 5′-CTCTATGTTTGAGTGGGCA-3′

Set up the below reactions in a RNase-free PCR tube.

Components Volume
In vitro spliced or unspliced RNA 2 μL
Primer P-Z-R4.1 (20 μM) 1 μL
RNase-free H2O 9 μL
Total 12 μL

Incubate the reaction at 70 °C for 5 min, and then cool on ice

immediately. Centrifuge the reaction tube briefly, and add the
following components:

Components Volume
5X M-MLV Buffer 4 μL
dNTP Mix (10 mM each) 1 μL
RNase inhibitor (40 U/μL) 1 μL
RTase M-MLV (RNase H-) (200 U/μL) 1 μL
RNase-free H2O 1 μL
Total (together with the RNA/primer mix) 20 μL

Perform the RT reaction at 42 °C for 1 h, followed by the

inactivation of the reverse transcriptase at 70 °C for 15 min.
Set up two PCR reactions using the spliced or unspliced cDNA
as template.

Components Volume
cDNA product 1 μL
Forward primer (20 μM) 1 μL
Reverse primer (20 μM) 1 μL
10× LA Taq Buffer 5 μL
dNTP mixture (2.5 mM each) 8 μL

(continued)
222 Zhong-Yu Liu et al.

Components Volume
LA Taq DNA polymerase (see Note 6) 0.5 μL
Nuclease-free water 33.5 μL
Total 50 μL

Run an 1% TAE/agarose gel to check the size of the PCR

product. Successful splicing will shift the PCR product from
1.35 kb to 0.73 kb.
5. Purify the RNA from the reaction by either column-based
cleanup protocol described in Subheading 3.4, step 9 or etha-
nol precipitation. Store the RNA at -80 °C for later use.

3.6 RNA Transfection 1. One day prior to transfection, plate BHK-21 cells into 24-well
plates at a density of 5 × 104 cells/well, and culture at 37 °C
with 5% CO2 for 12–14 h.
2. Perform the transfection by using the Lipofectamine 2000
reagent (Thermo Fisher Scientific). Use 500 ng RNA for each
well of BHK-21 cells. Perform the transfection in triplicates.
3. Dilute 2 μg spliced RNA in 200 μL of Opti-MEM I.
4. Dilute 4 μL Lipofectamine 2000 in 200 μL of Opti-MEM
I. Incubate at room temperature for 5 min.
5. Mix the diluted RNA and Lipofectamine 2000 by pipetting
gently. Do not centrifuge. Incubate at room temperature for
20 min.
6. Add 100 μL RNA/liposome complex to the cells in the 24-well
plate. Incubate for 6 h in a 37 °C CO2 incubator.
7. Discard the medium containing RNA/liposome complex at
6 h post-transfection, and culture the transfected cells in
DMEM with 5% FBS for 2–3 days.
8. Collect the culture supernatants of the transfected cells at 72 h
post-infection, and seed 100 μL onto C6/36 cell monolayer in
a 12-well plate.
9. Continue to culture the blind-infected C6/36 cells at 28 °C for
4–5 days, and then collect the supernatants for virus titer
determination.

3.7 Titration 1. One day prior to plaque assay, seed BHK-21 cells into 12-well
plates at a density of approximately 2.5 × 105 cells/well. Cul-
ture the cells in a CO2 incubator at 37 °C overnight.
2. At the next day, check the confluency of the BHK-21 cells
under microscopy. The confluency should be >90% at the
time of plaque assay. Perform a tenfold serial dilution of the
 A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 223

virus samples that are to be determined. DMEM containing 2%

FBS or FBS-free DMEM can be used as the medium for the
dilution.
3. Completely pipette and discard the supernatant of the BHK-21
monolayer, add 300 μL of serial diluted samples to the cells,
and incubate in a CO2 incubator at 37 °C for 1.5 h. Rotate the
plate for several times every 30 min.
4. Melt the 2% LMP agarose in a microwave oven. Set the power
to low or medium, and do not let the LMP agarose to boil for a
long time.
5. Mix an appropriate volume of the melted 2% LMP agarose with
an equal volume of 2× DMEM containing 4% FBS. Ensure that
the temperature of the DMEM/1% LMP agarose mixture does
not fall below 37 °C, which may result in its solidification.
6. Discard the virus inoculum completely (see Note 10). Add
1 mL of the DMEM/1% LMP agarose to the infected cells
carefully and rapidly to avoid the detachment and disruption of
the cell monolayer. Leave the plates at room temperature for
15–30 min to facilitate the solidification of the agarose matrix.
7. Culture the plates as described above for approximately 4 days.
Plaques can usually be observed at the 3rd day by the naked eye
under a lamp.
8. Fix the cells by adding 1.5 mL of 4% formaldehyde to each well.
The fixation should be performed in a fume hood for
30–60 min.
9. Remove the fixative and overlay by flushing under a tap water.
Add 0.5 mL of 1% crystal violet solution to the fixed cells, and
stain for 30–60 min.
10. Recover the staining solution for repeat use by a transfer
pipette. Wash the plate under tap water. Dry the plate
under air.
11. Count the plaques, and calculate the titer of the sample.

3.8 Mutagenesis Mutants targeting the cyclization sequences and 5′-SLA element
Analysis of cis-Acting will be analyzed in this protocol (see Note 11). For replication
RNA Elements Based analysis of (+)ssRNA viruses, a mutant containing an inactive
on pACNR-GZ01- vRNA polymerase usually serves as the negative control group.
Intron-IC 1. Perform the in vitro transcription and in vitro splicing as
described above.
2. Seed BHK-21 cells into 48-well plates at a density of 2 × 104
cells per well.
3. When the cells reach 50% confluency at the next day, perform
the transfection in triplicates using Lipofectamine 2000.
224 Zhong-Yu Liu et al.

4. Discard the medium containing RNA/liposome complexes at

6 h post-transfection, and then wash the cells using 0.5 mL per
well of FBS-free DMEM for three times. Using a compatible
multichannel pipette is recommended. Add 0.25 mL of
DMEM containing 5% FBS to each well. Put the plates into
incubator for culturing, and leave one plate for total RNA
isolation using an E.Z.N.A Total RNA Kit I.
5. Remove the culture supernatant, and wash the cells with sterile
PBS once. Add 350 μL of TRK lysis buffer supplemented with
fresh added 40 mM DTT, and lyse the cells for 2–3 min.
6. Transfer the lysate into an RNA Homogenizer Spin Column
supplied in the E.Z.N.A Total RNA Kit I. Centrifuge at
12,000 rpm for 2 min, and collect the flow-through.
7. Add 350 μL 70% ethanol to the flow-through, mix well by
pipetting, transfer the mixtures to HiBind RNA Mini Col-
umns, centrifuge at 12,000 rpm for 2 min, and discard the
flow-through.
8. Add 700 μL Wash Buffer I, and centrifuge at 12,000 rpm for
1 min. Discard the flow-through.
9. Add 500 μL Wash Buffer II containing 80% ethanol, and
centrifuge at 12,000 rpm for 30 s. Discard the flow-through.
10. Repeat the above step 9.
11. Centrifuge the column at 12,000 rpm for 2 min, transfer the
HiBind RNA Mini Column to a RNase-free 1.5 mL Eppendorf
tube, add 50 μL nuclease-free water to the bottom center of
the column, and do not let the tip of the pipette touch the silica
membrane. Centrifuge at 12,000 rpm for 1 min to elute RNA.
12. At 24, 48, and 72 h post-transfection, collect the supernatants,
and purify cellular RNA following the same procedure.

3.9 qRT-PCR The qRT-PCR will be performed using the One-Step PrimeScript
RT-PCR Kit (Perfect Real Time) on a LightCycler 2.0 real-time
PCR system.
The primer and probe sequence are listed in Table 2.

Table 2
Primer and probe for qRT-PCR

Primers Sequences(5′-3′) Binding regions

P-ZIKV-AS-F GGTCAGCGTCCTCTCTAATAAACG
P-ZIKV-AS-R GCACCCTAGTGTCCACTTTTTCC
ZIKV-AS-Probe FAM-AGCCATGACCGACACCACACCGT-BHQ1
A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 225

1. Generate a tenfold serial dilution of an in vitro transcribed

ZIKV RNA with known concentration. The diluted samples
will be used to generate the standard curve for absolute
quantification.
2. Set up the reactions as below:

Components Volume
2× One-Step RT-PCR Buffer III 10 μL
TaKaRa Ex Taq HS (5 U/μL) 0.4 μL
PrimeScript RT Enzyme Mix II 0.4 μL
P-ZIKV-AS-F (20 μM) 0.4 μL
P-ZIKV-AS-R (20 μM) 0.4 μL
ZIKV-AS-Probe (20 μM) 0.4 μL
RNA sample 2 μL
RNase-free water 6 μL
Total 20 μL

Prepare a mixture without the RNA sample, and dispense into

LightCycler capillaries (18 μL reaction mix per reaction) in the
LightCycler centrifuge adapters. Add the RNA samples, and close
the capillaries using the capping tool.
3. Load the capillaries onto LightCycler Sample Carousel. Cen-
trifuge briefly using the LC Carousel Centrifuge 2.0. Mount
the Sample Carousel in the LightCycler 2.0 instrument.
4. Set the reaction parameter in LightCycler software 4.1 as the
following:

Temperature
Cycles/ transition
Steps Temperatures Durations detection rates

Stage 1 ×1

1.1 Reverse 42 °C 5 min 20 °C/s

transcription

1.2 Inactivation of RT 95 °C 10 s 20 °C/s

Stage 2 ×40

2.1 Denaturation 95 °C 5s 20 °C/s

2.2 Annealing 60 °C 20 s Single 20 °C/s

and elongation acquisition

Stage 3 ×1

3.1 Cooling 42 °C 30 s 20 °C/s

226 Zhong-Yu Liu et al.

5. Check that the detection channel is set to Channel 1. Run the

protocol.
6. Perform an absolute quantification analysis in LightCycler soft-
ware 4.1 using the F′′max method. Retrieve the Ct values and
RNA copies. Remove the capillaries carefully using the
releaser tool.

3.10 IFA 1. Transfer the desired amounts of sterile cover slips into wells of
24-well plates using a sterile ophthalmic forceps.
2. Seed BHK-21 cells onto the cover slips, and perform RNA
transfection as described in Subheading 3.6.
3. At 24, 48, or 72 h post-transfection, wash the cover slips with
sterile PBS once, and add 1.5 mL fixation solution to each well
containing a cover slip. Fix at -20 °C for 1 h.
4. Aspire the fixation solution completely, and air-dry the fixed
cover slips.
5. Transfer the cover slips to a new 24-well plate. Add 160 μL of
1:2000 diluted mouse anti-ZIKV E mAb, and incubate at 37 °
C for 1 h. Make sure that the antibody work solution covers the
fixed cells completely and evenly.
6. Discard the primary antibody working solution, and wash the
cells with 1 mL of PBS for 5 min on a mini-shaker. Perform the
washing for three times. Air-dry the cover slips.
7. Add 160 μL of 1:200~1:400 diluted goat anti-mouse IgG
conjugated with Alexa Fluor 488. Incubate at 37 °C for 1 h.
Wash the cover slips as described in step 6.
8. Add 200 μL of 1:2000~1:5000 diluted DAPI (using deionized
water), and incubate at room temperature for 5–10 min. Dis-
card the DAPI dilutes, and wash once with 1 mL deionized
water.
9. Check the results using an Olympus BX51 fluorescence
microscope.

4 Notes

1. In our experience, using AR or GR grade of ethanol in nucleic

acid purification leads to satisfactory results; thus, it is not
necessary to utilize the expensive molecular-biology-grade eth-
anol in most of these applications.
2. DTT is a less toxic replacement for β-mercaptoethanol.
3. Determination of RNA concentration by a UV-Vis spectropho-
tometer is unnecessary for the samples isolated from culture
supernatant, as without the cellular RNAs, the concentration of
the isolated RNA is usually at a very low level.
A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing. . . 227

4. We have found that the RT reactions using a primer which

complements the last 19 nucleotides in ZIKV genome, failed
to generate a full-length cDNA as evidenced by subsequent
PCR analysis. However, the cDNA generated using the P-Z-
R14B primer, which binds to 10734–10753 region in GZ01
genome, supported the amplification of the four cDNA frag-
ments spanning most of the ZIKV genome.
5. Attempts to obtain full-length viral cDNA in as single amplicon
are not recommended, mostly due to the possible low efficiency
in the amplification.
6. In our experience, the LA Taq DNA polymerase is a good
choice for the amplification of 2–6-kb-length DNA fragments,
as it provides usually satisfactory fidelity, robust activity, and a
high cost–performance ratio. In addition, it is readily compati-
ble with T-A cloning. However, any other high-fidelity DNA
polymerase could be used, depending on the preference of
the user.
7. Touch-down PCR protocol is recommended. The annealing
temperature is based on the LA Taq DNA polymerase.
8. We have found that satisfactory results can be obtained by
dissolving the oligos in nuclease-free water and performing
the annealing without any annealing buffer.
9. The template for in vitro transcription can be prepared by PCR
using high-fidelity DNA polymerase like Phusion or Q5, which
does not need to perform a large-scale plasmid isolation
procedure.
10. Washing the cells with sterile PBS for one time is optional.
11. For the introduction of mutations into the infectious clones of
flavivirus, site-directed mutagenesis or overlapping PCR
approaches can be utilized, although we have successful experi-
ences in generating mutated infectious clones by a direct site-
directed mutagenesis based on the full-length infectious clone;
due to its relatively large size, the screening for the correct
clones can be painful. Thus, we highly recommend a two-step
approach to generate the infectious clones containing muta-
tions. Mutations can be introduced into a clone of the
corresponding sub-genomic fragment, either by site-directed
mutagenesis or overlapping PCR. The resultant mutated
sub-genomic fragment can be readily sub-cloned back to the
infectious clone by the same restriction endonuclease sites. In
vitro assembly methodologies are also applicable in principle.
228 Zhong-Yu Liu et al.

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 Chapter 14

Reverse Genetics of Dengue Virus

José Valter Joaquim Silva Júnior,
Andréa Nazaré Monteiro Rangel da Silva, Jefferson José da Silva Santos,
and Laura Helena Vega Gonzales Gil

Abstract
Dengue virus (DENV) is one of the most important and widespread arthropod-borne viruses, causing
millions of infections over the years. Considering its epidemiological importance, efforts have been directed
towards understanding various aspects of DENV biology, which have been facilitated by the development of
different molecular strategies for engineering viral genomes, such as reverse genetics approaches. Reverse
genetic systems are a powerful tool for investigating virus–host interaction, for vaccine development, and
for high-throughput screening of antiviral compounds. However, stable manipulation of DENV genomes is
a major molecular challenge, especially when using conventional cloning systems. To circumvent this issue,
we describe a simple and efficient yeast-based reverse genetics system to recover infectious DENV clones.

Key words Dengue, Reverse genetics, Infectious clone, Homologous recombination, Yeast, Shuttle
vector

1 Introduction

Dengue virus (DENV), a member of Flaviviridae family and Flavi-

virus genus, is an important arthropod-borne virus, with four
antigenically distinct serotypes (DENV-1 to DENV-4)
[1, 2]. The disease is a worldwide public-health concern, with
epidemics occurring in the Americas, Asia, Africa, and Australia
while also causing an important economic impact on the affected
countries [3, 4].
Dengue disease can range from asymptomatic infections and
mild febrile illness to severe manifestations, such as dengue hemor-
rhagic fever (DHF) and dengue shock syndrome (DSS), which are
related to coagulation abnormalities, vascular fragility, and plasma
leakage [5]. The disease severity may be associated with the
antibody-dependent enhancement (ADE) phenomenon, which is

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_14,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

231
232 José Valter Joaquim Silva Júnior et al.

related to the presence of non-neutralizing and cross-reactive anti-

bodies against a heterotypic secondary infection [6, 7].
DENV has a positive-sense single-stranded RNA genome
(~11,000 nucleotides) with a single long open reading frame
(ORF) flanked by 5′ and 3′ untranslated regions (UTRs). The
ORF encodes a polyprotein subsequently processed by cellular
and viral proteases into three structural proteins [capsid (C), pre-
membrane (prM), and envelope (E)] and seven nonstructural pro-
teins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [8, 9].
To date, there are no licensed antiviral therapies against den-
gue, and the development of an effective vaccine has been challeng-
ing, as neutralizing antibodies must be elicited to all DENV
serotypes to provide effective protection [10, 11]. Herein, reverse
genetics systems come as an important and timely tool for the
development of vaccines based on attenuated or chimeric viruses,
as well as for the generation of reporter viruses for screening and
identification of antiviral compounds. In addition, reverse genetics
can be used in studies on virus replication and transcription, path-
ogenicity, and virus–host interactions [12].
Viral reverse genetics is defined as the direct manipulation of
the virus genomes to generate wild-type-like or engineered viruses.
For RNA viruses, reverse genetics begins with the cloning of the
full-length cDNA, termed as “infectious clone” [13, 14]. Since
positive-sense virus RNAs can be directly translated without requir-
ing an intermediate transcription step, viruses with this genome can
be rescued by transfecting in vitro transcribed RNA from cloned
cDNA. Alternatively, positive RNA viruses can also be recovered by
direct transfection of the cDNA clone (DNA-launched infectious
cloning systems), provided there is a suitable promoter for viral
RNA transcription from plasmid cDNA in host cells [13, 15].
Although these two approaches are used in DENV reverse
genetics systems [16–19], the presence of cryptic prokaryotic pro-
moters in the DENV genome has been a major obstacle to its
manipulation. When DENV genomes are maintained in Escherichia
coli, these promoters can lead to the expression of proteins that are
toxic to the bacterium, resulting in unstable clones [20, 21]. To
overcome or minimize this obstacle, DENV genomes have been
cloned in yeast cells or in different strains of E. coli, using low-copy
number vectors, yeast E. coli shuttle vectors, bacterial artificial
chromosomes (BACs), or yeast artificial chromosomes (YACs)
[16, 18, 19, 22–24]. In addition, DENV reverse genetics systems
have been obtained by in vitro ligation of previously cloned geno-
mic fragments, resulting in a full-length infectious clone, which can
be transcribed or transfected without further maintenance into E.
coli [17, 25, 26]. Recombinant DENVs were also recovered by
inserting silent mutations into cryptic promoters [20] or by intro-
ducing linkers containing redundant stop codons [27].
 Reverse Genetics of Dengue Virus 233

Based on these different strategies, reverse genetics systems

have been successfully described for the four DENV serotypes,
providing insights into several aspects of DENV biology, such as
replication cycle and virus–host interaction. For example, using
site-directed mutagenesis in DENV infectious clones, Gebhard
et al. [28] analyzed the participation of different NS3 domains in
viral morphogenesis. The effect of NS3 mutations on viral replica-
tion has also been evaluated by reverse genetics [29, 30]. The
participation of DENV prM in viral egress was demonstrated by
the manipulation of infectious clone [31]. Furthermore, DENV
infectious clones can be used for studies of host specificity and
immune evasion [32–34].
In addition to molecular virology studies, reverse genetics sys-
tems have important biotechnological applications. DENV infec-
tious clone has contributed to the elucidation of resistance
mechanisms to antiviral compounds, as well as to the construction
of reporter viruses used in high-throughput screening platforms for
anti-DENV compounds [35, 36]. DENV reverse genetics systems
have also been used to generate attenuated viruses, which have been
evaluated for their vaccine potential [37–40].
Considering both the usefulness of viral reverse genetics and
the difficulties of manipulating the DENV genome, we describe a
simple and efficient DENV reverse genetics system based on
homologous recombination in yeast. The methodology reported
here allows a single-step cloning of the viral genome, as well as its
stable maintenance in yeast, and can be successfully used for the
recovery of recombinant DENV.

2 Materials

2.1 Dengue RNA 1. DENV-infected cell culture supernatant.

Extraction 2. QIAamp Viral RNA Mini Kit (QIAGEN).
3. Ethanol, molecular biology grade.

2.2 Amplification 1. DENV-specific primers (see Note 1).

and Purification of the 2. SuperScript III One-Step RT-PCR System with Platinum Taq
DENV Genome High-Fidelity DNA Polymerase (Thermo Fisher Scientific).
3. Thermocycler.
4. Agarose, molecular biology grade.
5. QIAquick PCR Purification Kit (QIAGEN).
6. QIAquick Gel Extraction Kit (QIAGEN).
7. 2-Propanol (isopropanol), molecular biology grade.
8. Water bath or thermal block at 50 °C.
234 José Valter Joaquim Silva Júnior et al.

2.3 Vector 1. Shuttle vector pSVJS01 [16], which carries the TRP1 gene
Preparation involved in the tryptophan biosynthesis pathway. TRP1 is
used as an auxotrophic marker for selection in trp1-Δ1yeast
strains (see Note 2).
2. NotI restriction enzyme (New England Biolabs).
3. Antarctic Phosphatase (New England Biolabs).
4. Agarose, molecular biology grade.
5. QIAquick Gel Extraction Kit (QIAGEN).
6. 2-Propanol (isopropanol), molecular biology grade.
7. Water bath or thermal block at 37 and 50 °C.

2.4 Yeast 1. Yeast Extract-Peptone-Dextrose (YPD) (Sigma-Aldrich).

Transformation 2. Bacteriological agar.
3. YPD broth (Thermo Fisher Scientific).
4. 30 °C incubator shaker.
5. S. cerevisiae YPH252 (MATα ura3-52 lys2-801 ade2-101 trp1-Δ
1 his3-Δ200 leu2-Δ1) strain [41] (ATCC) (see Note 3).
6. Dithiothreitol (DTT) (Thermo Fisher Scientific).
7. UltraPure Salmon Sperm DNA Solution (Thermo Fisher
Scientific).
8. Electropermeabilization buffer (EPB) (270 mM sucrose,
1 mM MgCl2, and 10 mM Tris–HCl, pH 7.5).
9. Yeast nitrogen base (YNB) media without amino acids (Sigma-
Aldrich).
10. Yeast synthetic dropout medium supplement (without trypto-
phan) (Sigma-Aldrich).
11. 1 mm cuvette (Bio-Rad).
12. ECM 830 electroporator (BTX Harvard Apparatus).
13. 30 °C microbiological incubator.

2.5 Yeast Plasmid 1. YNB media without amino acids (Sigma-Aldrich).

Isolation 2. Yeast synthetic dropout medium supplement (without trypto-
phan) (Sigma-Aldrich).
3. SCE buffer (1 M sorbitol, 0.1 M NaAc, and 60 mM EDTA,
pH 7.0).
4. Zymolyase (Zymo Research).
5. Ethanol, molecular biology grade.
6. 2-Propanol (isopropanol), molecular biology grade.
7. QIAprep Spin Miniprep Kit (QIAGEN).
8. QIAGEN Plasmid Midi Kit (QIAGEN).
9. Water bath or thermal block at 37 °C.
 Reverse Genetics of Dengue Virus 235

2.6 Full-Length PCR 1. Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific).
and In Vitro 2. UltraPure phenol–chloroform–isoamyl alcohol (25:24:1, v/v)
Transcription (Thermo Fisher Scientific).
3. Chloroform–isoamyl alcohol 24:1 (Sigma-Aldrich).
4. 3 M sodium acetate, pH 5.2.
5. Pellet Paint Co-Precipitant (Millipore).
6. Ethanol, molecular biology grade.
7. Cap Analog [m7G(5′)ppp(5′)G] (Thermo Fisher Scientific).
8. MEGAscript T7 Transcription Kit (Thermo Fisher Scientific).

2.7 Transfection into 1. Cytomix solution [120 mM KCl, 0.15 mM CaCl2, 10 mM

BHK-21 Cells and K2HPO4/KH2PO4 (pH 7.6), 25 mM HEPES (pH 7.6),
DENV Rescue 2.0 mM EGTA, 5.0 mM MgCl2] supplemented with ATP
(2 mM) (Thermo Fisher Scientific) and glutathione (5 mM)
(Thermo Fisher Scientific).
2. 2 mm cuvette (Bio-Rad).
3. ECM 830 electroporator (BTX Harvard Apparatus).
4. BHK-21 cells (ATCC).
5. Trypsin/EDTA solution (Thermo Fisher Scientific).
6. Minimum essential media (MEM) (Thermo Fisher Scientific)
supplemented with 10% fetal bovine serum (Thermo Fisher
Scientific).
7. Cell culture flasks (e.g., with 25 or 75 cm2).
8. 37 °C tissue culture CO2 incubator (5%).
9. Nunc Lab-Tek Chamber Slide System (Thermo Fisher
Scientific).

2.8 Indirect 1. Cold acetone.

Immunofluorescence 2. Wash solution: phosphate-buffered saline (PBS), pH 7.4:
138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 M
KH2PO4.
3. Primary antibody: anti-flavivirus group antigen antibody, clone
D1-4G2-4-15 (ATCC).
4. Secondary antibody: anti-mouse IgG (whole molecule)-FITC
antibody produced in goat (Sigma-Aldrich).
5. Evans blue dye (0.1%).
6. Mounting solution: phosphate-buffered glycerol (pH 7.4):
90 g glycerol in 10 mL PBS (pH 7.4).
7. Coplin jar.
8. Fluorescence microscope.
236 José Valter Joaquim Silva Júnior et al.

3 Methods

3.1 DENV RNA 1. Pipette 560 μL buffer AVL into a 1.5 mL microcentrifuge tube
Extraction (nuclease-free), and add 5.6 μL carrier RNA.
2. Add 140 μL of DENV-infected cell culture supernatant, and
mix by pulse-vortexing for 15 s.
3. Incubate at room temperature for 10 min.
4. Briefly centrifuge the microtube to remove drops from the lid.
5. Add 560 μL ethanol (96–100%) to the microtube, and mix by
pulse-vortexing (15 s). After, briefly centrifuge the tube to
remove drops from the lid.
6. Carefully apply 630 μL of the solution from the above step to
the QIAamp Mini Column (in a 2 mL vial collection tube).
Close the lid, and centrifuge at 6,000× g (8,000 rpm) for
1 min. Place the QIAamp Mini Column in a clean collection
tube, and discard the tube containing the filtrate.
7. Carefully open the QIAamp Mini Column, and repeat step 6
until all the lysate has been loaded onto the spin column.
8. Carefully open the QIAamp Mini Column, and add 500 μL
buffer AW1. Close the cap, and centrifuge at 6,000×
g (8,000 rpm) for 1 min. Discard the tube containing the
filtrate.
9. Carefully open the QIAamp Mini Column, and add 500 μL
buffer AW2. Close the lid, and centrifuge at a maximum speed
(20,000× g; 14,000 rpm) for 3 min.
10. It is recommended to place the QIAamp Mini Column in a
new 2 mL collection tube and centrifuge at full speed for
1 min.
11. Place the QIAamp Mini Column in a clean 1.5 mL microcen-
trifuge tube (nuclease-free), and add 60 μL AVE equilibrated
to room temperature. Close the lid, and incubate at room
temperature for 1 min.
12. Centrifuge at 6,000× g (8,000 rpm) for 1 min.
13. Viral RNA is stable for 1 year when stored at -80 °C.

3.2 Amplification of 1. After extracting virus RNA, amplify the viral genome into five
Genomic Fragments by complementary and partially homologous genomic fragments
RT-PCR using the primers shown in Table 1 (see Note 4).
2. Perform RT-PCR reactions using the SuperScript III One-Step
RT-PCR System with Platinum Taq High-Fidelity DNA
Polymerase.
(i) Set up the thermocycler with the following conditions:
1 cycle at 55 °C for 30 min and 94 °C for 2 min (for cDNA
Table 1
Primers to assemble the full-length infectious clone of DENV-2 by homologous recombination in yeast

Primer annealing
Genomic Amplicon
fragment Primer Sequence (5′-3′) size (bp) Position (nt)a Genomic region
F1 pSVJS01-F cggtccgtaatacgactcactataGAGTTGTTAR 2070 1–23 5′UTR
TCTACGTGGACCGA
DENV2-2045-R TCTGCTTCTATGTTGACTGGG 2025–2045 E
F2 DENV2-1810-F CTACAGCTCAAAGGAATGTCAT 1762 1810–1831 E
DENV2-3571-R CATGTTTCGTTCCTACTCGGGTCC 3548–3571 NS2A
F3 DENV2-3502-F TCACTAGGAGTCTTGGGAATGGC 2078 3502–3524 NS2A
DENV2-5579-R TCCGTGACCCATTCATGTCC 5560–5579 NS3
F4 DENV2-5492-F CATTTCCTCAGAGCAATGCACCAATC 1879 5492–5517 NS3
DENV2-7370-R AATACTTGAGTCACGCAGAGG 7350–7370 NS4B
F5 DENV2-7191-F GCCAGGACTTCAAGCAAAAGC 3560 7191–7211 NS4B
pSVJS01-R ttcaacatttccgtgtcgcgcggccgcAG 10,697– 3′UTR
AACCRGTTGATTCAACAGCACCATT 10,723
a
Positions referring to isolate 3311/BR-PE/95 (DENV-2) (GenBank access JX669481). The RsrII restriction site is in bold. The T7 RNA polymerase promoter is in italics. A
single G to start transcription is underlined. The homology region with the NotI-linearized pSVJS01 vector is in lowercase. E envelope, F fragment, NS nonstructural, UTR
untranslated region
Reverse Genetics of Dengue Virus
237
238 José Valter Joaquim Silva Júnior et al.

synthesis and pre-denaturation) and 40 cycles of 94 °C for

15 s (denaturation), 55 °C for 30 s (annealing), 68 °C for
4 min (extension), and a final extension at 68 °C for 5 min.
(ii) Add the following to a 0.2 mL nuclease-free PCR tube
on ice: 25 μL of 2X reaction mix, 1 μL of forward primer
(10 μM), 1 μL reverse primer (10 μM), 1 μL of Super-
Script III RT/Platinum Taq High-Fidelity enzyme mix,
5 μL of template RNA, and nuclease-free water
q.s.p. 50 μL.
(iii) Gently mix and centrifuge briefly to ensure all compo-
nents are at the bottom of the tube. Depending on the
thermocycler, overlay with silicone oil if necessary.
(iv) Place the reaction in the programmed preheated thermal
cycler described in step 2(i).

3.3 Purification and 1. Analyze the RT-PCR products on a 1% agarose gel.

Quality Control Check 2. Excise the amplicon with a clean scalpel. Remove excess aga-
of Amplicons Prior to rose from the slice (see Note 5).
Cloning
3. Weigh the gel slice into a colorless tube. Add 3 volumes of
buffer QG to 1 volume of gel (e.g., add 300 μL buffer QG to
each 100 mg of gel).
4. Incubate at 50 °C until the gel slice has completely dissolved.
To help, vortex the tube every 2–3 min.
5. After the gel is dissolved, check if the color of the mixture is
yellow. If the color is orange or violet, adjust the pH according
to the manufacturer’s instructions.
6. Add 1 gel volume of isopropanol and mix (e.g., for 100 mg of
gel slice, add 100 μL of isopropanol).
7. Place a QIAquick spin column in a provided 2 mL
collection tube.
8. Apply the sample to the QIAquick column, and centrifuge at
17,900× g (13,000 rpm) for 1 min.
9. Discard flow-through, and place QIAquick column back into
the same collection tube. Repeat step 8 until all the sample has
been loaded onto the spin column.
10. It is recommended to add 0.5 mL buffer QG to the QIAquick
column and centrifuge again for 1 min to remove all traces of
agarose.
11. Add 0.75 mL buffer PE into the QIAquick column, and let the
column stand 2–5 min.
12. Centrifuge for 1 min at 17,900× g (13,000 rpm).
13. Discard the flow-through, and centrifuge the QIAquick col-
umn for an additional 1 min at 17,900× g (13,000 rpm).
 Reverse Genetics of Dengue Virus 239

14. Place the QIAquick column in a clean 1.5 mL nuclease-free

microcentrifuge tube (nuclease-free).
15. To elute DNA, add 50 μL buffer EB or DNase/RNase-free
water (pH 7.0–8.5) to the center of the QIAquick column
membrane, and then centrifuge the column for 1 min at
17,900× g (13,000 rpm). To increase the purification yield,
consult the manufacturer’s instructions.
16. Analyze the purification product on 1% agarose gel.

3.4 Vector 1. Prepare the digestion reaction (vector linearization) by adding

Preparation 1 μg of pSVJS01, 10 U of NotI, 5 μL of 10X NEBufferTM
r3.1 (supplemented with Antarctic Phosphatase Reaction
Buffer), and nuclease-free water q.s.p. 50 μL.
2. Incubate the reaction at 37 °C for 8 h.
3. Inactivate the enzyme by heating at 65 °C for 20 min.
4. Add 12.5 U of Antarctic Phosphatase, and incubate at 37 °C
for 30 min.
5. Stop reaction by heat inactivation at 80 °C for 2 min.
6. Apply the digestion product to 1% agarose gel, and see if the
vector has been linearized.
7. Purify the vector from gel as described in Subheading 3.3.

3.5 Assembly of the 1. Seed S. cerevisiae YPH252 onto YPD agar plate, and incubate
Full-Length cDNA at 30 °C until individual colonies grow (2–3 days).
Clone in a Single-Step 2. Select a colony, and inoculate into 5 mL of YPD.
Cloning (Fig. 1)
3. Grow the yeast at 30 °C for 16–18 h at 130 rpm.
4. Count the yeast cells in a Neubauer chamber, and prepare a
20 mL suspension at a concentration of 5 × 106 cells/mL. Scale
up suspension volume based on the number of transformations
(see Note 6).
5. Grow the suspension described above to a concentration of 107
cells/mL (approximately for 2–4 h).
6. Centrifuge the cells at 522× g (or 1,500 rpm) for 10 min at 4 °
C.
7. Wash the cell pellet with 50 mL of deionized water.
8. Centrifuge the cells at 522× g (or 1,500 rpm) for 10 min at 4 °
C.
9. Resuspend the pellet with 20 mL of DTT (25 mM), and
incubate for 10 min at room temperature.
10. Centrifuge the cells at 522× g (or 1,500 rpm) at 4 °C.
11. Wash the pellet twice with 20 mL of EPB.
240 José Valter Joaquim Silva Júnior et al.

Fig. 1 Assembly of the full-length infectious clone of DENV-2 by homologous recombination in yeast. The
complete genome of DENV-2 can be assembled into the pSVJS01 vector in a single-step cloning by
homologous recombination of five complementary and partially homologous genomic fragments. The dotted
area corresponds to the region of overlap (homology) between fragment–fragment and fragment–vector. The
numbers refer to the initial and final nucleotides of each fragment, according to their position in the complete
genome (GeneBank access JX669481). The T7 RNA polymerase promoter, F fragment, UTR untranslated
region, C capsid, prM pre-membrane, E envelope, NS nonstructural. *Region of homology between F1-vector
and F5-vector inserted by primers pSVJS01-F and pSVJS01-R, respectively

12. Resuspend the pellet in EPB at a concentration of 5 × 107/

50 μL.
13. In parallel, heat the UltraPure Salmon Sperm DNA Solution at
100 °C for 5 min. Then leave on ice for 2 min before using it.
14. Combine 50 μL of the suspension described above with 20 μL
of the recombination mixture [i.e., vector, genomic fragments,
and 2 μL of carrier DNA (UltraPure Salmon Sperm DNA
Solution)].
15. Include a negative control reaction, replacing the genomic
fragments with the same volume of nuclease-free water.
16. Transfer the mixture to a 1 mm cuvette.
17. Electroporate cells under the following electroporation set-
tings: pulse length 11 msec, voltage 270 V, and 1 pulse (see
Note 7).
18. Resuspend the cells in 500 μL of YNB broth containing
yeast synthetic dropout medium supplement (without trypto-
phan), and incubate at 30 °C for 1 h at 130 rpm.
19. Seed yeast onto YNB plate containing yeast synthetic dropout
medium supplement (without tryptophan).
20. Incubate at 30 °C for 2–3 days.
 Reverse Genetics of Dengue Virus 241

3.6 Yeast Plasmid 1. Pick individual colonies into 10 mL of YNB broth containing
Isolation (for Cloning yeast synthetic dropout medium supplement (without
Screening) tryptophan).
2. Grow the yeast at 30 °C for 16–18 h at 130 rpm.
3. Centrifuge the cells at 2,000× g (3,000 rpm) for 5 min at 4 °C.
4. Wash the pellet twice in 20 mL of deionized water (centrifuge
at 2,000× g or 3,000 rpm for 5 min).
5. Resuspend the pellet in 400 μL buffer SCE, and transfer the
suspension to a clean 1.5 mL microfuge tube (nuclease-free).
6. Add 30 U of Zymolyase (Zymo Research), and incubate the
mixture at 37 °C for 1 h.
7. Centrifuge at 17,900×g (13,000 rpm) for 10 min.
8. Resuspend the pellet in 250 μL buffer P1 containing RNAse A.
9. Add 250 μL buffer P2, mix well by inverting vigorously four to
six times, and incubate at room temperature for up to 5 min.
10. Add 350 μL buffer N3, and mix immediately and thoroughly
by inverting the tube four to six times.
11. Centrifuge for 10 min at 17,900× g (13,000 rpm) in a
microcentrifuge.
12. Apply 800 μL supernatant from the step above to the QIAprep
2.0 spin column.
13. Centrifuge for 1 min at 17,900× g (13,000 rpm), and discard
the flow-through.
14. Wash the spin column by adding 0.5 mL buffer PB. Centrifuge
for 1 min at 17,900× g (13,000 rpm), and discard the flow-
through.
15. Wash the spin column by adding 0.75 mL buffer PE. Centri-
fuge for 1 min at 17,900× g (13,000 rpm), and discard the
flow-through.
16. Centrifuge for 1 min at 17,900× g (13,000 rpm) to remove
residual wash buffer.
17. Place the column in a clean 1.5 mL microcentrifuge tube
(nuclease-free).
18. To elute DNA, add 50 μL buffer EB (10 mM Tris-Cl, pH 8.5)
or water to the center of column, let stand for 1 min, and
centrifuge for 1 min at 17,900× g (13,000 rpm).
19. Use the eluted DNA for cloning screening.
(a) Confirm cloning by amplifying adjacent fragments
together, for instance, using primers pSVJS01-F and
DENV2-3571-R to amplify the F1-F2 cassette (Table 1).
Furthermore, full genome sequencing can be performed
from eluted DNA to confirm the identity of the assembled
amplicons and the lack of unwanted mutations.
242 José Valter Joaquim Silva Júnior et al.

3.7 Yeast Plasmid 1. Inoculate selected clones into 20 mL of YNB broth containing
Isolation (for Full- yeast synthetic dropout medium supplement (without
Length PCR Template) tryptophan).
2. Incubate the yeast at 30 °C and 130 rpm for 16–18 h.
3. Inoculate all overnight cultured yeasts into 500 mL of YNB
broth containing yeast synthetic dropout medium supplement
(without tryptophan).
4. Incubate the yeast at 30 °C and 130 rpm for 48 h.
5. Centrifuge the cells at 6,000× g for 15 min at 4 °C.
6. Wash the pellet twice in 50 mL of deionized water (centrifuge
at 6,000× g for 15 min at 4 °C).
7. Resuspend the pellet in 4 mL buffer SCE.
8. Add 200 U of Zymolyase, and incubate the mixture at 37 °C
for 2 h.
9. Centrifuge at 6,000× g for 15 min at 4 °C.
10. Resuspend the pellet in 10 mL buffer P1 containing RNAse A.
11. Add 10 mL buffer P2, mix thoroughly by vigorously inverting
four to six times, and incubate at room temperature for up to
5 min.
12. Add 10 mL chilled buffer P3, mix immediately and thoroughly
by vigorously inverting four to six times, and incubate on ice
for 20 min.
13. Centrifuge at ≥20,000× g for 30 min at 4 °C.
14. Promptly remove supernatant containing plasmid DNA.
15. Centrifuge the supernatant again at ≥20,000× g for 15 min at
4 °C. Again, remove supernatant containing plasmid DNA
promptly.
16. Equilibrate a QIAGEN-tip 100 by applying 4 mL buffer QBT,
and allow the column to empty by gravity flow.
17. Apply the supernatant from step 15 to the QIAGEN-tip, and
allow it to enter by gravity flow.
18. Wash the QIAGEN-tip with 2 × 10 mL buffer QC.
19. Add 15 mL buffer QF, and allow it to elute by gravity.
20. Precipitate DNA by adding 10.5 mL room-temperature iso-
propanol to the eluted DNA. Mix and centrifuge immediately
at ≥15,000× g for 30 min at 4 °C. Carefully decant the
supernatant.
21. Wash DNA pellet with 5 mL of room-temperature 70% etha-
nol, and centrifuge at ≥15,000× g for 10 min. Carefully decant
the supernatant without disturbing the pellet.
22. Air-dry the pellet for 5–10 min, and redissolve the DNA in a
suitable volume of buffer (TE buffer (pH 8.0) or 10 mM Tris-
Cl (pH 8.5)).
 Reverse Genetics of Dengue Virus 243

3.8 Full-Length PCR 1. Thaw, mix, and briefly centrifuge PCR reagents.
2. Prepare the master mix: 10 μL of 5X SuperFiTM Buffer, 1 μL of
10 mM dNTPs mix, 0.5 μL of PlatinumTM SuperFiTM DNA
Polymerase, and water (nuclease-free) q.s.p 50 μL (here, con-
sider additional volume of primers and template).
3. Add 2.5 μL of primer pSVJS01-F (Table 1) and 2.5 μL of
primer pSVJS01-R2 (5′- AGAACCRGTTGATTCAACAG
CACCATT-3′) (both at 10 μM) and 200 ng of template.
4. Set up the thermocycler with the following conditions: 95 °C
for 2 min (initial denaturation), 35 cycles of 95 °C for 10 s
(denaturation), 56 °C for 10 s (annealing), 68 °C for 7 min
(extension), and final extension at 68 °C for 5 min. Proceed
with the PCR reaction.
5. Analyze amplicon on 1% agarose gel.

3.9 Purification and 1. Measure full-length PCR product volume, and complete to
Precipitation of the 200 μL with nuclease-free water.
Full Amplicon 2. Add 200 μL of UltraPure phenol–chloroform–isoamyl alcohol
(25:24:1), and vortex for 10 s.
3. Centrifuge for 5 min at 17,900× g (13,000 rpm).
4. Carefully collect the upper phase, and measure its volume.
5. Add the same volume of chloroform–isoamyl alcohol (24:1),
and vortex for 10 s.
6. Centrifuge for 5 min at 17,900× g (13,000 rpm).
7. Carefully collect the upper phase, and measure its volume.
8. Add 1/10 of the volume of 3 M sodium acetate, 2 μL of Pellet
Paint Co-Precipitant, and 2 volumes of ethanol.
9. Vortex and incubate for 30 min at -20 °C.
10. Wash the pellet twice with 80% ethanol.
11. Centrifuge for 5 min at 17,900× g (13,000 rpm).
12. Carefully remove all ethanol with a pipette.
13. Air-dry pellet at 37 °C for 5 min.
14. Proceed with in vitro transcription.

3.10 In Vitro 1. Perform in vitro transcription with the DNA from Subheading
Transcription 3.9, step 13.
2. Resuspend the DNA pellet in 1.25 μL of nuclease-free water
(see Note 8).
3. Add in vitro transcription mix: 0.5 μL of 10X Reaction Buffer,
2 μL of rNTPs (75 mM ATP, CTP, UTP, and 15 mM GTP),
0.75 μL of cap analog [m7G(5′)ppp(5′)G] (40 mM), and
0.5 μL of enzyme mix.
244 José Valter Joaquim Silva Júnior et al.

4. Incubate the reaction at 37 °C for 4 h.

5. Briefly centrifuge after every 1 h of incubation.
6. Store RNA at -80 °C until use (see Note 9).

3.11 Cell 1. Split BHK-21 cells the day before transfection, and maintain
Transfection the cell under routine culture conditions (e.g., in MEM sup-
plemented with 10% heat-inactivated FBS, at 37 °C and 5%
CO2).
2. On the day of transfection, harvest BHK-21 cells, and prepare a
suspension with 2 × 106 cells per transfection.
3. Wash the cell suspension twice, using ice-cold PBS or pure
MEM (i.e., without antibiotic and FBS).
4. For washes, centrifuge the cells at 334× g (1,200 rpm) for
5 min at 4 °C.
5. After the last wash, resuspend cells in 100 μL ice-cold cytomix
solution.
6. Add 5 μg of in vitro transcribed RNA. As a negative control,
transfect BHK-21 under the same conditions without RNA.
7. Transfer the transfection mixture into a 2 mm cuvette.
8. Proceed with transfection using the following electroporation
settings: 2 pulses of 100 μs, 1,200 V, and 1 s interval (see Note
10).
9. After electroporation, allow cells to recover for 10 min at room
temperature, then resuspend in complete growth medium, and
seed as recommended below:
(i) Plate approximately 104 cells on NuncTM Lab-TekTM
chamber slide to monitor viral rescue by IFA as described
in the next section.
(ii) Seed the rest of the cells into 25 cm2 culture flasks for virus
rescue amplification.

3.12 Viral Rescue 1. Three days after transfection, proceed with IFA of the seeded
Monitoring by Indirect cells on NuncTM Lab-TekTM chamber slide.
Immunofluorescence (a) Include negative control IFA for cell background
differentiation.
(b) Include DENV-infected BHK-21 as a positive control.
2. Fix cells with cold acetone for 5 min at 4 °C and air-dry.
3. Incubate cells with the monoclonal antibody D1-4G2-4-15 (1:
100 dilution in PBS) as the primary antibody for 1 h at 37 °C,
using a humid chamber.
4. Wash the slides twice with PBS (pH 7.4) using a Coplin jar. It is
recommended to let the slide rest in PBS for 10 min with
each wash.
 Reverse Genetics of Dengue Virus 245

5. Wash slides with distilled water, and allow to air-dry at room

temperature.
6. Incubate for 1 h at 37 °C with a 1:100 dilution (PBS) of
fluorescein isothiocyanate-conjugated goat anti-mouse IgG
antibody as the secondary antibody.
7. Wash the slides twice with PBS (pH 7.4), according to step 4.
8. Air-dry the slides.
9. Stain the slides with counterstain (e.g., 0.1% Evans blue for
10 min).
10. Wash slides with distilled water, and allow to air-dry at room
temperature.
11. Pipette a sufficient volume of the mounting solution to the
middle of the slide, to cover it.
12. Read in a fluorescence microscope:
(i) If IFA from transfected cells is negative: keep monitoring
the cell by performing IFA every 1–2 days for a week.
(ii) If IFA from transfected cells is positive: repeat the assay in
1–2 days to assess the increase in positive cells, which
indicates the generation of infectious particles.

4 Notes

1. The recombination primers were designed based on the

genome of isolate 3311/BR-PE/95 (DENV-2) (GenBank
accession JX669481).
2. Plasmid pSVJS01 is available upon request from Dr. Laura Gil
at the Aggeu Magalhães Institute, Oswaldo Cruz Foundation,
PE, Brazil (laura.gil@fiocruz.br).
3. YPH252 strain colonies are typically white in color, but because
this strain also carries the ade2-101 mutation, the colony can
look pinkish to red in color. This is normal but can be pre-
vented by adding adenine to the medium.
4. One of the most critical requirements for homologous recom-
bination in yeast is the presence of sufficient sequence homol-
ogy flanking the fragments and vector to be assembled. When
designing primers, include 15–25 bp of homology length. For
a larger number of DNA fragments (≥10), it is recommended
to increase the homology length between DNA fragments
(30–50 bp).
5. If nonspecific amplifications are not observed, purification can
be performed using the QIAquick PCR Purification Kit
(QIAGEN).
246 José Valter Joaquim Silva Júnior et al.

6. Alternatively, there are protocols to produce frozen competent

yeast cells with a high efficiency of transformation [42].
7. When using a different electroporation system, transformation
conditions need to be optimized. Alternatively, transformation
can be performed using the lithium acetate/single-stranded
carrier DNA/polyethylene glycol (LiAc/SS carrier DNA/-
PEG) method [43].
8. The final volume of 5 μL is sufficient for one transfection. For
further assays, adjust the volume while maintaining the reagent
ratio.
9. It is recommended to analyze the RNA by formaldehyde–aga-
rose gel electrophoresis before proceeding with cell
transfection.
10. When using a different electroporation system, transformation
conditions need to be optimized.

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 Chapter 15

Recovery of Recombinant Rotaviruses by Reverse Genetics

Chantal A. Agbemabiese, Asha A. Philip, and John T. Patton

Abstract
Rotaviruses are the primary cause of severe gastroenteritis in infants and young children throughout the
world. To combat rotavirus illness, several live oral vaccines have been developed, or are under develop-
ment, that are formulated from attenuated human or human–animal reassortant strains of rotavirus. While
the effectiveness of these vaccines is generally high in developed countries, the same vaccines are signifi-
cantly less effective in many developing countries, where the need for rotavirus vaccines is greatest.
Recently, reverse genetics systems have been developed that allow modification of the segmented double-
stranded (ds)RNA genome of rotavirus, including reprogramming the genome to allow expression of
additional proteins that may stimulate expanded neutralizing antibody responses in vaccinated children.
The use of reverse genetics systems may not only lead to the development of more potent classes of vaccines
but can be used to better explore the intricacies of rotavirus molecular biology and pathogenesis. In this
article, we share protocols that can be used to generate recombinant rotaviruses, including modified strains
that express foreign proteins.

Key words Rotavirus, Reverse genetics, Double-stranded RNA, dsRNA, Segmented genome,
Gastroenteritis

1 Introduction

Rotaviruses are a large and genetically diverse group of RNA viruses

that belong to the newly recognized family Sedoreoviridae
[1]. Viruses within the family are primary causes of acute gastroen-
teritis in many mammalian and avian species [2]. In humans, rota-
viruses represent the primary cause of severe dehydrating diarrhea
in infants and young children, leading to as many as 200,000 deaths
per year. The extensive morbidity and mortality associated with
rotavirus infection have stimulated the development of a number
of live oral rotavirus vaccines, including those that are monovalent
and formulated from attenuated human rotaviruses (e.g., Rotarix/
GSK, Rotavac/Bharat Biotech, and RV3-BB/Bio Farma) and
those that are multivalent and formulated from human–animal
reassortant rotaviruses (e.g., RotaTeq/Merck and Rotasiil/Serum

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9_15,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

249
250 Chantal A. Agbemabiese et al.

Institute of India) [3]. While the effectiveness of these vaccines is

generally high in developed countries (80–90%), the same vaccines
are significantly less effective in many developing countries
(40–70%), where the need for rotavirus vaccines is greatest
[4]. The recent development of reverse genetics systems opens
the possibility of genetically altering rotavirus vaccine strains in
such a way as to not only improve their effectiveness but also to
advance the development of rotavirus-based combination vaccines
that may protect children against other enteric or mucosal patho-
gens (e.g., rotavirus–norovirus vaccines) [5–9]. Moreover, the
development of rotavirus reverse genetics system enables the ratio-
nal design of mutant viruses that can be used to probe all aspects of
rotavirus biology, including virus entry, replication, and egress, the
structure and function of rotavirus proteins, rotavirus mechanisms
of escaping antiviral responses, and the basis of rotavirus pathogen-
esis [10, 11]. Significant progress in these areas has already been
made through the generation and study of mutant recombinant
rotaviruses.
The rotavirus virion is a triple-layered non-enveloped icosahe-
dral particle with a genome consisting of 11 segments of dsRNA
that have a total size of ~18.5 kilobase pair (kbp) [12, 13]. During
rotavirus entry, the virion undergoes partial disassembly, yielding a
transcriptionally active subviral particle that catalyzes the synthesis
of 11 capped plus-sense +RNAs [2, 14]. These +RNAs not only
direct the synthesis of rotaviral proteins but also act as templates for
dsRNA synthesis. The +RNAs encode six structural proteins (VP1–
VP4, VP6–VP7) and six nonstructural proteins (NSP1–NSP6).
Two of the nonstructural proteins, NSP2 and NSP5, are critical
to the formation of viral factories (viroplasms) in infected cells, sites
where +RNAs are packaged and replicated to produce progeny
subviral particles [2]. Once formed, progeny subviral particles
migrate from the viroplasm to the endoplasmic reticulum, where
they engage viral membrane-associated proteins. The mature virion
is formed by budding of the subviral particle through the endoplas-
mic reticulum, a process accompanied by the assembly of VP7-VP4
outer capsid protein layer [2, 14]. Sequences of the outer capsid
proteins VP7 (glycoprotein) and VP4 (protease-sensitive attach-
ment protein) are used in defining the G and P genotypes of
rotavirus strains [15].
Reverse genetics systems have been generated for several strains
of rotavirus, including those infecting humans (KU, CDC-9, Ode-
lia, RIX4414), nonhuman primates (SA11, RRV), cattle (UK, RF),
and birds (PO-13) [16–19]. The efficiencies of the systems vary
markedly with the SA11 reverse genetics system remaining the
most commonly used and reliable in producing recombinant
virus. Although there is some variation in the reverse genetics
systems used by various research laboratories, all begin with trans-
fection of T7 transcription vectors containing full-length cDNAs of
 Rotavirus Reverse Genetics 251

rotavirus genome segments into baby hamster kidney cells expres-

sing T7 RNA polymerase (BHK-T7 cells) (Fig. 1a, b). A T7 pro-
moter positioned at the 5′ end of the cDNA and a hepatitis D
virus (HDV) ribozyme positioned at the 3′ end of the cDNA
allow transcription by T7 RNA polymerase to produce viral
+RNAs with authentic 5′- and 3′-terminal sequences (Fig. 1a). To
stimulate 5′ capping of the viral +RNAs, a plasmid directing the
synthesis of an RNA capping enzyme is usually co-transfected with
the T7 transcription vectors (Fig. 1b). In our laboratory, we rou-
tinely co-transfect the pCMV-NP868R plasmid, which encodes the
African swine fever virus (NP868R) capping enzyme [20]. To pro-
mote the formation of viral factories, the T7 transcription vectors
expressing NSP2 and NSP5 +RNAs are transfected into BHK-T7
cells at levels disproportionately greater (3–5X) greater than the
other T7 transcription vectors. Although transfection of BHK-T7
cells with rotavirus T7 transcription vectors and a plasmid expres-
sing a capping enzyme may generate recombinant rotaviruses, the
BHK-T7 cell line is a poor growth substrate for most rotavirus
strains. As a result, 1–3 days following transfection of BHK-T7
cells, the cells are overseeded with MA104 cells which are highly
permissive for rotavirus growth (Fig. 1b). Recombinant rotaviruses
can generally be recovered from the BHK-T7/MA104 cell mix-
tures about 1 week after transfection.
Although the typical rotavirus has a 18.5-kbp genome, mutant
strains have been recovered with genomes that are significantly
smaller or larger due to the presence of sequence duplications or
deletions. Indeed, mutant strains have been described with gen-
omes reaching 21 kbp, reflecting the ability of the rotavirus genome
to accommodate additional RNA sequence [21]. This genome-size
flexibility has provided the basis for the generation of recombinant
rotaviruses that, in addition to expressing rotaviral proteins, are
able to express foreign reporter proteins [20, 22, 23]. Such recom-
binant rotaviruses expressing fluorescent proteins (e.g., UnaG,
mCherry, mRuby) have enabled the study of rotavirus replication
and spread by live cell imagining [24] (Fig. 2). The genome-size
flexibility has also led to the generation of recombinant viruses
expressing the immunogenic capsid proteins of other enteric and
mucosal viruses, a development that may allow the development of
rotavirus-based combination vaccines that protect children against
a second pathogen [7, 8]. A commonly used approach for generat-
ing recombinant rotaviruses that express foreign proteins is
through modification of genome segment 7 (Figs. 1 and 2). The
rotavirus segment 7 RNA encodes NSP3, a protein with
RNA-binding activity that promotes translation of rotavirus
+RNAs [25]. The NSP3 protein remains functional even when a
fluorescent protein is fused to its C terminus, providing a pathway
for generating fully functional rotaviruses that express a reporter
protein during replication [20] (Figs. 1 and 2). By inserting a
252 Chantal A. Agbemabiese et al.

Fig. 1 Rotavirus reverse genetics system. (a) T7 (pT7) plasmids used in generating recombinant rotaviruses
contain a full-length cDNA of a rotavirus genome segment, including its 5′- and 3′-untranslated region (UTR)
and open reading frame. Positioned upstream of the cDNA is a T7 promoter (prm) sequence, and downstream
is an HDV ribozyme (Rz) sequence and a T7 termination (term) signal. The T7 transcriptional start site and HDV
ribozyme cleavage site are indicated, along with origin of plasmid replication (Ori) and ampicillin resistance
gene (AmpR). (b) Recombinant rotavirus is prepared by transfecting BHK-T7 cells with the pT7 plasmids, along
with a plasmid expressing the pCMV-NP868R capping enzyme. The BHK-T7 cells are overseeded 3 days post-
transfection with MA104 cells to facilitate the spread and amplification of recombinant rotavirus. Five days
 Rotavirus Reverse Genetics 253

translational 2A stop–restart (“self-cleaving”) element [26]

between the coding sequences for NSP3 and the fluorescent pro-
tein, recombinant viruses can be generated that express the two
proteins as separate products [11, 12].
Below we provide the protocol routinely used in our laboratory
to generate recombinant SA11 (G3P[2]) isolates. By replacing T7
vectors with homologs containing modified sequences, the proto-
col can be used to recover mutant rotaviruses, including those
expressing foreign proteins. Because mutations may impact rotavi-
rus growth and plaque size, adjustment should be made in the
reverse genetics protocols to allow for the recovery of virus strains
that may have replication defects.

2 Materials

2.1 Cell Lines 1. Baby hamster kidney (BHK-21) cells constitutively expressing
bacteriophage T7 RNA polymerase (BHK-T7) can be obtained
from Dr. Ulla Buchholz at the Laboratory of Infectious Dis-
eases, NIAID, NIH.
2. African green monkey kidney MA104 cells (American Type
Culture Collection).

2.2 Cell Culture 1. Cell culture medium is prepared under sterile conditions in a
Media biological safety cabinet and stored at 4 °C.
2. Prepare GMEM incomplete medium by combining 500 mL of
Glasgow minimal essential medium (MEM) with 50 mL of
tryptose–phosphate broth (TPB) (Gibco), 5 mL of 100X non-
essential amino acids (NEAA) (Gibco), 5 mL of 100X
L-glutamine (Gibco), and 5 mL of 100X penicillin–streptomy-
cin solution (Corning).
3. Prepare GMEM complete medium by adding 25 mL of heat-
inactivated fetal bovine serum to 500 mL of GMEM incom-
plete medium. To supplement with Geneticin, add 10 mL of a
50 mg/mL stock of Geneticin G418 (Gibco) to 500 mL of
GMEM complete medium.

Fig. 1 (continued) after overseeding, recombinant virus in cell lysates is amplified on MA104 cells and the
dsRNA genome of the viruses analyzed by RNA gel electrophoresis and sequencing. Subsequently, the
recombinant virus is plaque isolated and further amplified on MA104 cells. (c) Schematics of T7 plasmids
used to recover recombinant wild-type SA11 (pT7/SA11-NSP3-wt) and mutant form of the virus expressing
NSP3 fused to FLAG-tagged (3X FL) UnaG fluorescent protein (pT7/SA11-NSP3-fUnaG). The NSP3 +RNA
produced from pT7/SA11-NSP3-wt is 1.1 kb, while the modified NSP3 +RNA produced is 1.6 kb
254 Chantal A. Agbemabiese et al.

Fig. 2 Analysis of recombinant SA11 rotaviruses. (a) Electrophoretic profiles of the genomes of wild-type
recombinant SA11 rotavirus (rSA11-wt) and rSA11 with a modified segment 7 (NSP3) RNA expressing FLAG-
tagged UnaG (rSA11-NSP3-fUnaG). The RNAs were recovered by TRIzol extraction, analyzed by electrophore-
sis on a 10% polyacrylamide gel, and detected by ethidium–bromide staining. The 11 genome segments are
numbered with the position of the segment 7 RNAs indicated with an arrowhead. (b) Detection of UnaG
fluorescence by rSA11-NSP3-fUnaG-infected cells at 8 h post-infection using a Zoe fluorescent digital cell
imaging system (Bio-Rad), at 20× magnification (Scale bar = 100 μm). (c) Plaques produced on MA104 cell
monolayers (4 days post-infection) by rSA11-wt and rSA11-NSP3-fUnaG viruses and detected by neutral red
 Rotavirus Reverse Genetics 255

4. Prepare DMEM incomplete medium by combining 500 mL of

Dulbecco’s modified Eagle’s MEM containing 4.5 g/L glu-
cose with 5 mL of 100X L-glutamine and 5 mL of 100X
penicillin–streptomycin solution.
5. Prepare DMEM complete medium by adding 25 mL of FBS
(Gibco) to 500 mL of DMEM incomplete medium.
6. Prepare SMEM incomplete medium by combining 500 mL of
Joklik’s modified Eagles’ MEM (Lonza), 50 mL of TPB, 5 mL
of 100X L-glutamine, 10 mL of 100X NEAA, and 5 mL of
100X penicillin–streptomycin solution.
7. Prepare 2X EMEM incomplete medium by combining 500 mL
Eagle’s MEM (phenol red-free, Quality Biological) with 5 mL
of 100X L-glutamine and 5 mL of 100X penicillin–streptomy-
cin solution.
8. Opti-MEM I (Invitrogen)-reduced serum medium.

2.3 Reverse Genetics 1. The 11 pT7 plasmids expressing rotavirus SA11 (+)RNA
Plasmids (pT7/VP1SA11, pT7/VP2SA11, pT7/VP3SA11,
pT7/VP4SA11, pT7/VP6SA11, pT7/VP7SA11,
pT7/NSP1SA11, pT7/NSP2SA11, pT7/NSP3SA11,
pT7/NSP4SA11, and pT7/NSP5SA11) can be obtained
from Addgene [19] or commercially synthesized (see Note 1).
{https://www.addgene.org/browse/article/25158/}
2. The plasmid pCMV/NP868R expressing the African swine
fever virus (ASFV) capping enzyme (GenBank MH212166)
can be synthesized commercially or obtained from the
authors [20].
3. Modified pT7/NSP3SA11 plasmids that express NSP3 fused
to FLAG-tagged UnaG (MK868472), or NSP3 and FLAG-
tagged UnaG (MK851042), as separate protein products, can
be synthesized commercially or obtained from the authors.
Modified pT7/NSP3SA11 plasmids expressing NSP3 and
other fluorescent reporter proteins (e.g., mRUBY,
mCHERRY, etc.), as separate products, are described by Philip
et al. [22, 23]. Sequences of modified pT7 plasmids should be
confirmed prior to use in reverse genetics experiments.
4. Plasmids are prepared using endo-free Qiagen or equivalent
plasmid purification kits, diluted to final concentrations of
1 mg/mL in ultrapure water, and stored at -20 °C in 5 μL
aliquots in 0.2 mL microfuge tubes.
ä

Fig. 2 (continued) staining. (d) Detection of VP6 and NSP3 in rSA11-wt-infected MA104 cells and VP6 and the
fused product NSP3-3XFLAG-UnaG in rSA11-NSP3-fUnaG-infected MA104 cells by immunoblot analysis with
anti-VP6, anti-NSP3, and anti-FLAG antibodies [21]. The positions of molecular weight markers (MWM) are
indicated
256 Chantal A. Agbemabiese et al.

2.4 Other Reagents 1. TransIT-LT1 (Mirus) transfection reagent.

2. Trypsin (0.05%) – EDTA (0.1%) solution (Quality Biological)
used for subculturing BHK-T7 and MA104 cells.
3. Porcine trypsin, type IX-S (Sigma-Aldrich), for activating rota-
virus. One mg per mL stocks prepared in 1X phosphate-
buffered saline (PBS) and stored at -20 °C. Because trypsin
loses activity during cycles of freeze–thaw, trypsin stocks should
be aliquoted into small portions (10 μL per 0.2 μL microfuge
tube) and individual tubes not used more than twice.
4. Neutral red solution (0.33%) (Sigma-Aldrich) for detecting
rotavirus plaques.

3 Methods

3.1 Maintaining Cell 1. BHK-T7 cells are propagated in T75 flasks containing 15 mL
Lines of GMEM complete media. Geneticin is included in the cell
culture media with every other passage. To passage BHK-T7
cells, monolayers are rinsed with PBS, disrupted using trypsin–
EDTA solution, and resuspended in 5 mL of complete
GMEM/FBS. Four drops of resuspended cells are placed in
fresh T75 flasks containing 15 mL of complete GMEM/FBS.
The cells typically reach confluency in 3 days. BHK-T7 cells
beyond passage 20 are not used in reverse genetics experiments
(see Note 2).
2. MA104 cells are propagated in T75 flasks containing 15 mL of
DMEM complete medium. To passage MA104 cells, mono-
layers are rinsed with PBS, disrupted using trypsin–EDTA
solution, and resuspended in 5 mL of DMEM complete
medium. One mL of resuspended MA104 cells is added to a
T75 flask containing 15 mol of DMEM complete medium. The
newly seeded cells will reach confluency in 4–5 days. MA104
cells beyond passage 25 are not used in reverse genetics
experiments.
3. Cell lines are grown in humidified 37 °C incubators in the
presence of 5% CO2 (see Note 3).

3.2 Generation of The generation and characterization of recombinant rotavirus

Recombinant strains must be handled under biosafety level 2 (BSL-2) conditions.
Rotavirus Such experiments will also require prior approval by the Institu-
tional Biosafety Committee/Institutional Review Board.
1. One day prior to transfection, seed 5.0 × 105 per well of
BHK-T7 cells in GMEM complete medium (Geneticin-free)
(2 mL per well) into 12-well cell culture plates. The following
day, the cells should have reached 80–90% confluency by the
time of transfection.
 Rotavirus Reverse Genetics 257

2. Prepare plasmid transfection mixtures in 0.5 mL microfuge

tubes by combining 0.8 μL each of SA11 pT7 plasmid stocks
(1 mg/mL) for VP1, VP2, VP3, VP4, VP6, VP7, NSP1,
NSP3, and NSP4, 2.4 μL each of the SA11 pT7 plasmid stocks
(1 mg/mL) for NSP2 and NSP5, and 800 ng of pCMV/
NP868R. Gently mix the plasmid mixtures by pipetting up
and down two to three times, followed by a brief low-spin
centrifugation to collect the contents in the bottom of the
tubes (see Note 4).
3. Add 110 μL of prewarmed (37 °C) Opti-MEM I medium to
each plasmid mixture, and mix tube contents by gently pipet-
ting up and down three to four times. Add 25 μL of Mirus
Transit-LT1 transfection reagent to plasmid mixtures, gently
mix tube contents, and incubate tubes for 20 min at room
temperature (see Note 5).
4. While plasmid mixtures are incubating, remove media from
BHK-T7 cell monolayers. Wash the monolayers twice with
1 mL per well prewarmed (37 °C) GMEM incomplete media.
Place 1 mL of SMEM incomplete media on the monolayers.
5. Transfect the BHK-T7 monolayers by adding the plasmid mix-
tures to SMEM incomplete media overlaying the cells in a slow,
dropwise manner. Incubate the plates at 37 °C for 3 days in a
humidified CO2 incubator.
6. Three days after transfection, overseed the BHK-T7 cell mono-
layers with 2 × 105 of suspended MA104 cells in 250 μL serum-
free DMEM complete media. Gently rock the plate to evenly
distribute the MA104 cells in the wells.
7. Bring the concentration of trypsin in the medium to ~0.5 μg/
mL by adding the necessary amount of 1 mg/mL porcine
trypsin stock. Incubate the cells for an additional 3 days at
37 °C in a CO2 incubator (see Note 6).
8. To recover and amplify recombinant viruses in the transfected
BHK-T7/MA104 cell mixtures, subject the 12-well plates to
three rounds of freeze-thawing. Afterwards, transfer the cell
lysates to 1.5 mL microfuge tubes, and clarify the lysates by
centrifuging the tubes at 500× g for 10 min at 4 °C. Transfer
the supernatant (clarified lysate) to a new tube.
9. Trypsin-activate rotavirus contained in 300 μL portions of the
clarified lysates by adding 3 μL of a 1 mg/mL stock of porcine
trypsin and incubating for 1 h at 37 °C.
10. Prepare MA104 monolayers in six-well plates for infection by
rinsing twice with 2 mL per well prewarmed DMEM incom-
plete media. Overlay the cells with 300 μL of the trypsin-
treated clarified lysate. Allow for virus in lysates to adsorb to
MA104 cells by incubating plates at 37 °C in a humidified 5%
CO2 incubator for 1 h, gently rocking the plates every 15 min.
258 Chantal A. Agbemabiese et al.

11. After the 1-h virus adsorption, add 1.7 mL of prewarmed

DMEM incomplete medium containing 0.5 μg/mL porcine
trypsin to wells of the plate. Incubate plates for 7 days or until
complete CPE is observed.
12. Subject the six-well plates to three cycles of freeze-thawing.
Transfer the MA104 cell lysates to 2 mL microfuge tubes, and
clarify the lysates by centrifugation at 500× g for 10 min at 4 °
C.
13. Transfer clarified MA104 cell lysates into new 2.0 mL micro-
fuge tubes, and store at -20 °C.

3.3 Detection of 1. To recover viral RNA from clarified MA104 cell lysates, com-
Recombinant bine 600 μL volumes of lysates with 400 μL of TRIzol reagent
Rotavirus RNA by Gel (Invitrogen) in 1.5 mL microfuge tubes, vortex the mixtures
Electrophoresis for 30 s, and then incubate at room temperature for 5 min.
2. Add 200 μL of chloroform to the TRIzol-treated mixtures,
vortex for 30 s, and then incubate for 3 min at room
temperature.
3. Centrifuge mixtures at 12,000× g for 15 min at 4 °C, and
transfer the upper aqueous phase into new microfuge tubes.
Add 2 volumes of cold isopropanol to the aqueous phase, mix
by inverting the tubes six to eight times, and incubate the
mixtures at room temperature for 10 min.
4. Centrifuge mixtures for 15 min at 12,000× g to pellet viral
RNA. Look for a white pellet at the bottom of the microfuge
tube, and carefully discard the supernatant.
5. Rinse RNA pellets by adding 500 μL of 75% ethanol to the
tubes, gently inverting the tubes a single time, followed by
centrifugation at 7500× g for 5 min at 4 °C. Discard the
supernatant, and air-dry the pellet at room temperature for
5–10 min.
6. Thoroughly dissolve the RNA pellet in 20 μL of nuclease-free
water, and store at -20 °C.
7. Mix 10 μL of extracted RNA with 2 μL of 6X DNA loading
buffer (Thermo Scientific). Carefully load the mixture onto
10% pre-cast polyacrylamide minigels (e.g., Invitrogen Novex
XV00100PK20), or hand-cast equivalents, and resolve the
RNA by electrophoresis at constant current (16 mA for one
gel, 32 mA for two gels) in Tris–glycine running buffer for 2 h
(see Note 7).
8. Stain gels by incubating in ultrapure water containing ~1 μg/
mL ethidium bromide for 5-10 min. Detect the rotavirus
genome segments using a UV transilluminator or equivalent
gel imaging system (e.g., BioRad ChemiDoc system) (see
Note 8).
 Rotavirus Reverse Genetics 259

3.4 Plaque Isolation 1. Seed six-well plates with 1.5 × 105 MA104 cells in 2 mL
of Recombinant DMEM complete medium per well. Incubate cells at 37 °C in
Rotaviruses a humidified 5% CO2 incubator until MA104 monolayers have
fully formed (typically 3–4 days).
2. Activate rotavirus in 100 μL portions of clarified cell lysates by
adding 1 μL of 1 mg/mL porcine trypsin stock and incubating
mixtures for 1 h at 37 °C.
3. Prepare tenfold (10-1–10-7) dilution series of trypsin-treated
lysates by mixing 100 uL volumes of lysate samples into 900 μL
of prewarmed DMEM incomplete medium in 1.5 mL micro-
fuge tubes. Vortex to mix serial dilutions at each step before
subsequent transfer. Use fresh pipettor tips to prepare each
dilution.
4. Rinse confluent monolayers in six-well plates twice with 2 mL
of prewarmed 1X PBS. Rinse monolayers one additional time
with 2 mL of prewarmed DMEM incomplete medium, leaving
100 μL of medium in the wells to prevent the cells from drying.
5. Gently add 400 μL of the 10-1–10-7 serially diluted virus
samples to the MA104 monolayers in duplicate starting from
the most dilute to the least dilute. For the negative control, add
400 μL of DMEM incomplete media to wells.
6. Adsorb the virus to the cells by incubating the plates in an
incubator for 1 h, accompanied by gentle rocking of the inoc-
ulum covering the cells every 15 min.
7. While virus is adsorbing, prepare a solution of 1.5% SeaPlaque
agarose (Lonza) by melting in water using a microwave. Place
melted agar in a 56 °C water bath until ready to use. Prepare
the necessary volume of 1:1 agar–EMEM mixture (3 mL per
well) by mixing equal volumes of the melted agarose and pre-
warmed 2X EMEM incomplete medium, for a final concentra-
tion of 0.75% agar. Place agar–EMEM mixture in a 42 °C water
bath until ready to use. Adjust agar–EMEM solution to a final
concentration of 0.5 μg/mL porcine trypsin immediately
before placing on monolayers.
8. Carefully pipet off inoculum from the MA104 monolayers, and
rinse once with 2 mL of prewarmed DMEM. Adjust the dis-
charge setting of a pipet-aid to slow speed, and use it to
carefully overlay the MA104 monolayers with 3 mL of the
agar–EMEM mixture. Briefly and slowly swirl agar–EMEM
mixture over monolayer, and then let the six-well plates rest
at room temperature until agar has solidified.
9. Place the plates in a humidified 37 °C CO2 incubator for
3–4 days, and leave until plaques are visible as whitish/opaque
circular patches on the MA104 monolayer when the plate is
observed using a light box.
260 Chantal A. Agbemabiese et al.

10. Prepare an agarose–EMEM mixture (0.75% final agarose con-

centration) as in step 7 above, and place in a 42 °C water bath.
Supplement the agarose–EMEM mixture with 0.33% neutral-
red solution stock (Sigma-Aldrich) to a final concentration of
50 μg/mL.
11. Slowly overlay the clear solid agarose layer already present on
the MA104 monolayers with 2 mL per well of agarose–EMEM
containing neutral red. Allow the overlay to solidify at room
temperature away from light, as neutral red is light sensitive.
12. Return the plates to the incubator, and leave until plaques can
be observed (typically 4–24 h for SA11 viruses). Detection of
plaques can be aided through the use of a light box. Plaques
will appear as peach-colored circular zones on a brownish-red
monolayer of cells, while the negative control will have a
uniform brownish-red monolayer.
13. Pick clearly defined plaques using sterile disposable glass Pas-
teur pipets by completely inserting the tip through the agarose
layer until touching the cell monolayer.
14. Retrieve the agarose plug, and expel into 500 μL of DMEM
incomplete medium contained in a 1.5 mL microfuge tube.
15. Vortex the sample, and store the mixture at 4 °C for >8 h,
providing time for the virus to diffuse from the agarose plug
into the media.

3.5 Amplification of 1. Trypsin activate 250 μL portions of virus samples eluted from
Plaque Isolated Virus agarose plugs by adjusting to 10 μg/mL porcine trypsin and
incubating for 1 h at 37 °C.
2. Following procedures detailed in Subheading 3.4 above, pre-
pare MA104 monolayers in six-well plates. After rinsing twice
with prewarmed 1X PBS and once with prewarmed DMEM
incomplete media, add activated virus samples to the mono-
layers, and incubate the plates for 1 h, rocking every 15 min.
Afterwards, add 2 mL of DMEM incomplete medium contain-
ing 0.5 μg/mL porcine trypsin to the wells.
3. Monitor monolayers daily for the presence of CPE. Once CPE
has reached 100%, subject the plates to three rounds of freeze-
thawing, and then collect the lysates in 2 mL microfuge tubes.
Clarify the lysates by centrifugation at 500× g for 10 min at 4 °
C.
4. Aliquot the clarified lysates (0.5 mL per 1.5 mL microfuge
tube), and freeze at -20 °C.
5. Viruses contained in the samples can be further amplified in
T75 flasks containing MA104 cell monolayers, prepared as
described in Subheading 3.1.
6. The titer of viruses contained in lysates can be determined by
plaque assay following procedures provided in Subheading 3.4.
 Rotavirus Reverse Genetics 261

7. Viral dsRNA can be recovered from virus samples by TRIzol

extraction and analyzed by RNA gel electrophoresis (see Sub-
heading 3.3). Confirm the RNA sequences of recombinant
rotaviruses using a one-step RT-PCR kit (Invitrogen Super-
script III RT-PCR System) and rotavirus segment-specific pri-
mers. The complete sequence of any modified segment should
be determined (see Note 9).

4 Notes

1. When using commercial sources to produce pT7 plasmids

containing synthetically made rotavirus cDNA inserts, we
have the plasmids made using pUC19 as the backbone vector
that includes an ampicillin resistance gene (Azenta Life
Sciences).
2. In our experience, the single most important factor affecting
efficiency and reproducibility in the generation of recombinant
rotaviruses by reverse genetics is the quality of the BHK-T7
cells. We passage BHK-T7 cells as soon as they reach con-
fluency. If allowed to overgrow, the cells will lose viability and
will perform poorly in reverse genetics experiments. We main-
tain BHK-T7 cells in GMEM media containing TPB and
NEAA, even for just routine maintenance. We freeze down
aliquots of BHK-T7 cells in liquid nitrogen that are known to
support efficient recovery of recombinant viruses.
3. We routinely monitor our cell lines for possible mycoplasma
contamination using a MycoAlert Detection Kit (Lonza). Such
contamination is suspected when the rate of cell division is
decreased, detached cells are seen floating in the medium, or
virus titers do not reach expected levels in cells.
4. In generating common strains of rotavirus by reverse genetics,
it may be important to include genetic markers in one or more
of the viral cDNAs of the pT7 vectors to allow differentiation
between recombinant strains and laboratory strains.
5. In SA11 reverse genetics experiments, we typically add 2.0 μL
of TransIT-LT per μg of plasmid present in transfection mix-
tures. In reverse genetics experiments with other rotavirus
strains or for mutant SA11 strains where the efficiency of
recovery of recombinant viruses is poor, improvement may be
seen if the amount of transfection reagent is increased to
2.5 μL/ug of plasmid. Similarly, efficiency may be improved
by increasing the amount of pT7 plasmids for NSP2 and NSP5
to 5X concentrations (4.0 μg per transfection mixture). Also,
efficiency may be improved by adding twice the amount of the
plasmid (pCMV-NP8686R) for the capping enzyme (1.6 μg
per transfection mixture).
262 Chantal A. Agbemabiese et al.

6. Recombinant SA11 viruses with modified NSP3 genome seg-

ments designed to express foreign protein will typically grow
slower and have smaller plaque sizes than wild-type SA11 virus.
These modified viruses may also grow to lower titers. It is
important to make allowances for these phenotype changes
when generating, isolating, and amplifying recombinant
viruses.
7. In our hands, modified NSP3 genome segments that contain
insertions of foreign sequences greater than 1 kbp in size can be
genetically unstable. During serial passage of such viruses, most
of the foreign sequence insertion is often deleted, producing a
segment 7 RNA that is close to wild-type in its size. RNA gel
electrophoresis and sequencing can be used to monitor for
genetic instability.
8. Instead of staining with ethidium bromide, rotavirus genome
segments can be detected in polyacrylamide gels using a
Bio-Rad silver staining kit. Silver staining is more sensitive
than ethidium bromide and especially useful for analyzing
poorly growing viruses or viruses with genetic instability.
9. To reduce the levels of background cellular RNAs that may
impede visualization of rotavirus dsRNAs on polyacrylamide
gels, treat infected cell lysates with 100 U of RNase T1 stock
(1000 U/μL, Thermo Fisher EN0541) for 15 min at 37 °C
prior to TRIzol extraction.

Acknowledgment

Our appreciation goes out to the many past and present members
of the laboratory for their efforts in developing an efficient rotavi-
rus reverse genetics system. This work was supported by funds
provided by the National Institutes of Health (R21AI144881),
GIVAX, Indiana Clinical and Translational Sciences Institute, and
the Lawrence M. Blatt Endowment.

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 INDEX

0-9, and Symbols Artificial intron-contained ............................................ 208

Asia........................................................................ 207, 231
0.5% turkey or chicken red blood cells ............. 55, 56, 68
Atlantic salmon............................................ 88, 89, 91, 92
2A cleavage site ............................................................. 169
Atlantic salmon kidney (ASK) .............. 89, 91, 92, 95–97
229E .............................................................................. 133
Australia ......................................................................... 231
3’UTR .................................................................. 155, 171
5’UTR .................................................155, 159, 167, 173 B
6-well plates................................................................... 5, 6
78-kDa.................................................................. 103, 104 Baby hamster kidney (BHK-21) cell......... 105, 121–124,
8 + 4-plasmid rescue ....................................................... 76 126, 127, 208, 211, 222, 223, 226, 235, 244
BAC-based................................................... 136, 137, 141
A Bacterial artificial chromosomes (BACs) ........... 133–151,
187, 188, 190, 192–195, 201, 202, 232
Abortion storms ............................................................ 101 Bacteriological ............................................................... 234
Accessory protein .......................................................... 103 Bacteriophage ....................................................... 103, 136
Acetone ........................................................ 178, 235, 244 Bacto agar ...................................................................... 138
Aedes.............................................................................. 207 Bacto Tryptone ............................................................. 138
Africa ....................................................101, 116, 207, 231
Bacto yeast extract......................................................... 138
African green monkey kidney epithelial....................... 121 Bat influenza A viruses..............................................75–85
Agar Noble .................................................................... 191 Bat mumps virus (BatMuV) .....................................16, 24
Agarose ............................................ 89, 91, 94, 209, 210,
β-actin promoter ........................................................... 120
214, 215, 217–223, 233, 234 β-mercaptoethanol ....................................................89, 91
Agarose Gel Electrophoresis......................................... 158 B/Brisbane/60/2008 .................................................... 64
AgeI ............................................................. 158, 160, 165
BioEdit............................................................................. 40
Alexa Fluor 488............................................................. 181 Biosafety level 2............................................................... 41
Alpha-dystroglycan (αDG) ........................................... 118 Biosafety level 3............................................................. 137
Ambisense............................................................. 102, 103 Bi-segmented negative-sense............................... 116, 117
Ambisense coding strategy .................................. 116, 117
Bombali virus .................................................................... 1
American Type Culture Collection (ATCC) ..... 121, 137, Bovine growth hormone (bGH).................................. 141
138, 191 Bovine serum albumin (BSA)...........................41, 78, 80,
Americas ...................................................... 116, 207, 231
83, 84, 122, 124, 126, 128, 139, 140
Ampicillin ...............................................40, 95, 109, 120, BSL-2...................................................................... 41, 211
158, 164–166, 172, 176, 179, 209 BSL-3.................................................................... 137, 150
Annealing.............................................214, 216, 217, 225
BSL-4........................................3, 6, 7, 11, 104, 122, 128
Antarctic alkaline phosphatase...................................... 234 BsmBI ............................................. 80, 84, 178, 179, 181
Antennavirus................................................................. 115 BSR-T7/5 cells .............................. 25, 28, 104–106, 110
Antibiotics ....................................................................... 41
BstE II ........................................................................... 210
Antibody-dependent enhancement (ADE) ................. 231 Buffer RLT ........................................................... 212, 220
Antigenome ...............................................................38, 40 Buffer RPE ........................................................... 212, 220
Antigenomic .................................................102–104, 109 Buffer RW1 ................................................................... 212
ApaI ................................................................................. 80
Buffer TRK ................................................................89, 92
A/Puerto Rico/08/34 (H1N1) ................................... 64 Bundibugyo virus .............................................................. 1
Arenaviridae ................................................................. 115 Bunyavirales .................................................................. 102
Argentinean Pampas ..................................................... 116

Daniel R. Pérez (ed.), Reverse Genetics of RNA Viruses: Methods and Protocols, Methods in Molecular Biology, vol. 2733,
https://doi.org/10.1007/978-1-0716-3533-9,
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer
Nature 2024

265
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
266 Index
C DH5α........................................................... 120, 176, 179
4′,6′-Diamidino-2-2phenylindole
Canada ........................................................................... 116 (DAPI) ............................................. 191, 197, 200
Canine RIE1495 cells ..................................................... 78 Dianlovirus ........................................................................ 1
Cap Analog .................................................................... 235 Direct-zol RNA kit ...................................................77, 80
Capsid (C) ............................................................ 208, 232 DI RNAs........................................................................ 110
Carbenicillin ..............................................................25, 26 Dithiothreitol (DTT).......................................... 5, 7, 158,
CCL-81 ......................................................................... 191 159, 209, 212, 224, 226, 234
cDNA clone................................................ 136, 178, 179, Double-stranded RNA (dsRNAs) .............. 250, 261, 262
188, 189, 192–196, 202, 232 DMEM/F-12....................................................... 139, 149
cDNA synthesis ............................................................. 156 DMEM10%FCS/PS/Q ...................................................... 6, 7
Chemocompetent Escherichia coli.................................. 91 DMSO ........................................................................... 5, 9
Chicken Pol-II β-actin promoter ................................... 76 DNA-dependent RNA polymerase ................................ 18
Chicken polymerase II .................................................. 120 DNA ladder ................................................................... 158
Chicken polymerase II-driven β-actin promoter......... 104 DNA-launch plasmids.......................................... 156, 170
Chloramphenicol............... 138, 142, 188–190, 193, 194 DNA ligation................................................................. 158
Chloroform-isoamyl...................................................... 235 DNA polymerase .................................................. 190, 194
Chronic liver disease ..................................................... 175 dNTP mix ........................................................................ 81
Cirrhosis ........................................................................ 175 dNTPs.......... 5, 7, 9, 158, 159, 209, 212, 213, 216, 221
Cis-elements .................................................................. 175 Dulbecco’s Modified Eagle Medium (DMEM).......5, 25,
Codon optimization ....................................................... 45 28, 31, 41, 44, 78, 80, 105, 121, 122, 124, 127,
Colony PCR .................................................................... 61 138, 139, 147, 149, 159, 164, 167, 169, 177,
Competent cells.................................................... 176, 179 178, 180, 190, 208, 210, 211, 222–224
Complementary DNA (cDNA)........................ 16, 18–21, Dulbecco’s modified minimum essential medium ...... 105
27–29, 37, 38, 40, 41, 43, 76, 80–82, 87, 94, 103,
156, 157, 159–161, 163, 164, 166, 167, E
170–172, 187–189, 192–196, 201, 202, 208,
209, 212, 213, 215–217, 221, 227 E. coli DH5α cells ........................................................... 82
Complementary RNA (cRNA)..................................... 119 E70........................................................................ 209, 215
Congenital Zika syndrome ........................................... 208 Ebola virus (EBOV)................................................1–3, 11
Coronaviridae................................................................ 134 EcoRI...................................................158, 160, 165, 166
Coronavirus ................................................. 133, 134, 185 Effective serum dilution 50 ............................................ 70
Coronavirus disease 19 (COVID-19)................ 133, 134, eGFP .................................................................................. 6
137, 185 Elongation ...........................................214, 216, 217, 225
Cricetid rodents ............................................................ 116 Embryonated chicken eggs ............................... 38, 41–44
Crystal violet........................................191, 201, 210, 223 Encephalomyocarditis virus (EMCV) .......................... 104
Cuevavirus ......................................................................... 1 Enhanced BSL-3 ........................................................... 104
Cytomegalovirus (CMV) ..................................... 141, 156 Enteroviruses (EVs) .....................................155–157, 171
Cytomix solution .......................................................... 235 Envelope (E) ..............................102, 134, 136, 208, 232
Cytopathic effect (CPE) ...................................65, 66, 96, Envelope glycoproteins........................................ 103, 104
97, 109, 111, 124, 136, 143–145, 148, 150 Escherichia coli (E. coli) ..................... 137, 142, 148, 149,
Cytotoxic effects.............................................................. 96 156, 164–166, 176, 208, 209, 215, 217, 232
ESD50 ............................................................................. 70
D Ethanol .......................................... 5, 7, 11, 89, 209, 210,
212, 214, 218, 220, 222, 224, 226, 233–235
DAB Substrate............................................. 139, 191, 199 Ethidium bromide (EtBr).................................... 158, 209
DEAE-Dextran.............................................................. 139 EtOH ............................................................................... 93
Denaturation ............................................... 214, 216, 225 Eukaryotic promoter..................................................... 156
Dengue ................................................................. 231–246 Europe ........................................................................... 116
Dengue hemorrhagic fever (DHF) .............................. 231 European Medicines Agency (EMA) ............................... 2
Dengue shock syndrome (DSS) ................................... 231 EV-D68 ..............................156–161, 163–167, 169–173
Dengue virus (DENV) ........................................ 231–246 Exon-binding sequences (EBS).................................... 217
DENV-1 ........................................................................ 231 Extract-Peptone-Dextrose ............................................ 234
DENV-4 ........................................................................ 231 E.Z.N.A. ............................................................... 209–211
DH10B ............ 137, 138, 142, 148, 149, 189, 193, 201 E.Z.N.A. Total RNA Kit I.............................................. 89
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
Index 267
F HDV-Rbz ..................................................................20, 21
HEK293T constitutively expressing hACE2............... 149
FastAP alkaline phosphatase ........................................... 81 HEK293T-hACE2 ........................................................ 149
Fetal bovine serum (FBS) ........................ 5, 7, 25, 41, 91, Hemagglutination assay (HA)..........................41, 44, 48,
105, 108, 122, 124, 127, 138, 159, 164, 167, 51, 52, 64–68, 71, 72, 75, 79
169, 177, 178, 180, 208, 209, 211, 222–224, 235 Hemagglutinin (HA) ...................................................... 48
Fetal calf serum (FCS) .................................................... 78 Hemorrhagic fever (HF) ..................................... 101, 116
Filoviruses ..................................................................1–4, 9 Henipavirus ...............................................................15, 22
Flaviviridae ................................................. 175, 207, 231 HEp2 cells ....................................................................... 41
Flavivirus.....................................185, 207, 208, 227, 231 Hepacivirus .................................................................... 175
FLUAV-NLuc .................................................... 64, 67, 71 Hepatitis C virus (HCV) ..................................... 175–182
FLUBV-NLuc ..........................55, 56, 64, 65, 67–69, 71 Hepatitis delta virus (HDV)................................. 40, 104,
Fluorescence reporter gene ........................ 157, 159, 167 141, 157, 161, 165, 171
Fluorescent microscope ................................................ 192 Hepatocellular carcinoma ............................................. 175
Focus-forming units (FFU).......................................... 181 HEPES........................................................................... 208
Food and Drug Administration (FDA) ...................2, 116 HiBind RNA column...................................................... 93
Formaldehyde.............................................. 210, 221, 223 High-fidelity DNA polymerase .................................... 177
Formalin ........................................................................ 122 Highly pathogenic avian influenza (HPAI) .............39, 40
Fugene 6 ....................................................................91, 96 Hind III-HF.................................................................. 210
Full-length cDNA ....................................... 192, 232, 239 HKU1 ............................................................................ 133
Full-length vRNA ......................................................... 208 Homologous recombination ...................... 233, 240, 245
Fusion PCR .........................................161, 162, 168, 169 Huh-7 ...............................................................3, 177, 180
Huh7.5.1 .............................................................. 177, 180
G
Human angiotensin converting enzyme 2
G418 .............................................................................. 105 (hACE2) ............................................................ 149
Gastroentiritis................................................................ 249 Human cytomegalovirus (CMV) ................................... 76
Gc.......................................................................... 102–104 Human embryonic kidney.............................................. 78
GC buffer .......................................................................... 9 Human embryonic kidney 293T cells (HEK293T)
Gel Extraction Kit ....................................... 190, 193, 202 cells................................................ 3, 9, 25, 28, 54,
Gene end (GE)................................................... 39, 40, 42 78, 82, 159, 164, 169, 170, 173
Gene start (GS) .........................................................39, 42 Human enteroviruses........................................... 155–173
Geneticin ....................................................................... 105 Human parainfluenza viruses (HPIV) ........15, 16, 22–24
Gentamicin ................................................................91, 92 Human polymerase I promoter (hPol-I).................50, 76
Glasgow’s minimal essential medium (GMEM) ......... 105 Human rhabdomyosarcoma (RD) cells ....................... 159
Glycoprotein (GP) ......................................................2, 11 Human RNA polymerase I............................................. 76
Glycoprotein precursor (GPC)............................ 117–119 Human transferrin receptor I ....................................... 118
Gn ......................................................................... 102–104
Gn/Gc .................................................................. 102, 103 I
GoTaq ................................................................... 209, 216 Immediate-early promoter.............................................. 76
GP complex ................................................................... 117 Immunofluorescence .................................................... 191
GP1 ....................................................................... 117, 119 Immunofluorescent assay (IFA) .........136, 143, 144, 150
GP2 ....................................................................... 117, 118 Immunofocus assay ....................................................... 199
Guillain-Barre syndrome (GBS) .......................... 186, 208 Immunostaining...........................................191, 196–198
GZ01 ............... 208, 209, 211, 213, 215, 217, 223, 227 Infection medium ........................................................... 92
Infectious cDNA clone ........................................ 156, 176
H
Infectious clones ........................................ 143, 156, 157,
H17............................................................................75, 76 169, 187, 188, 192, 193, 201, 208, 209, 215–
H17N10 ............................................................. 76, 77, 79 218, 227, 232, 233, 240
H18..................................................................... 75, 76, 84 Infectious DNA............................................................. 208
H18N11 ............................................................. 76, 77, 79 Infectious Salmon Anemia Virus (ISAV) ................87–90,
H5 ..............................................................................39–44 92, 94, 95, 97, 98
Haemagglutinin .............................................................. 75 Influenza .................................. 39, 41–43, 47–50, 52–55,
Hammerhead ribozyme (Hh-Rbz) ..........................20, 21 57, 64, 65, 70, 73, 75, 78–85, 87, 150, 151, 176
Hartmanivirus .............................................................. 115
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
268 Index
Influenza A (FLUAV) .......................................47, 48, 51, LPF2000...................................................... 122, 124, 127
52, 54–56, 58–61, 63–65, 67–69, 71, 72 Luciferase..................................................... 48, 56, 69, 70
Influenza B (FLUBV) ................................ 47, 48, 51, 52, Lujo virus (LUJV)......................................................... 116
54–56, 58, 59, 61, 63–65, 67, 69, 71, 72 Luria Bertani (LB) .......................................................... 40
Influenza viruses........................................................47, 49 Luria broth (LB) ............................... 109, 120, 138, 142,
In-Fusion Snap Assembly Master Mix ........................... 40 158, 164–166, 172, 176, 179, 209, 215, 217
Interferon (IFN) ............................................................... 2 Lymphocytic choriomeningitis virus (LCMV)............ 116
Intergenic (IG).................................................39, 42, 103 Lysogeny broth (LB) .................... 25–28, 109, 138, 142,
Intergenic region (IGR) ............ 102, 103, 116–119, 122 158, 164–166, 172, 176, 179, 209, 215, 217
Internal ribosomal entry site (IRES) ................. 104, 106,
109, 155, 175 M
Internal transcribed spacer region 1 (ITS-1) ..........88, 89
Madagascar .................................................................... 101
iProof DNA polymerase ........................................ 5, 9, 12 Madin-Darby canine kidney ........................................... 54
IPTG ..................................................................... 209, 215 Madin-Darby canine kidney cells II ............................... 78
IRES-vL ................................................................ 104, 106
Mammarenavirus .........................................115–120, 128
IRES-vN ............................................................... 104, 106 Mammarenaviruses .............................115, 116, 118, 119
ISAV901_09 ....................................................... 91, 92, 94 Marburg virus.................................................................... 1
ITS-1................................................................................ 88
Matrix ............................................................................ 117
Max-Efficiency Stbl2 competent cells ......................25, 27
J
Maxiprep........................................................................ 120
Japan .............................................................................. 116 Mayinga ............................................................................. 9
jetPRIME ............................................................. 159, 164 MDCK HLA-DR cells .................................................... 84
JFH-1 strain .................................................................. 176 MDCK II............................................................ 78, 80, 83
Josiah strain ................................................................... 120 Measles virus (MeV) ................................... 15, 18, 20, 22
Junin virus (JUNV)....................................................... 116 Media without amino acids .......................................... 234
MEGAscript T7 Transcription ..................................... 235
K MEM Amino acids solution ......................................... 105
Kas I ...................................................................... 210, 215 Membrane ............................................................ 134, 136
Kpn I-HF.............................................................. 210, 215 Měnglà virus ...................................................................... 1
Mesogenic........................................................................ 41
L Methanol ....................................................................... 178
MgCl2 ............................................................................ 5, 9
L-15 ...........................................................................91, 92 M7GpppG analogue ..................................................... 210
LaSota ........................................................................40–42 Microneutralization assays ........................................48, 71
Lassa fever (LF) ............................................................. 116 Middle East respiratory syndrome coronavirus
Lassa–lymphocytic choriomeningitis serocomplex ..... 116 (MERS-CoV)............................................ 133, 134
Lassa virus (LASV) .............................................. 116–118, Minimum essential media (MEM)...................... 235, 244
120–122, 124, 126–128 Minireplicon ..............................................................16, 29
Lassa virus rescue systems............................................. 122 Mirus TransIT-LT1...................................................25, 41
Lassa virus reverse genetics........................................... 120 Mitochondrial rRNA intron 2...................................... 217
LA Taq DNA polymerase ................................... 209, 210, MluI ...........................................................................91, 92
213, 216, 222, 227 M-MLV Reverse transcriptase ...................................... 210
Late-onset encephalitis ................................................. 101 Modified Eagle Media .................................................. 105
LB agar ............................................................... 25, 27, 28 Monarch DNA Gel Extraction Kit............................... 178
Leibovitz medium ........................................................... 91 Monoclonal antibody (mAb) .................... 139, 140, 143,
Lentogenic.................................................................40, 41 144, 146, 147, 149
L-glutamine ........................................................ 5, 91, 138 Mononegavirales .............................................1, 15, 37, 39
Ligase-T4......................................................................... 94 Morbillivirus ..............................................................16, 22
Lipofectamine 2000..................................... 82, 122, 136, Mosquito-borne ................................................... 101, 207
139, 191, 210, 211, 222, 223 MP-12...........................................................104, 106–109
Lipofectamine LTX/PLUS ............................................ 25 mPol-I............................................................................ 127
Live-attenuated RVFV .................................................. 104 mPol-I L ...................................................... 120, 122, 126
Live-attenuated vaccines (LAV) .......................... 119, 134 mPol-I S......................................................................... 120
Lloviu virus........................................................................ 1 mRNAs ..................................................3, 4, 76, 102, 103
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
Index 269
Mumps-like virus............................................................. 16 O
Mumps virus (MuV) .................................................15, 24
Murid rodents ............................................................... 116 OC43 ............................................................................. 133
Murine polymerase I transcription terminator .............. 50 Ocular damage .............................................................. 101
Murine terminator sequence .......................................... 76 Old World (OW)......................................... 115, 116, 118
MVA-T7 ............................................................. 38, 41, 42 Oligonucleotide primer ................................................ 158
Oligonucleotides ........................................................... 215
N One Shot TOP10.......................................................... 109
OneStep RT-PCR .............................................. 77, 80, 81
NanoDrop ..................................................................... 192 Open reading frames (ORFs) ...........................76, 78, 80,
NanoDrop 2000 .................................158, 159, 163, 164 102, 104, 134, 136, 155, 208, 232
Nano Glo ......................................................................... 70 Opti-MEM ........................................ 5, 6, 25, 28, 41, 42,
NanoLuc (NLuc) ........48, 49, 54–56, 64, 65, 67, 69, 71 54–56, 64–66, 68, 69, 78, 82, 139, 143, 180
NcoI ...........................................................................91, 92 Opti-MEM I............................................... 105, 106, 121,
NEBuffer ....................................................................... 164 122, 124, 127, 177, 191, 195, 210, 211
Negative-sense................................................................... 2 ORF1a ........................................................................... 134
Neuraminidase (NA)................48, 51, 52, 64, 72, 75, 79 ORF1b.................................................................. 134, 136
Neutral buffered formalin ..................139, 143, 147, 150 Orthomyxoviridae ............................................... 47, 75, 87
Neutralizing antibodies ........................48, 49, 56, 69, 71
Neutral red solution...................................................... 105 P
Newcastle disease virus (NDVs)...............................37–44
New World (NW) ................................................ 116, 118 PA.................................................... 76, 78–80, 91, 92, 95
NheI.....................................................158, 159, 164, 167 PACNR ................................................209, 215, 217, 223
Nidovirales..................................................................... 134 Panhandle structure ...................................................... 103
Nitrogen Base................................................................ 234 Paramyxoviridae .................................... 15–17, 29, 37, 38
NL63 ............................................................................. 133 Paramyxoviruses ............................. 15–23, 25, 27, 29, 39
Noble agar ................................................... 105, 108, 110 Pararubulavirus ........................................................15, 24
Non-coding intergenic region...................................... 116 PB1 ................................................. 76, 78–80, 91, 92, 95
Non-coding regions (NCRs)................................. 76, 102 PB1-NLuc ....................................................................... 64
Nonessential amino acids..................................... 177, 180 PB2 ................................................. 76, 78–80, 91, 92, 95
Non-polyadenylated viral mRNAs ............................... 119 pBeloBAC11 .............................................. 137, 141–143,
Non-structural proteins .............................. 103, 134, 232 149, 151, 187, 188, 192, 193, 201
North America .............................................................. 101 PBS + BSA +P/S.......................................................41, 42
NotI ................................... 158–161, 163–165, 234, 239 pCAGGS...........3, 6, 76, 78, 80–82, 104, 106, 109, 120
Not I-HF ..................................................... 210, 215, 217 pCAGGs-T7CoOpt ..................................................27, 28
NR-50327 ............................................................ 191, 203 pcDNA3.1 .................................................................95, 97
NS1 ....................................................................... 208, 232 pcDNA3.1(+) ............................................. 156, 158, 159,
NS2A .................................................................... 208, 232 161, 163, 166, 167, 169, 170, 172, 173
NS2B .................................................................... 208, 232 pCI-neo .....................................................................91, 95
NS3 .............................................................. 208, 232, 233 PCR...............................................................89–95, 97, 98
NS4A .................................................................... 208, 232 PCR Clean-Up ..................................................... 209, 210
NS4B .................................................................... 208, 232 PCR polymerase ............................................................ 156
NS5 ....................................................................... 208, 232 pDP2002 ............................................... 50–54, 59–61, 64
NSm ...................................................................... 102–104 pEGFP .................................................................. 158, 167
nsp.................................................................................. 134 Pellet Paint ........................................................... 235, 243
Nuclease-free water .................................... 158, 209–211, Penicillin ................. 41, 78, 80, 105, 177, 178, 180, 208
213, 216, 218, 220, 222 Penicillin/streptomycin (PS)............................5, 77, 105,
Nucleobond Xtra Midiprep .......................................... 109 122, 124, 127, 138
Nucleocapsid ..................... 102, 104, 134, 136, 139, 140 Peroxidase (HRP) ......................................................... 139
Nucleoprotein (NP)................ 2, 4, 6, 17, 37–39, 41–43, Peroxidase mouse IgG .................................................. 139
76, 78–80, 84, 91, 92, 95, 117–122, 127, 128 PGEM-Teasy ............................................... 209, 215, 217
Nunc Lab-Tek Chamber Slide ..................................... 235 Phage T7 ......................................................................... 38
Phenuiviridae ................................................................ 102
pHH21 ................................................176, 178, 179, 181
Phlebovirus ..................................................................... 102
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
270 Index
Phosphate buffered saline (PBS)......................41, 42, 91, Q
92, 96, 97, 122, 124, 126, 128, 139, 140, 143,
144, 146, 147, 159, 177, 181, 209, 211, 224, Q5 high fidelity DNA polymerase ...................... 178, 210
226, 227 Q5 Hot Start High-Fidelity Master Mix ... 160, 161, 169
Phosphoprotein............................................................... 17 QIAamp Viral RNA Mini Kit (QIAGEN)..................5, 7,
pHW2000........................................................... 76, 80–82 158, 159, 233
Picornaviridae ............................................................... 155 QIAGEN Plasmid midi ................................................ 234
Plaque assays...........................................30, 97, 107, 109, QIAGEN Plasmid Plus Midi Kit......................... 158, 166
110, 128, 136, 137, 139, 143, 144, 146–150, QIAprep Spin Miniprep Kit ........ 26, 158, 164, 165, 234
164, 167, 169, 191, 196–200, 222, 260 QIAquick Gel Extraction kit .....158, 161, 169, 233, 234
Plasmid-based................................................................ 119 QIAquick PCR purification kit .................. 158, 164, 233
Plasmid-based reverse genetics..................................... 120
R
Plasmid DNA ................................................................ 218
Plasmid Midi Kit .................................................. 190, 194 Rabbit β-globin ............................................................... 89
Platinum Pfx DNA Polymerase ...................................... 91 Rabbit β-globin polyadenylation.................................. 120
Platinum SuperFi DNA Polymerase ............................ 235 Rabbit β-globin polyadenylation signal ......................... 77
Platinum Taq High Fidelity DNA polymerase... 233, 236 Rabies virus (RV) ............................................................ 37
Pneumoviridae................................................................. 18 Receptor-destroying enzyme.......................................... 69
Pol I ..............................................................176–179, 181 Recombinant ISA virus (rISAV)..................................... 88
Pol I-T ......................................................... 176, 178, 179 Recombinant Newcastle Disease Virus....................37–45
Poly (A) tail ................................................................... 155 Recombinant Rift Valley fever virus............................. 102
Polyadenylation ............................................................. 141 Recombinant virus ......................... 4, 142, 146, 250, 253
Polyadenylation signal .................................................... 76 Red blood cells at 50%.................................................... 69
Positive-sense........................................................ 208, 232 Reed-Muench method.................................................... 68
Positive-sense RNA ....................................................... 155 Regular growth medium ..........................................95, 97
pProT7......................................................... 104, 106, 109 Rhabdomyosarcoma (RD).................................. 157, 159,
Pre-denaturation .................................................. 214, 216 164, 167, 169, 171, 173
Pre-membrane (prM).................................................... 232 Reporter genes ................................................................ 48
Premembrane/membrane ............................................ 208 Reptarenavirus .............................................................. 115
PrM/M.......................................................................... 208 Respiratory syncytial virus (RSV).............................16, 18
Promoters ............................................. 16–21, 24, 29, 37, Reston virus....................................................................... 1
38, 40, 48, 50, 76, 87–89, 103, 118, 120–122, Restriction endonuclease ...........158, 163, 169, 177, 210
126, 127, 136, 141, 156, 176, 177, 179, 187, Reverse genetics .............................................. 1–4, 16–18,
188, 192, 232, 240, 251, 252 20, 21, 25, 37, 39, 48–50, 54, 59, 61, 64, 70, 71,
Propanol ............................................................... 233, 234 82, 87, 88, 92, 103, 104, 106–107, 110,
Proprotein-convertase (PC) ......................................... 117 119–121, 127, 128, 134, 136, 137, 141, 151,
Protein expression vector ............................................... 95 175–182, 187, 201, 207–227, 231–246, 249–262
P-S4ter-1 ....................................................................... 216 Reverse transcriptase ................................... 158, 177, 178
P-S4TER-2 .................................................................... 216 Reverse transcriptase and polymerase chain reaction
pSS-URG ...............................................89, 92, 94, 95, 97 (RT-PCR) ............................................................ 80
PSVJS01 ..................................... 234, 239–241, 243, 245 Reverse transcription.......................................80, 81, 156,
pT7............................................................... 104, 106, 109 158, 159, 164, 166, 167, 170
pTriex-3 .....................................................................91, 95 Ribavirin ........................................................................ 116
pUC57............................................................................. 94 RiboMax ........................................................................ 210
PureLink HiPure Plasmid Filter Maxiprep ......... 210, 218 Ribonuclease inhibitor ......................................... 158, 159
PureLink HiPure Plasmid Midiprep Kit ........................ 26 Ribonucleoprotein ............................................... 2, 4, 102
Pure Yield TM Plasmid Miniprep system ................91, 95 Ribonucleoprotein complex (RNP).................... 2–4, 102
Pylaiella littoralis .......................................................... 217 Ribosomal frameshift .................................................... 134
Pyrobest high fidelity DNA polymerase ...................... 209 Ribozyme........................................ 40, 95, 104, 141, 208
P-Z-F4 ........................................................................... 221 RIE1495 ...................................................... 78, 80, 83, 84
P-Z-R14B .................................................... 212, 213, 227 Rift Valley Fever (RVFV) .................... 101–104, 106–111
P-Z-R4........................................................................... 221 rLASV .................................................. 120–122, 124–127
P-ZSK-1......................................................................... 215 RNA ........................................93, 95, 208–212, 220–226
P-ZSK-2......................................................................... 215
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
Index 271
RNA-dependent RNA polymerase SP6 ................................................................................. 156
(RdRp) ................................................76, 102, 116 Spike...................................................................... 134, 136
RNA genome ................................. 2, 116, 117, 175, 232 SSIV Buffer.................................................................... 5, 7
RNA mPol-I /Pol-II .................................................... 120 SSP/GP1/GP2 .................................................... 117, 119
RNA polymerase ......................................... 38, 40, 41, 43 Stable signal peptide (SSP) .................................. 117, 119
RNA polymerase I........... 48, 87–89, 103, 120, 175–182 STBL3..................................................156, 158, 165, 172
RNA polymerase I-mediated transcription......... 175–182 Stellar ............................................................................... 40
RNA polymerase I terminator........................................ 48 Stellar competent cells ..............................................25, 27
RNA polymerase I/II system ....................................... 103 Stop codon ................................................................40, 42
RNA polymerase II ............... 18, 24, 38, 48, 76, 87, 187 Streptomycin .......... 41, 78, 80, 105, 177, 178, 180, 208
RNase free H2O ................................................... 212, 221 Subgenomic mRNAs .................................................... 134
RNase-free water .......................................................93, 94 Sub-Saharan Africa ........................................................ 101
RNase H ............................................................... 5, 7, 210 Subtilisin kexin isozyme-1/site-1 protease ................. 117
RNase inhibitor .................................................... 212, 221 Sudan virus ........................................................................ 1
RNA transcription promoter ........................................ 156 Super optimal broth with catabolite repression ............ 25
RNaseOUT ................................................................... 5, 7 Supernatant ...............................................................96, 97
RNAses ............................................................................ 80 SuperScript III one-step RT-PCR system .......... 233, 236
RNeasy Mini Kit................................................... 209, 210 Superscript III reverse transcriptase .................... 209, 212
rNTPs ............................................................................ 243 SuperScript IV ................................................................... 7
Rotavirus..................................... 249–257, 259, 261, 262 SuperScript IV First-Strand Synthesis System ............. 178
rSARS-CoV-2 .....................136–138, 141–146, 148–151 Superscript IV Reverse Transcriptase ............................... 5
Rule of six ..................................................................38, 42 SuperScript™ III One-Step RT-PCR ............................ 93
Ruminants ..................................................................... 101 Synthetic drop-out medium ........................234, 240–242

S T
SacII ............................................40, 42, 43, 45, 210, 217 Tacaribe serocomplex.................................................... 116
Saccharomyces cerevisiae........................................ 234, 239 TAE buffer........158, 161, 171, 209, 210, 214, 218–222
SalI ................................................................................... 80 Taı̈ Forest virus.................................................................. 1
Salmo salar ...................................................................... 88 Termination ................................................................... 141
Salmon cells ...............................................................91, 95 Terminator.........................................................21, 76, 89,
SapI ..................................................................... 89, 94, 98 120–122, 167, 170, 176, 179
SARS-CoV ........ 133–138, 141–144, 146, 147, 149–151 Terrific broth (TB) ....................................................26, 28
Saudi Arabia................................................................... 101 T4 DNA ligase ............................................. 81, 158, 164,
SCE .............................................................. 234, 241, 242 165, 177, 179, 190, 193, 202, 209, 215
SDS ....................................................................... 210, 220 TMPRSS2...................................................................... 149
Segmented RNA virus .................................................. 102 T150 tissue culture flasks.................................................. 5
Self-cleaving ribozyme .................................................... 38 TOP10 cells...........................................61, 209, 215, 217
Self-splicing group II intron................................ 207–227 TPCK-treated trypsin ........................................ 77, 80, 83
Sendai virus (SeV) .............................................. 16, 18, 23 TPCK-trypsin ................................. 54–56, 65, 66, 68, 69
Severe acute respiratory syndrome coronavirus 2 Transcription promoter .................................................. 88
(SARS-CoV-2)...................................40, 133–138, Transfection medium ...................................................... 91
141–144, 146, 149–151, 185 Transfection mixture....................................................... 96
Sexual intercourse ......................................................... 207 Transfection of cDNAs ........................................ 103, 250
Shuttle vector ................................................................ 234 Transfections .............................................. 5–7, 9, 16, 25,
Sialic acid ......................................................................... 76 28, 29, 41, 42, 44, 49, 50, 54, 64, 65, 67, 72, 82,
Signal peptidases ........................................................... 117 83, 91, 92, 95, 96, 98, 105–108, 110, 121–124,
Single-stranded.......................................2, 102, 116, 117, 126, 127, 136, 138, 139, 142, 143, 149, 156,
155, 156, 175, 208, 232, 246 157, 159, 164, 173, 176, 178–181, 191,
SKI-1/S1P..................................................................... 117 195–197, 202, 210, 222, 223, 226, 232, 235,
SmaI ...........................................................................91, 92 244, 246, 250, 251, 256, 257, 261
SnapGene Viewer ............................................................ 40 Transferrin receptor I (Tfr1) ........................................ 118
SOC ...... 25, 27, 40, 138, 142, 158, 164, 165, 189, 193 TransIT-LT1..................5, 6, 9, 105, 106, 110, 178, 180
Sodium acetate ..................................................... 235, 243 Transmembrane serine protease 2................................ 149
Sodium bicarbonate ...................................................... 105 Triton-X100 .................................................................. 139
 REVERSE GENETICS OF RNA VIRUSES: METHODS AND PROTOCOLS
272 Index
TRIzol...............................................................77, 80, 122 155–159, 167, 170, 171, 175, 177, 178, 188,
TRP1.............................................................................. 234 208, 211, 212, 220, 223, 232, 233, 236, 257
Trp1-Δ1yeast strains ...................................................... 234 Viral RNA-dependent RNA polymerase
Trypsin ........................................................................... 209 (vRdRp) ................................................16, 19, 116
Trypsin-EDTA....................... 25, 29, 123, 138, 178, 235 Virus amplification ............................................... 105, 111
Tryptone ........................................................................ 158 Virus rescues.........................................11, 25, 28, 65, 72,
T7......................................................................3, 4, 6, 156 88, 103, 105, 109–111, 140, 201, 244
T7 bacteriophage ............................................................ 18 Virus vectored vaccine .................................................... 39
T7 polymerase .................................................... 18–20, 25 Vision loss...................................................................... 101
T7 polymerase-driven rescue system............................ 103 VP1 ................................................................................ 155
T7 promoter.............................38, 41, 43, 103, 104, 136 VP2 ................................................................................ 155
T7 RNA polymerase .................... 3, 4, 38, 103, 105, 110 VP24 .................................................................................. 2
T7 terminator ......................................................... 40, 104 VP3 ................................................................................ 155
Type-I interferon (IFN-I) ............................................ 117 VP30 ..........................................................................2, 4, 6
Typtose phosphate broth (TPB) ......................... 105, 108 VP35 ..........................................................................2, 4, 6
VP4 ....................................................................... 155, 167
U VPg ................................................................................ 155
Uganda .......................................................................... 207
W
UltraPure phenol-chloroform-isoamyl
alcohol....................................................... 235, 243 WA-1 strain.................................136, 137, 141, 146, 150
UltraPure Salmon Sperm DNA Solution ........... 234, 240 Western Africa ............................................................... 116
United States ................................................................. 116 Wizard Plus SV Minipreps............................................ 209
Untranslated regions (UTRs)....117, 118, 175, 208, 232 Wizard SV Gel............................209, 210, 214, 215, 218
USA................................................................................ 102 Wizard SV Gel and PCR Clean-Up System ..... 89, 91, 94
USA-WA1/2020........................................................... 136
X
V
XbaI ...........................................................................91, 92
Variants of concern (VoC) ................................... 134, 136 X-gal...................................................................... 209, 215
Variants of interest (VoI) ..................................... 134, 136 XhoI ....................................... 91, 92, 210, 215, 218, 219
Vectastain ABC Kit..............................139, 147, 191, 198
Velogenic ......................................................................... 41 Y
Vero. 121, 123, 124, 126–128, 187, 191, 195–200, 202 Yeast artificial chromosomes (YACs) ........................... 232
Vero CCL81 cells ......................................................25, 29
Yeast-E. coli shuttle vectors .......................................... 232
Vero E6 (African green monkey kidney epithelial) cells 3, Yeast extract................................................................... 158
9, 104, 105, 107–109, 111, 136, 138, 142–144, Yeast Extract-Peptone-Dextrose (YPD) .... 234, 239, 240
146, 148–150
Yeast nitrogen base (YNB) ..........................234, 240–242
Vertical transmission ..................................................... 207 Yeasts.................................................. 138, 158, 189, 209,
Vesicular stomatitis virus................................................... 2 232–234, 239–242, 245, 246
vG.......................................................................... 104, 106 Yemen ............................................................................ 101
Viral cDNAs .................................................................... 95 YPH252....................................................... 234, 239, 245
Viral-like particles (VLPs)............................................... 16
Viral nucleoprotein ....................................................... 117 Z
Viral polymerase L ............................................................ 2
Viral protein 30 ................................................................. 2 Zero Blunt TOPO PCR Cloning ................................ 178
Viral replication complex ................................... 17, 18, 20 ZH501........................................................................... 104
Viral ribonucleoprotein complex ZH548........................................................................... 104
(vRNP)................................................76, 117, 118 Zika forest...................................................................... 207
Viral ribonucleoproteins ............................................... 117 Zika virus (ZIKV) ............................................... 185–189,
Viral RNA (vRNA)............2–5, 7, 18–20, 29, 44, 48–50, 191–197, 199–202, 207–227
57, 58, 64, 71, 72, 76, 80, 89, 92, 94–96, 98, Zymoclean .................................................................77, 81
102–104, 110, 116–122, 127, 134, 136, Zymolyase.................................................... 234, 241, 242
ZymoPURE...............................................................77, 82