Viruses

12 pages, 1879 KiB

Open AccessArticle

Characterization of the Immune Response of MERS-CoV Vaccine Candidates Derived from Two Different Vectors in Mice

by Entao Li, Feihu Yan, Pei Huang, Hang Chi, Shengnan Xu, Guohua Li, Chuanyu Liu, Na Feng, Hualei Wang, Yongkun Zhao, Songtao Yang and Xianzhu Xia

Viruses 2020, 12(1), 125; https://doi.org/10.3390/v12010125 - 20 Jan 2020

Cited by 30 | Viewed by 7468

Abstract

Middle East respiratory syndrome (MERS) is an acute, high-mortality-rate, severe infectious disease caused by an emerging MERS coronavirus (MERS-CoV) that causes severe respiratory diseases. The continuous spread and great pandemic potential of MERS-CoV make it necessarily important to develop effective vaccines. We previously [...] Read more.

Middle East respiratory syndrome (MERS) is an acute, high-mortality-rate, severe infectious disease caused by an emerging MERS coronavirus (MERS-CoV) that causes severe respiratory diseases. The continuous spread and great pandemic potential of MERS-CoV make it necessarily important to develop effective vaccines. We previously demonstrated that the application of Gram-positive enhancer matrix (GEM) particles as a bacterial vector displaying the MERS-CoV receptor-binding domain (RBD) is a very promising MERS vaccine candidate that is capable of producing potential neutralization antibodies. We have also used the rabies virus (RV) as a viral vector to design a recombinant vaccine by expressing the MERS-CoV S1 (spike) protein on the surface of the RV. In this study, we compared the immunological efficacy of the vaccine candidates in BALB/c mice in terms of the levels of humoral and cellular immune responses. The results show that the rabies virus vector-based vaccine can induce remarkably earlier antibody response and higher levels of cellular immunity than the GEM particles vector. However, the GEM particles vector-based vaccine candidate can induce remarkably higher antibody response, even at a very low dose of 1 µg. These results indicate that vaccines constructed using different vaccine vector platforms for the same pathogen have different rates and trends in humoral and cellular immune responses in the same animal model. This discovery not only provides more alternative vaccine development platforms for MERS-CoV vaccine development, but also provides a theoretical basis for our future selection of vaccine vector platforms for other specific pathogens. Full article

(This article belongs to the Section SARS-CoV-2 and COVID-19)

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Graphical abstract

Graphical abstract
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The particle size of recombinant RV expressing MERS-CoV S1 protein (RV/MERS) and MERS bacterium-like particle (MERS BLP). The particle size distribution of RV/MERS (a) and MERS BLP (b). The particle size and polydispersity index (PDI) of RV/MERS and MERS BLP (c). Data was shown as mean ± SD (n = 3). a PDI, polydispersity index from dynamic light scattering (DLS). Full article ">Figure 2
Schematic diagram of immunization and neutralizing antibody responses. (a) The vaccination schedule and characterization of immunologic responses in BALB/c mice. A total of five groups of mice were immunized and detected for virus-neutralizing antibody, IgG subtypes, and cytokines release. Group 1 (G1) was negative controls; group 2 (G2) was vaccinated with inactivated and purified rabies virus (RV)/Middle East respiratory syndrome (MERS); group 3, group 4, and group 5 (G3, G4, G5) were vaccinated with varying doses of the MERS bacterium (Lactococcus lactis)-like particle (BLP). All groups received a second and third identical vaccination boost with a combined adjuvant at 3-week intervals after the primary immunization. (b) Neutralizing activity was detected by using MERS coronavirus (MERS-CoV) pseudovirus, and the data are shown as the mean ± SD from five mice in each group and were analyzed by Student’s t-test (* p < 0.05, *** p < 0.001, **** p < 0.0001, n = 5). Full article ">Figure 3
MERS-CoV receptor-binding domain (RBD)-specific IgG antibodies. Sera from 2 weeks after the last immunization were tested for MERS-CoV RBD-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA). The data are shown as the mean ± SD from five mice in each group and were analyzed by Student’s t-test (* p < 0.05, n = 5). (a) The total IgG antibodies specific to MERS-CoV RBD protein were assessed by ELISA. The level of the MERS-CoV RBD-specific IgG (b) antibodies was determined by ELISA and is shown as end-point dilution titers. Full article ">Figure 4
MERS-CoV RBD-specific antibody subtypes response. Sera from 2 weeks after the last immunization were tested for MERS-CoV RBD-specific IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 antibodies by ELISA. The data are shown as the mean ± SD from five mice in each group and were analyzed by Student’s t-test (* p < 0.05, ** p < 0.01, n = 5). The level of the MERS-CoV RBD-specific IgG1 (a), IgG2a (b), IgG2b (c), IgG2c (d), and IgG3 (e) antibodies were determined by ELISA and are shown as end-point dilution titers. The ratios between IgG1 and IgG2a antibody responses were also calculated (f). Full article ">Figure 5
Immunization induced a significant cytokine response in mice. In order to evaluate antigen-specific T-cell responses, splenocytes were harvested from five mice in each group and were stimulated by MERS-CoV peptide S291 for 24 h. The level of secreted IFN-γ (a), IL-2 (b), and TNF-α (c) in the supernatants was detected by mouse ELISA kits. The data are shown as the mean ± SD and were analyzed by Student’s t-test (* p < 0.05, **** p < 0.0001, n = 5). Full article ">

9 pages, 2939 KiB

Open AccessArticle

Identification of a Novel Papillomavirus Associated with Squamous Cell Carcinoma in a Domestic Cat

by Maura Carrai, Kate Van Brussel, Mang Shi, Ci-Xiu Li, Wei-Shan Chang, John S. Munday, Katja Voss, Alicia McLuckie, David Taylor, Andrew Laws, Edward C. Holmes, Vanessa R. Barrs and Julia A. Beatty

Viruses 2020, 12(1), 124; https://doi.org/10.3390/v12010124 - 20 Jan 2020

Cited by 24 | Viewed by 11062

Abstract

Papillomaviruses infect the skin and mucosal surfaces of diverse animal hosts with consequences ranging from asymptomatic colonization to highly malignant epithelial cancers. Increasing evidence suggests a role for papillomaviruses in the most common cutaneous malignancy of domestic cats, squamous cell carcinoma (SCC). Using [...] Read more.

Papillomaviruses infect the skin and mucosal surfaces of diverse animal hosts with consequences ranging from asymptomatic colonization to highly malignant epithelial cancers. Increasing evidence suggests a role for papillomaviruses in the most common cutaneous malignancy of domestic cats, squamous cell carcinoma (SCC). Using total DNA sequencing we identified a novel feline papillomavirus in a nasal biopsy taken from a cat presenting with both nasal cavity lymphoma and recurrent squamous cell carcinoma affecting the nasal planum. We designate this novel virus as Felis catus papillomavirus 6 (FcaPV6). The complete FcaPV6 7453 bp genome was similar to those of other feline papillomaviruses and phylogenetic analysis revealed that it was most closely related to FcaPV3, although was distinct enough to represent a new viral type. Classification of FcaPV6 in a new genus alongside FcaPVs 3, 4 and 5 is supported. Archived excisional biopsy of the SCC, taken 20 months prior to presentation, was intensely positive on p16 immunostaining. FcaPV6, amplified using virus-specific, but not consensus, PCR, was the only papillomavirus detected in DNA extracted from the SCC. Conversely, renal lymphoma, sampled at necropsy two months after presentation, tested negative on FcaPV6-specific PCR. In sum, using metagenomics we demonstrate the presence of a novel feline papillomavirus in association with cutaneous squamous cell carcinoma. Full article

(This article belongs to the Special Issue Feline Viruses and Viral Diseases)

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Figure 1
A recurrent invasive squamous cell carcinoma on the nasal planum (horizontal arrow) was adjacent the site of a biopsy (vertical arrow) from which high-grade lymphoma of the nasal cavity was diagnosed. Full article ">Figure 2
Felis catus Papillomavirus 6 (FcaPV6) genome configuration using metadata. The predicted ORFs are represented by the coloured inner segments. GC content is displayed in blue, percentage nucleotide polymorphism in orange and read coverage in yellow. Full article ">Figure 3
Evolutionary relationships of FcaPV6 to other mammalian papillomaviruses, including those previously identified in cats (Felis catus). Those papillomaviruses previously identified in cats are marked with animal symbols and in red, along with their respective genera. FcaPV6 is proposed to fall as part of a proposed new genus. All virus names contain their associated GenBank accession numbers. The tree is rooted using six nonmammalian papillomaviruses that have been removed to improve clarity. All horizontal branch lengths are scaled according to the number of amino acid substitutions per site, and the * denotes nodes with SH-like branch support values >0.95. Full article ">Figure 4
Photomicrograph of a nasal planum squamous cell carcinoma that contained FcaPV6 DNA. This biopsy was obtained 20 months prior to the SCC recurrence shown in <a href="#viruses-12-00124-f001" class="html-fig">Figure 1</a>. Intense nuclear and cytoplasmic immunostaining using anti-p16CDKN2A protein (p16) antibodies is visible diffusely within the neoplastic cells. There is minimal immunostaining within adjacent non-neoplastic epidermis (arrows). Hematoxylin counterstain. Full article ">

12 pages, 3441 KiB

Open AccessBrief Report

Multi-Approach Investigation Regarding the West Nile Virus Situation in Hungary, 2018

by Brigitta Zana, Károly Erdélyi, Anna Nagy, Eszter Mezei, Orsolya Nagy, Mária Takács, Tamás Bakonyi, Petra Forgách, Orsolya Korbacska-Kutasi, Orsolya Fehér, Péter Malik, Krisztina Ursu, Péter Kertész, Anett Kepner, Máté Martina, Tamás Süli, Zsófia Lanszki, Gábor Endre Tóth, Anett Kuczmog, Balázs Somogyi, Ferenc Jakab and Gábor Kemenesi Show full author list Hide full author list

Viruses 2020, 12(1), 123; https://doi.org/10.3390/v12010123 - 20 Jan 2020

Cited by 11 | Viewed by 6525

Abstract

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year [...] Read more.

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics. Full article

(This article belongs to the Special Issue West Nile Virus 2019)

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Geographical distribution of West Nile virus (WNV) polymerase chain reaction (PCR)-positive cases at the LAU 2 level and cumulative incidence rates of autochthonous and imported human WNV infections at NUTS 3 level of Hungary, from 2014 through 2017. The total number of human WNV cases between 2014 and 2017: n = 103. Black dots indicate PCR-positive samples containing histidine at amino acid position NS3249. Yellow dots indicate PCR-positive samples containing proline at amino acid position NS3249. LAU 2: Local Administrative Units level 2. NUTS 3: Nomenclature of territorial units for statistics level 3. IR: Incidence rates (number of WNV human infections/100,000 inhabitants). Full article ">Figure 2
Geographical distribution of West Nile virus (WNV) PCR-positive cases at LAU 2 level and cumulative incidence rates of autochthonous human WNV infections at NUTS 3 level of Hungary, 2018. The total number of autochthonous human WNV cases in 2018: n = 215. Black dots indicate PCR-positive samples containing histidine at amino acid position NS3249. Yellow dots indicate PCR-positive samples containing proline at amino acid position NS3249. LAU 2: Local Administrative Units level 2. NUTS 3: Nomenclature of territorial units for statistics level 3. IR: Incidence rates (number of WNV human infections/100,000 inhabitants). Full article ">Figure 3
Geographical distribution of enzootic WNV cases. Color background of each county represents the number of IgM seropositive horses in that specific region from 2018 as annotated on the sidebar. Individual PCR-positive animal cases are indicated with respective pictograms to each case in that specific region. Full article ">Figure 4
Phylogenetic representation of partial NS3 (left side) and NS5 (right side) gene sequences of Hungarian horse and wild bird samples compared to cognate sequences from the region, collected between 2010 and 2018. Progenitor African, Hungarian, and a Serbian WNV lineage 2 strains are highlighted with blue outline, while the two major subclades are indicated with green (Balkanian subclade) and orange (Central/South-West European subgroup) outline. Samples from 2018, Hungary, are highlighted with red color. Note MH021189 as an imported case from Hungary to Belgium [<a href="#B36-viruses-12-00123" class="html-bibr">36</a>]. Full article ">Figure 5
Human-derived, partial WNV NS3 sequences from the 2018 season. The figure represents similarity scores of all available human-derived WNV partial NS3 sequences from 2018. The two dominant genetic subclades are highlighted on the dendrogram with green and peach color. Full article ">Figure 6
Time-calibrated Bayesian maximum clade credibility phylogenetic reconstruction of the evolution of Hungarian wild bird and horse samples. Partial NS5 gene sequences of this study, along with cognate sequences, were included in this dataset. Samples from 2018 are highlighted with red color, while the timeframe of origin is indicated with blue background. Full article ">

9 pages, 2286 KiB

Open AccessCommunication

5,6-Dichloro-2-Phenyl-Benzotriazoles: New Potent Inhibitors of Orthohantavirus

by Giuseppina Sanna, Sandra Piras, Silvia Madeddu, Bernardetta Busonera, Boris Klempa, Paola Corona, Roberta Ibba, Gabriele Murineddu, Antonio Carta and Roberta Loddo

Viruses 2020, 12(1), 122; https://doi.org/10.3390/v12010122 - 20 Jan 2020

Cited by 14 | Viewed by 4808

Abstract

Orthohantaviruses, previously known as hantaviruses (family Hantaviridae, order Bunyavirales), are emerging zoonoses hosted by different rodent and insectivore species. Orthohantaviruses are transmitted by aerosolized excreta (urine, saliva and feces) of their reservoir hosts. When transmitted to humans, they cause hemorrhagic fever with renal [...] Read more.

Orthohantaviruses, previously known as hantaviruses (family Hantaviridae, order Bunyavirales), are emerging zoonoses hosted by different rodent and insectivore species. Orthohantaviruses are transmitted by aerosolized excreta (urine, saliva and feces) of their reservoir hosts. When transmitted to humans, they cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and hantavirus (cardio) pulmonary syndrome (HPS) in the Americas. Clinical studies have shown that early treatments of HFRS patients with ribavirin (RBV) improve prognosis. Nevertheless, there is the need for urgent development of specific antiviral drugs. In the search for new RNA virus inhibitors, we recently identified a series of variously substituted 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazole derivatives active against the human respiratory syncytial virus (HRSV). Interestingly, several 2-phenyl-benzotriazoles resulted in fairly potent inhibitors of the Hantaan virus in a chemiluminescence focus reduction assay (C-FRA) showing an EC₅₀ = 4–5 µM, ten-fold more active than ribavirin. Currently, there are no FDA approved drugs for the treatment of orthohantavirus infections. Antiviral activities and cytotoxicity profiles suggest that 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazoles could be promising candidates for further investigation as a potential treatment of hantaviral diseases. Full article

(This article belongs to the Special Issue Antiviral Agents)

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Graphical abstract

Graphical abstract
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5,6-dichloro-1-phenyl-1H-benzo[d][1,2,3]triazoles (1a-c, e-j) and 5,6-dichloro-2-phenyl-2H-benzo[d][1,2,3]triazoles (2a-n). Full article ">Figure 2
2-phenyl-2H-benzo[d][1,2,3]triazoles (3f, j-n) and 5,6-dimethyl-2-phenyl-2H-benzo[d][1,2,3]triazoles (4f, j-n). Full article ">Figure 3
Anti-hantaviral effect of 2n, 2l, 2j, 2f, 2k, and RBV in vitro. Each symbol shows mean values for three independent determinations + SD. Full article ">Figure 4
Focus reduction assay for 2n and ribavirin.VeroE6 cells were infected with HTNV (m.o.i. 0.05). The infected cultures were treated with 2n (panel A) and RBV (panel B; used as reference) at indicated doses. Ci = untreated control; CC = uninfected control. Antiviral activity was determined by focus reduction assay at day 7 post-infection. Full article ">Figure 5
Yield of infectious Hantaan (HTNV) viruses produced in infected VeroE6 cells treated with selected benzotriazoles (2j, 2l, 2n) (20 µM) and RBV (50 µM). Results are expressed as means ± standard deviations from 3 separate experiments done in triplicate. * indicates the p-value <0.05. Full article ">Scheme 1
Synthesis the amide derivatives (3f, j and 4f, j). Full article ">

19 pages, 1687 KiB

Open AccessReview

Microtubules in Polyomavirus Infection

by Lenka Horníková, Kateřina Bruštíková and Jitka Forstová

Viruses 2020, 12(1), 121; https://doi.org/10.3390/v12010121 - 18 Jan 2020

Cited by 12 | Viewed by 4206

Abstract

Microtubules, part of the cytoskeleton, are indispensable for intracellular movement, cell division, and maintaining cell shape and polarity. In addition, microtubules play an important role in viral infection. In this review, we summarize the role of the microtubules’ network during polyomavirus infection. Polyomaviruses [...] Read more.

Microtubules, part of the cytoskeleton, are indispensable for intracellular movement, cell division, and maintaining cell shape and polarity. In addition, microtubules play an important role in viral infection. In this review, we summarize the role of the microtubules’ network during polyomavirus infection. Polyomaviruses usurp microtubules and their motors to travel via early and late acidic endosomes to the endoplasmic reticulum. As shown for SV40, kinesin-1 and microtubules are engaged in the release of partially disassembled virus from the endoplasmic reticulum to the cytosol, and dynein apparently assists in the further disassembly of virions prior to their translocation to the cell nucleus—the place of their replication. Polyomavirus gene products affect the regulation of microtubule dynamics. Early T antigens destabilize microtubules and cause aberrant mitosis. The role of these activities in tumorigenesis has been documented. However, its importance for productive infection remains elusive. On the other hand, in the late phase of infection, the major capsid protein, VP1, of the mouse polyomavirus, counteracts T-antigen-induced destabilization. It physically binds microtubules and stabilizes them. The interaction results in the G2/M block of the cell cycle and prolonged S phase, which is apparently required for successful completion of the viral replication cycle. Full article

(This article belongs to the Special Issue Regulation and Exploitation of Microtubules by Viruses)

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Figure 1

Figure 1
Mouse polyomavirus moves along microtubules. 3T6 cells expressing tubulin fused with enhanced green fluorescent protein (green) were infected with Alexa Fluor 546-labeled mouse polyomavirus (MPyV) (red). Cells were scanned with ΔT = 2 s. Arrowheads point to MPyV virion. Cells were examined with an Olympus IX81 CellR microscope equipped with an MT20 illumination system. Full article ">Figure 2
Polyomaviruses utilize microtubules for trafficking towards the cell nucleus. Individual polyomaviruses are, after receptor binding, internalized into monopinocytic vesicles derived from lipid rafts, including the caveolae or by clathrin-coated pits (JC virus). The productive pathway leads through early endosomes (EE), late endosomal compartments (LE) to the endoplasmic reticulum (ER). For the transport along microtubules, polyomaviruses use both dynein and kinesin motors (right lower inset). All polyomaviruses exploit microtubules for their appearance in mature late endosomes and further trafficking to the ER. Dynamic actin propels PyV-carrying vesicles to multicaveolar endosomes (CAV1), representing probably non-productive pathways. For some polyomaviruses, the actin-dependent appearance of virions in early endosomes was suggested. Partially disassembled virions are released from the ER to the cytosol, and virions are consequently further disassembled by dynein. Then disassembled virions translocate, with the help of importins, to the nucleus. Right upper inset: From the ER, the virus is released by the action of cytoplasmic and ER chaperons, including J protein B14 and exposed hydrophobic virus, minor-capsid proteins. The kinesin-1 motor, together with hyperacetylated microtubules, push the B12 chaperon to create large exit foci on the ER membrane. Dashed lines represent hypothetic pathways. MPyV—mouse polyomavirus, SV40—simian vacuolating virus 40, BKPyV—BK virus, JCPyV—JC virus, MCPyV—Merkel cell polyomavirus. Full article ">Figure 3
Gene products of polyomaviruses affect microtubule dynamics. Early T antigens affect microtubules indirectly. Small T antigen (ST) and membrane-bound middle T (MT) antigens interact with PP2A phosphatase and alter its redistribution within the cell. The ST antigen is able to replace the regulation unit of PP2A phosphatase and affect its substrate specificity. ST antigen of Merkel cell polyomavirus also binds PP4C phosphatase and SCFFbw7 ubiquitin ligase. As a consequence, microtubule-associated proteins’ (MAPs) functions become deregulated. Mainly, the commutation of dephosphorylated stathmin, which binds tubulin dimers, thus preventing their polymerization, leads to microtubular instability and numeric chromosomal instability. Simian vacuolating virus 40 large T antigen (SV40 LT) disrupts intermediate filaments (vimentin), resulting in microtubule rearrangement. Further, SV40 binds centrosome-binding protein transforming acidic coiled-coil protein 2 (TACC2), and this interaction results in spindle instability. Binding of Bub1 kinase to SV40 LT results in spindle assembly checkpoint failure, resulting in numeric chromosomal instability. In contrast, the late gene product, the VP1 capsid protein of MPyV, binds microtubules directly, causing their stabilization leading to the cell cycle block in G2/M. MPyV—mouse polyomavirus, SV40—simian vacuolating virus 40, MCPyV—Merkel cell polyomavirus. Full article ">

11 pages, 2057 KiB

Open AccessCase Report

Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

by Rene Edgar Condori, Michael Niezgoda, Griselda Lopez, Carmen Acosta Matos, Elinna Diaz Mateo, Crystal Gigante, Claire Hartloge, Altagracia Pereira Filpo, Joseph Haim, Panayampalli Subbian Satheshkumar, Brett Petersen, Ryan Wallace, Victoria Olson and Yu Li

Viruses 2020, 12(1), 120; https://doi.org/10.3390/v12010120 - 18 Jan 2020

Cited by 9 | Viewed by 3923

Abstract

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis [...] Read more.

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing. Full article

(This article belongs to the Special Issue Rabies Virus: Knowledge Gaps and Challenges to Elimination)

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Figure 1
Immunohistochemical (IHC) staining for RABV antigen in fixed brain tissue from the Dominican Republic (DOM) patient (A18-2173). (A) RABV antigen detection at 400× magnification. (B) Neuronal cytoplasmic inclusions at 630× magnification. (C) Negative control brain at 400× magnification. Streptavidin–biotin complex staining method using rabbit antibodies against RABV nucleoprotein, signal development with AEC chromogen (magna red), and Gills hematoxylin counterstain. Full article ">Figure 2
Sequence alignment (115 bp) from LN34 amplicons link the human case A18-2173 (labelled in red) to the RABV strain (blue) circulating mainly in the Ouest Department of Haiti (HTI). Three groups of RABV were identified based on the sequence alignment: HTI contains two groups (blue and green) and DOM has three groups (blue, green, and purple). Full article ">Figure 3
Molecular phylogenetic analysis using 115 bp sequences generated from LN34 amplicons. (A) The Bayesian evolutionary analysis sampling tree (BEAST) showed that the human rabies case (A18_2173) grouped with samples from HTI. The major nodes are labelled with the posterior probabilities. * Posterior values > 0.5 are shown at each node. (B) The neighbor joining tree agreed with the BEAST tree for the major clades and showed strains with identical sequences. The selected nodes are labelled with bootstrap values. (C) The geographic location of samples from HTI and DOM. The blue star is the location where the patient was infected. Full article ">

21 pages, 454 KiB

Open AccessEditorial

Acknowledgement to Reviewers of Viruses in 2019

by Viruses Editorial Office

Viruses 2020, 12(1), 119; https://doi.org/10.3390/v12010119 - 17 Jan 2020

Cited by 1 | Viewed by 3843

Abstract

The editorial team greatly appreciates the reviewers who have dedicated their considerable time and expertise to the journal’s rigorous editorial process over the past 12 months, regardless of whether the papers are finally published or not [...] Full article

18 pages, 4440 KiB

Open AccessArticle

Integrated Analysis of Differentially Expressed miRNAs and mRNAs in Goat Skin Fibroblast Cells in Response to Orf Virus Infection Reveals That cfa-let-7a Regulates Thrombospondin 1 Expression

by Feng Pang, Xinying Wang, Zhen Chen, Zhenxing Zhang, Mengmeng Zhang, Chengqiang Wang, Xiaohong Yang, Qi An, Li Du and Fengyang Wang

Viruses 2020, 12(1), 118; https://doi.org/10.3390/v12010118 - 17 Jan 2020

Cited by 7 | Viewed by 3613

Abstract

Orf is a zoonotic disease that has caused huge economic losses globally. Systematical analysis of dysregulated cellular micro RNAs (miRNAs) in response to Orf virus (ORFV) infection has not been reported. In the current study, miRNA sequencing and RNA sequencing (RNA-seq) were performed [...] Read more.

Orf is a zoonotic disease that has caused huge economic losses globally. Systematical analysis of dysregulated cellular micro RNAs (miRNAs) in response to Orf virus (ORFV) infection has not been reported. In the current study, miRNA sequencing and RNA sequencing (RNA-seq) were performed in goat skin fibroblast (GSF) cells at 0, 18, and 30 h post infection (h.p.i). We identified 140 and 221 differentially expressed (DE) miRNAs at 18 and 30 h.p.i, respectively. We also identified 729 and 3961 DE genes (DEGs) at 18 and 30 h.p.i, respectively. GO enrichment analysis indicates enrichment of apoptotic regulation, defense response to virus, immune response, and inflammatory response at both time points. DE miRNAs and DEGs with reverse expression were used to construct miRNA-gene networks. Seven DE miRNAs and seven DEGs related to “negative regulation of viral genome replication” were identified. These were validated by RT-qPCR. Cfa-let-7a, a significantly upregulated miRNA, was found to repress Thrombospondin 1 (THBS1) mRNA and protein expression by directly targeting the THBS1 3′ untranslated region. THBS1 has been reported to induce apoptosis; therefore, the cfa-let-7a-THBS1 axis may play an important role in cellular apoptosis during ORFV infection. This study provides new insights into ORFV and host cell interaction mechanisms. Full article

(This article belongs to the Section Animal Viruses)

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20 pages, 3332 KiB

Open AccessReview

Microtubules in Influenza Virus Entry and Egress

by Caitlin Simpson and Yohei Yamauchi

Viruses 2020, 12(1), 117; https://doi.org/10.3390/v12010117 - 17 Jan 2020

Cited by 30 | Viewed by 14255

Abstract

Influenza viruses are respiratory pathogens that represent a significant threat to public health, despite the large-scale implementation of vaccination programs. It is necessary to understand the detailed and complex interactions between influenza virus and its host cells in order to identify successful strategies [...] Read more.

Influenza viruses are respiratory pathogens that represent a significant threat to public health, despite the large-scale implementation of vaccination programs. It is necessary to understand the detailed and complex interactions between influenza virus and its host cells in order to identify successful strategies for therapeutic intervention. During viral entry, the cellular microenvironment presents invading pathogens with a series of obstacles that must be overcome to infect permissive cells. Influenza hijacks numerous host cell proteins and associated biological pathways during its journey into the cell, responding to environmental cues in order to successfully replicate. The cellular cytoskeleton and its constituent microtubules represent a heavily exploited network during viral infection. Cytoskeletal filaments provide a dynamic scaffold for subcellular viral trafficking, as well as virus-host interactions with cellular machineries that are essential for efficient uncoating, replication, and egress. In addition, influenza virus infection results in structural changes in the microtubule network, which itself has consequences for viral replication. Microtubules, their functional roles in normal cell biology, and their exploitation by influenza viruses will be the focus of this review. Full article

(This article belongs to the Special Issue Regulation and Exploitation of Microtubules by Viruses)

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Figure 1

Figure 1
Structure and organisation of microtubules. (A) Microtubule filaments are comprised of multiple dimeric complexes of α- and β-tubulin, assembled around a hollow core. Thirteen protofilaments assemble to form a microtubule. Microtubules are anchored at their minus ends at MTOCs, which is mediated by γ-tubulin. (B) Microtubules form dynamic networks in the cytoplasm which are stably anchored at MTOCs, including the centrosome and Golgi apparatus. Three-dimensional structural data: PDB ID tubulin dimer (1TUB). Full article ">Figure 2
Influenza A virus endocytosis and early trafficking through the cell. IAV is a single-stranded negative sense RNA virus, belonging to the Orthomyxoviridae family. Viral particles are composed of an outer envelope containing the glycoproteins hemagglutinin (HA) and neuraminidase (NA) and M2 ion channels. An M1 shell constitutes the viral shell, within which are 8 viral gene segments each in association with nucleoprotein (NP) and an RNA polymerase. The influenza polymerase itself is formed from three subunits; PA, PB1 and PB2. Following attachment of IAV to permissive cells via sialylated cell-surface receptors, the virus is endocytosed via clathrin-mediated endocytosis and macropinocytosis. After initial association with the actin-myosin network, early endosomes containing IAV virions interact with microtubules via dynein motor proteins for retrograde traffic towards the MTOC, in close proximity to the cellular nucleus. Upon reaching the perinuclear region, IAVs undergo low-pH mediated fusion with the late endosomal membrane. M1 shell uncoating is dependent on microtubules, actin, and the motors dynein and myosin II. Release of vRNPs into the cytosol and uptake into the nucleus precedes viral genome replication. Three-dimensional structural data: PDB ID HA (2IBX); NA (6CRD); M2 (3BKD); M1 (1EA3) [<a href="#B65-viruses-12-00117" class="html-bibr">65</a>]. Full article ">Figure 3
Influenza A virus egress. Following replication of viral RNA, newly synthesised vRNPs are exported from the nucleus and accumulate at the MTOC, before trafficking towards the cellular periphery in a microtubule dependent manner for assembly and budding. Influenza viruses utilise components of the endocytic recycling and secretory pathways for apical transport; associations between Rab11-positive recycling endocytic vesicles and influenza viruses allow viral traffic along microtubules. In addition, vRNPs induce formation of liquid organelles which associate with Rab11 for vRNP traffic via the secretory pathway. Following microtubule-dependent anterograde traffic to the cellular periphery, vRNPs assemble to form new virions and bud from the cell surface to trigger secondary infection in permissive cells. Three-dimensional structural data: PDB ID HA (2IBX); NA (6CRD); M2 (3BKD); M1 (1EA3) [<a href="#B65-viruses-12-00117" class="html-bibr">65</a>]. Full article ">

13 pages, 2324 KiB

Open AccessArticle

Real Time Analysis of Bovine Viral Diarrhea Virus (BVDV) Infection and Its Dependence on Bovine CD46

by Christiane Riedel, Hann-Wei Chen, Ursula Reichart, Benjamin Lamp, Vibor Laketa and Till Rümenapf

Viruses 2020, 12(1), 116; https://doi.org/10.3390/v12010116 - 17 Jan 2020

Cited by 10 | Viewed by 4569

Abstract

Virus attachment and entry is a complex interplay of viral and cellular interaction partners. Employing bovine viral diarrhea virus (BVDV) encoding an mCherry-E2 fusion protein (BVDV_E2-mCherry), being the first genetically labelled member of the family Flaviviridae applicable for the analysis of [...] Read more.

Virus attachment and entry is a complex interplay of viral and cellular interaction partners. Employing bovine viral diarrhea virus (BVDV) encoding an mCherry-E2 fusion protein (BVDV_E2-mCherry), being the first genetically labelled member of the family Flaviviridae applicable for the analysis of virus particles, the early events of infection—attachment, particle surface transport, and endocytosis—were monitored to better understand the mechanisms underlying virus entry and their dependence on the virus receptor, bovine CD46. The analysis of 801 tracks on the surface of SK6 cells inducibly expressing fluorophore labelled bovine CD46 (CD46_fluo) demonstrated the presence of directed, diffusive, and confined motion. 26 entry events could be identified, with the majority being associated with a CD46_fluo positive structure during endocytosis and occurring more than 20 min after virus addition. Deletion of the CD46_fluo E2 binding domain (CD46_fluo∆E2bind) did not affect the types of motions observed on the cell surface but resulted in a decreased number of observable entry events (2 out of 1081 tracks). Mean squared displacement analysis revealed a significantly increased velocity of particle transport for directed motions on CD46_fluo∆E2bind expressing cells in comparison to CD46_fluo. These results indicate that the presence of bovine CD46 is only affecting the speed of directed transport, but otherwise not influencing BVDV cell surface motility. Instead, bovine CD46 seems to be an important factor during uptake, suggesting the presence of additional cellular proteins interacting with the virus which are able to support its transport on the virus surface. Full article

(This article belongs to the Special Issue Bovine Viral Diarrhea Virus and Related Pestiviruses)

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Figure 1

Figure 1
Characterization of BVDVE2-mCherry entry into SK6 CD46fluo cells. (A) Characterization of 26 entry events of BVDVE2-mCherry in SK6 CD46fluo cells with regard to occurrence after virus addition, association with CD46fluo signal, intracellular persistence of the E2-mCherry signal and intracellular distance travelled. Outliers (Q3 + 1.5-times interquartile range) are indicated by dots. (B,C) Examples of entry events of BVDVE2-mCherry (red) into SK6 CD46bov (green) cells. The full field of view at the time of the start of acquisition (time after virus addition is specified in the top right corner) is shown and the area of interest is indicated by grey squares. Frames as depicted in the detail images were acquired every 10 s for up to 10 min after the start of acquisition. Times indicated in s refer to the start of acquisition. The length of the scale bar in the detail images in (B) = 2.5 µm and in (C) = 5 µm. Full article ">Figure 2
E2-mCherry signal development but not signal distribution is depending on the MOI. (A) Occurrence of E2-mCherry signal in min after addition of BVDVE2-mCherry at an MOI of 1 (blue) or 10 (green) to SK6 CD46fluo cells. SK6 CD46fluo cells were imaged recording one frame every 10 min at one z-level for 16 h. The mean is indicated by x and outliers (Q3 + 1.5-times interquartile range) are indicated by dots. (B) Distribution of E2-mCherry (red) and CD46fluo (green) signal 20 h after infection with BVDVE2-mCherry in SK6 CD46fluo cells. SK6 CD46fluo cells were imaged for 10 min recording 3 z-levels (0.8 µm) every 10 s (<a href="#app1-viruses-12-00116" class="html-app">movie 6</a>). Points of high E2-mCherry intensity that are colocalizing with CD46fluo are indicated by white circles. Full article ">Figure 3
Deletion of CCP-1 reduces CD46fluo effect of the susceptibility of SK6 cells to the level of GFP-expressing controls. Susceptibility of SK6 CD46fluo (blue), CD46fluo∆E2bind (green) and GFP-expressing (grey) SK6 cells to infection with BVDVE2-mCherry in % of MDBK cells (=100%) with and without the induction of expression by the addition of doxycycline (Doxy) (n = 6). Cells were infected with serial dilutions of BVDVE2-mCherry for 4 h. Subsequently, medium was exchanged to medium containing 1% carboxymethycellulose and E2-mCherry positive foci were detected 48 h after infection by fluorescence microscopy to determine the focus forming units (ffu)/mL. Susceptibility of a given cell line was calculated as the percentage of ffu/mL determined for MDBK cells, which was set to 100%. Full article ">Figure 4
Movements of BVDVE2-mCherry on the surface of SK6 CD46fluo (blue) or CD46fluo∆E2bind cells (green) are comparable. Box and Whisker blots of maximum and mean velocity, directionality, real distance and direct distance of particles tracked on the surface of SK6 CD46fluo (blue, n = 801) or CD46fluo∆E2bind cells (green, n = 1081), respectively. The mean is indicated by x and outliers (Q3 + 1.5-times interquartile range) are indicated by dots. Full article ">Figure 5
The average velocity of directed motion of BVDVE2-mCherry particles is increased on the surface of SK6 CD46fluo∆E2bind cells. MSD analysis of particle movement on SK6 CD46fluo and CD46fluo∆E2bind cells. (A) Slopes of tracks on SK6 CD46fluo (blue) and CD46fluo∆E2bind (green) cells displaying a linear increase and distribution of the calculated diffusion coefficient D0 depicted as box and whisker blot. The mean is indicated by x and outliers (Q3 + 1.5 times interquartile range) are depicted as dots. Average slopes are depicted as thick black lines. (B) Slopes of tracks on SK6 CD46fluo (blue) and CD46fluo∆E2bind (green) cells displaying an exponential increase and distribution of the calculated particle velocities V, depicted as box and whisker blot. The mean is indicated by x and outliers (Q3 + 1.5 times interquartile range) are depicted as dots. Average slopes are depicted as thick black lines. Full article ">

12 pages, 1413 KiB

Open AccessArticle

Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns

by Sten Calvelage, Marcin Smreczak, Anna Orłowska, Conrad Martin Freuling, Thomas Müller, Christine Fehlner-Gardiner, Susan Nadin-Davis, Dirk Höper and Paweł Trębas

Viruses 2020, 12(1), 115; https://doi.org/10.3390/v12010115 - 17 Jan 2020

Cited by 7 | Viewed by 3177

Abstract

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and [...] Read more.

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bern_orig)-derived rabies cases, the in-depth variant analysis revealed three distinct combinations of selected variants for the ERA vaccine-induced cases, suggesting the presence of multiple replication-competent haplotypes in the investigated ERA-BHK21 vaccine. Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level. Full article

(This article belongs to the Special Issue Rabies Virus: Knowledge Gaps and Challenges to Elimination)

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Figure 1
Population-based analysis of ERA-BHK21 (left) and SAD Bernorig-derived vaccine batches (B/…) (right) and viruses of their vaccine-induced rabies cases (C/…) displayed by a fitted pairwise Manhattan distances plot. Data sets of samples that were added in this study (<a href="#viruses-12-00115-t001" class="html-table">Table 1</a> and <a href="#viruses-12-00115-t002" class="html-table">Table 2</a>) are highlighted in bold, whereas data sets of vaccine batches and their related induced rabies cases are displayed in different colors: blue—SAD B19 vaccine batches; green—SAD Bern vaccine batches, dark blue—SAD Bernorig-derived vaccine-induced rabies cases; orange—ERA-BHK21 vaccine batch; and dark orange—ERA-derived vaccine-induced rabies cases (orange/dark orange). For the ERA vaccine-induced rabies cases, three distinct combinations of single nucleotide variants were selected that originate from the progenitor ERA-BHK21 vaccine (see <a href="#sec3dot2-viruses-12-00115" class="html-sec">Section 3.2</a>), forming groups of samples with specific differences that are represented by the colors of the respective sample names (1–3). Full article ">Figure 2
Schematic illustration of all variants found for the ERA vaccine-induced rabies cases as well as nucleotide exchanges between viruses from vaccine-induced rabies cases and the ERA-BHK21 vaccine virus strain. Colored dotted lines display differences at the consensus level for viruses from vaccine-induced rabies cases that derived from selected combinations of single nucleotide variants (SNVs) found in the progenitor ERA-BHK21 vaccine virus (group 1–3; <a href="#viruses-12-00115-t004" class="html-table">Table 4</a>, for variant frequencies, see <a href="#app1-viruses-12-00115" class="html-app">Table S7, Supplementary File</a>). Nucleotide bases illustrated on the genome sequence represent spontaneous mutations found for viruses of vaccine-induced rabies cases. Each of these three groups were characterized by a unique set of differences at the consensus level (<a href="#viruses-12-00115-t004" class="html-table">Table 4</a>). The only exception was the sample C/CAN/1994N35116 which lacked one difference (position 3587, <a href="#app1-viruses-12-00115" class="html-app">Table S7</a>) and had one additional difference in close proximity that cannot be found in any of the other samples (position 3734, <a href="#app1-viruses-12-00115" class="html-app">Table S7</a>). Full article ">Figure 3
Population-based analysis of ERA-BHK21- and SAD-Bernorig-derived vaccine strains and viruses of their vaccine-induced rabies cases in relation to selected field RABV strains (F/…). Full article ">

15 pages, 7297 KiB

Open AccessArticle

Systematic Identification of Host Immune Key Factors Influencing Viral Infection in PBL of ALV-J Infected SPF Chicken

by Manman Dai, Shibing Li, Keyi Shi, Jiayu Liao, Hui Sun and Ming Liao

Viruses 2020, 12(1), 114; https://doi.org/10.3390/v12010114 - 16 Jan 2020

Cited by 21 | Viewed by 3803

Abstract

Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old [...] Read more.

Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old SPF chickens are infected with ALV-J strain CHN06, after which the host immune response is detected. We found that the expression of two antiviral interferon-stimulated genes (ISGs) (Mx1 and IFIT5) were increased in ALV-J infected peripheral blood lymphocytes (PBL). A significant CD8⁺ T cell response induced by ALV-J appeared as early as seven days post-infection (DPI), and humoral immunity starting from 21 DPI differed greatly in the time scale of induction level. Meanwhile, the ALV-J viremia was significantly decreased before antibody production at 14 DPI, and eliminated at 21 DPI under a very low antibody level. The up-regulated CD8⁺ T cell in the thymus (14DPI) and PBL (7 DPI and 21 DPI) was detected, indicating that the thymus may provide the output of CD8⁺ T cell to PBL, which was related to virus clearance. Besides, up-regulated chemokine CXCLi1 at 7 DPI in PBL was observed, which may be related to the migration of the CD8⁺ T cell from the thymus to PBL. More importantly, the CD8 ^high+ T cell response of the CD8αβ phenotype may produce granzyme K, NK lysin, or IFN-γ for clearing viruses. These findings provide novel insights and direction for developing effective ALV-J vaccines. Full article

(This article belongs to the Special Issue T Cell-Mediated Antiviral Immunity)

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Figure 1
Dynamic detection of the avian leukosis virus subgroup J (ALV-J) shedding, ALV-J viremia, and ALV-J antibody. Five chickens were randomly selected for sampling every seven days post-infection (DPI). (A) ALV-J shedding was monitored via detecting the p27 expression levels in cloacal swabs. S/P value below 0.2 (red) indicated negative ALV-J shedding. (B) ALV-J viremia was monitored via virus isolation with the routine method. An S/P value above 0.2 (red) indicated positive ALV-J viremia. (C) The ALV-J antibody level in the serum was monitored using the commercial ALV-J antibody test kit. An S/P value above 0.6 (red) was considered ALV-J antibody positive. (D) ALV-J viremia and antibody level of chicken number 12 were analyzed from 7 DPI to 63 DPI. The paired t-test was used for statistical comparison. * p < 0.05, ** p < 0.01. Full article ">Figure 2
Dynamic detection of T lymphocyte percentage in peripheral blood lymphocytes (PBL). Five days before infection (DBI) and each week after infection, PBL derived from five chickens of infected and control groups were isolated to detect the T lymphocyte percentage, including the percentage of (A) the CD3+CD8+ T cell, (B) CD3+CD4+ T cell, and (C) CD3+CD4+CD8+. (D) The ratio of CD3+CD4+/CD3+CD8+ was detected. Each sample collected 1 × 105 cells for flow cytometric analysis. The two-way ANOVA was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01. Full article ">Figure 3
Analysis of T lymphocyte percentage in the thymus. Thymus single-cell suspensions derived from chickens of infected and control groups were isolated to detect the T lymphocyte percentage. Every dot stands for one chicken. The unpaired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01. Each sample collected 2 × 105 cells for flow cytometric analysis. Full article ">Figure 4
Analysis of T lymphocyte percentage in spleen. Spleen single-cell suspensions derived from chickens of infected and control groups were isolated to detect the T lymphocyte percentage. Every dot stands for one chicken. The unpaired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05.. Each sample collected 2 × 105 cells for flow cytometric analysis. Full article ">Figure 5
Analysis expression of immune-related genes in PBL by qRT-PCR. Expressions of immune-related genes in PBL were detected by qRT-PCR. The total RNA of PBL was extracted from three chickens of the infected and control groups, respectively. The data was collected from three biological samples in each group, each sample performed in triplicate. The results were presented as means ± SEM and the paired t-test was used for statistical comparison. ns p > 0.05, * p < 0.05, ** p < 0.01. Full article ">Figure 6
T cell phenotype analysis. (A) Gating of CD8α+, CD4+, and CD4+CD8α+ T cells with the CD3+(APC), CD4+(FITC), and CD8α+(PE) antibodies. (B) Analysis of the percentage and phenotype of T cells in PBL of ALV-J infected chicken number 12 and control chicken number 29 at various time points. Histogram of the CD3+CD8α+ T cells in PBL of (C) ALV-J infected group and (D) control group at 21 DPI. Each sample collected 1 × 105 cells for flow cytometric analysis. Full article ">Figure 7
CD8+ T cell phenotype identification in PBL of ALV-J infected chicken number 12. (A) Gating strategy for analysis of CD8highα+, CD8mediumα+, CD4+, and CD4+CD8lowα+ T cells with the CD3+(APC), CD4+(FITC), and CD8α+(PE) antibodies. (B) Gating strategy for analysis of CD8+ αα and CD8+αβ phenotype in three CD8+ populations with the CD4+ (APC), CD8β+ (FITC), and CD8α+ (PE) antibodies. (C) Gating strategy for analysis of CD8+ αα and CD8+αβ phenotype in three CD8+ populations with the CD3+ (APC), CD8β+ (FITC), and CD8α+ (PE) antibodies. Each dot indicated each collected cell. And the circle or fame indicated the gated target cell population labelled by various antibodies. Each sample collected 1 × 105 cells for flow cytometric analysis. Full article ">Figure 8
CD8+ T cell phenotype analysis. Contour plot of CD8+ αα and CD8+αβ T cells in PBL of (A) control chicken number 34 and (B) ALV-J infected chicken number 12 with the CD3+ (APC), CD8β+ (FITC), and CD8α+ (PE) antibodies. Each sample collected 1 × 105 cells for flow cytometric analysis. The circle or fame indicated the gated target cell population labelled by various antibodies. And the red arrow indicated that each CD8α+T cell population was further subdivided to CD8αα and CD8αβ phenotype. Full article ">

25 pages, 3900 KiB

Open AccessArticle

Interferon-Gamma Modulation of the Local T Cell Response to Alphavirus Encephalomyelitis

by Victoria K. Baxter and Diane E. Griffin

Viruses 2020, 12(1), 113; https://doi.org/10.3390/v12010113 - 16 Jan 2020

Cited by 19 | Viewed by 5517

Abstract

Infection of mice with Sindbis virus (SINV) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (CNS). Interferon-gamma (IFN-γ) is an important component of this response, and we show that SINV-infected differentiated neurons [...] Read more.

Infection of mice with Sindbis virus (SINV) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (CNS). Interferon-gamma (IFN-γ) is an important component of this response, and we show that SINV-infected differentiated neurons respond to IFN-γ in vitro by induction of antiviral genes and suppression of virus replication. To determine the in vivo effects of IFN-γ on SINV clearance and T cell responses, C57BL/6 mice lacking IFN-γ or IFN-γ receptor-1 were compared to wild-type (WT) mice after intracranial SINV infection. In WT mice, IFN-γ was first produced in the CNS by natural killer cells and then by CD4⁺ and CD8⁺ T cells. Mice with impaired IFN-γ signaling initiated clearance of viral RNA earlier than WT mice associated with CNS entry of more granzyme B-producing CD8⁺ T cells. However, these mice established fewer CD8⁺ tissue-resident memory T (T_RM) cells and were more likely to experience reactivation of viral RNA synthesis late after infection. Therefore, IFN-γ suppresses the local development of granzyme B-expressing CD8⁺ T cells and slows viral RNA clearance but promotes CD8⁺ T_RM cell establishment. Full article

(This article belongs to the Special Issue T Cell-Mediated Antiviral Immunity)

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28 pages, 8155 KiB

Open AccessArticle

Herpes Simplex Virus Type-2 Paralyzes the Function of Monocyte-Derived Dendritic Cells

by Linda Grosche, Petra Mühl-Zürbes, Barbara Ciblis, Adalbert Krawczyk, Christine Kuhnt, Lisa Kamm, Alexander Steinkasserer and Christiane Silke Heilingloh

Viruses 2020, 12(1), 112; https://doi.org/10.3390/v12010112 - 16 Jan 2020

Cited by 11 | Viewed by 5096

Abstract

Herpes simplex viruses not only infect a variety of different cell types, including dendritic cells (DCs), but also modulate important cellular functions in benefit of the virus. Given the relevance of directed immune cell migration during the initiation of potent antiviral immune responses, [...] Read more.

Herpes simplex viruses not only infect a variety of different cell types, including dendritic cells (DCs), but also modulate important cellular functions in benefit of the virus. Given the relevance of directed immune cell migration during the initiation of potent antiviral immune responses, interference with DC migration constitutes a sophisticated strategy to hamper antiviral immunity. Notably, recent reports revealed that HSV-1 significantly inhibits DC migration in vitro. Thus, we aimed to investigate whether HSV-2 also modulates distinct hallmarks of DC biology. Here, we demonstrate that HSV-2 negatively interferes with chemokine-dependent in vitro migration capacity of mature DCs (mDCs). Interestingly, rather than mediating the reduction of the cognate chemokine receptor expression early during infection, HSV-2 rapidly induces β2 integrin (LFA-1)-mediated mDC adhesion and thereby blocks mDC migration. Mechanistically, HSV-2 triggers the proteasomal degradation of the negative regulator of β2 integrin activity, CYTIP, which causes the constitutive activation of LFA-1 and thus mDC adhesion. In conclusion, our data extend and strengthen recent findings reporting the reduction of mDC migration in the context of a herpesviral infection. We thus hypothesize that hampering antigen delivery to secondary lymphoid organs by inhibition of mDC migration is an evolutionary conserved strategy among distinct members of Herpesviridae. Full article

(This article belongs to the Special Issue Dendritic Cells and Antiviral Defense)

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14 pages, 636 KiB

Open AccessArticle

Host Range Evolution of Potyviruses: A Global Phylogenetic Analysis

by Benoît Moury and Cécile Desbiez

Viruses 2020, 12(1), 111; https://doi.org/10.3390/v12010111 - 16 Jan 2020

Cited by 35 | Viewed by 5528

Abstract

Virus host range, i.e., the number and diversity of host species of viruses, is an important determinant of disease emergence and of the efficiency of disease control strategies. However, for plant viruses, little is known about the genetic or ecological factors involved in [...] Read more.

Virus host range, i.e., the number and diversity of host species of viruses, is an important determinant of disease emergence and of the efficiency of disease control strategies. However, for plant viruses, little is known about the genetic or ecological factors involved in the evolution of host range. Using available genome sequences and host range data, we performed a phylogenetic analysis of host range evolution in the genus Potyvirus, a large group of plant RNA viruses that has undergone a radiative evolution circa 7000 years ago, contemporaneously with agriculture intensification in mid Holocene. Maximum likelihood inference based on a set of 59 potyviruses and 38 plant species showed frequent host range changes during potyvirus evolution, with 4.6 changes per plant species on average, including 3.1 host gains and 1.5 host loss. These changes were quite recent, 74% of them being inferred on the terminal branches of the potyvirus tree. The most striking result was the high frequency of correlated host gains occurring repeatedly in different branches of the potyvirus tree, which raises the question of the dependence of the molecular and/or ecological mechanisms involved in adaptation to different plant species. Full article

(This article belongs to the Special Issue The Complexity of the Potyviral Interaction Network)

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Figure 1
Phylogenetic tree of potyviruses and rymoviruses based on alignment of amino acid sequences of full-length genomes with MAFFT and the LG model with gamma-distributed rates among sites, invariant sites, and amino acid frequencies. Internal branches are numbered from 63 to 123. Terminal branches are named after the acronym of the corresponding virus species. Branches with bootstrap values above 0.7 are indicated by gray circles. Arrows indicate branches with a significantly higher number of host gains than expected by chance (<a href="#app1-viruses-12-00111" class="html-app">Supplementary Table S3</a>). Virus species corresponding to acronyms and GenBank accession numbers are as follows: Agropyron mosaic virus (AgMV; accession NC_005903), Amaranthus leaf mottle virus (AmLMV; MN709786), Asparagus virus 1 (AV-1; NC_025821), Bean common mosaic necrosis virus (BCMNV; NC_004047), Bean common mosaic virus (BCMV; MK069988), Bidens mottle virus (BiMoV; NC_014325), Bidens mosaic virus (BiMV; NC_023014), Beet mosaic virus (BtMV; NC_005304), Bean yellow mosaic virus (BYMV; NC_003492), Colombian datura virus (CDV; NC_020072), Celery mosaic virus (CeMV; NC_015393), Chilli veinal mottle virus (ChiVMV; NC_005778), Clover yellow vein virus (ClYVV; NC_003536), Cocksfoot streak virus (CSV; NC_003742), Carrot thin leaf virus (CTLV; NC_025254), Dasheen mosaic virus (DsMV; MG602234), Euphorbia ringspot virus (EuRSV; NC_031339), Freesia mosaic virus (FreMV; NC_014064), Hippeastrum mosaic virus (HiMV; NC_017967), Henbane mosaic virus (HMV; MH779476), Hordeum mosaic virus (HoMV; NC_005904), Hyacinth mosaic virus (HyaMV; NC_037051), Iris mild mosaic virus (IMMV; JF320812), Iris severe mosaic virus (ISMV; NC_029076), Johnsongrass mosaic virus (JGMV; NC_003606), Konjac mosaic virus (KoMV; NC_007913), Lettuce mosaic virus (LMV; NC_003605), Leek yellow stripe virus (LYSV; NC_004011), Maize dwarf mosaic virus (MDMV; NC_003377), Moroccan watermelon mosaic virus (MWMV; NC_009995), Narcissus degeneration virus (NDV; NC_008824), Narcissus late season yellows virus (NLSYV; NC_023628), Narcissus yellow stripe virus (NYSV; NC_011541), Ornithogalum mosaic virus (OrMV; NC_019409), Onion yellow dwarf virus (OYDV; NC_005029), Peanut mottle virus (PeMoV; NC_002600), Pepper mottle virus (PepMoV; NC_001517), Pepper severe mosaic virus (PepSMV; NC_008393), Pokeweed mosaic virus (PkMV; NC_018872), Plum pox virus (PPV; NC_001445), Papaya ringspot virus (PRSV; NC_001785), Pea seed-borne mosaic virus (PSbMV; NC_001671), Peru tomato mosaic virus (PTV; NC_004573), Potato virus A (PVA; NC_004039), Pepper veinal mottle virus (PVMV; NC_011918), Potato virus V (PVV; NC_004010), Potato virus Y (PVY; NC_001616), Passionfruit woodiness virus (PWV; NC_014790), Ryegrass mosaic virus (RgMV; NC_001814), Sugarcane mosaic virus (SCMV; NC_003398), Soybean mosaic virus (SMV; NC_002634), Sweet potato feathery mottle virus (SPFMV; NC_001841), Sweet potato latent virus (SPLV; NC_020896), Sorghum mosaic virus (SrMV; NC_004035), Tobacco etch virus (TEV; NC_001555), Turnip mosaic virus (TuMV; KF595121), Tobacco vein mottling virus (TVMV; NC_001768), Watermelon mosaic virus (WMV; NC_006262), Wild potato mosaic virus (WPMV; NC_004426), Wisteria vein mosaic virus (WVMV; NC_007216), Yam mosaic virus (YMV; NC_004752), and Zucchini yellow mosaic virus (ZYMV; NC_003224). Full article ">

41 pages, 1120 KiB

Open AccessReview

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

by Catherine S. Adamson and Michael M. Nevels

Viruses 2020, 12(1), 110; https://doi.org/10.3390/v12010110 - 16 Jan 2020

Cited by 41 | Viewed by 8841

Abstract

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved [...] Read more.

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. “Bright and early” events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors. Full article

(This article belongs to the Special Issue Antiviral Agents)

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15 pages, 796 KiB

Open AccessReview

Prophylactic Hepatitis E Vaccines: Antigenic Analysis and Serological Evaluation

by Yike Li, Xiaofen Huang, Zhigang Zhang, Shaowei Li, Jun Zhang, Ningshao Xia and Qinjian Zhao

Viruses 2020, 12(1), 109; https://doi.org/10.3390/v12010109 - 16 Jan 2020

Cited by 20 | Viewed by 5393

Abstract

Hepatitis E virus (HEV) infection causes sporadic outbreaks of acute hepatitis worldwide. HEV was previously considered to be restricted to resource-limited countries with poor sanitary conditions, but increasing evidence implies that HEV is also a public health problem in developed countries and regions. [...] Read more.

Hepatitis E virus (HEV) infection causes sporadic outbreaks of acute hepatitis worldwide. HEV was previously considered to be restricted to resource-limited countries with poor sanitary conditions, but increasing evidence implies that HEV is also a public health problem in developed countries and regions. Fortunately, several vaccine candidates based on virus-like particles (VLPs) have progressed into the clinical development stage, and one of them has been approved in China. This review provides an overview of the current HEV vaccine pipeline and future development with the emphasis on defining the critical quality attributes for the well-characterized vaccines. The presence of clinically relevant epitopes on the VLP surface is critical for eliciting functional antibodies against HEV infection, which is the key to the mechanism of action of the prophylactic vaccines against viral infections. Therefore, the epitope-specific immunochemical assays based on monoclonal antibodies (mAbs) for HEV vaccine antigen are critical methods in the toolbox for epitope characterization and for in vitro potency assessment. Moreover, serological evaluation methods after immunization are also discussed as biomarkers for clinical performance. The vaccine efficacy surrogate assays are critical in the preclinical and clinical stages of VLP-based vaccine development. Full article

(This article belongs to the Special Issue Virus-Like Particle Vaccines)

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14 pages, 1869 KiB

Open AccessArticle

In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism

by Kaelan Gobeil Odai, Conor O’Dwyer, Rineke Steenbergen, Tyler A. Shaw, Tyler M. Renner, Peyman Ghorbani, Mojgan Rezaaifar, Shauna Han, Marc-André Langlois, Angela M. Crawley, Rodney S. Russell, John P. Pezacki, D. Lorne Tyrrell and Morgan D. Fullerton

Viruses 2020, 12(1), 108; https://doi.org/10.3390/v12010108 - 16 Jan 2020

Cited by 9 | Viewed by 4217

Abstract

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C [...] Read more.

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

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Graphical abstract

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Characterization of Huh7.5 cell choline uptake. (A) Choline uptake saturation kinetics fit to a Michaelis-Menten curve. (B) Inhibition of choline uptake in response to hemicholinium-3 (HC3). (C) Choline uptake inhibition in response to 200 μM quinine (OCT inhibitor). Data are mean ± SEM and represent 3–4 independent experiments. Full article ">Figure 2
Choline transporter mRNA expression at 24 and 96 h post-HCV infection. FBS- and HS-cultured Huh7.5 cells were infected at an MOI of 1 for (A) 24 or (B) 96 h before cells were washed and RNA isolated to measure choline transporter (SLC44A1; choline transporter-like 1, SLC44A2; choline transporter-like 2 and SLC22A1; organic cation transporter 1) as well as CDP-choline pathway (CHKa; choline kinase alpha and PCYT1a; phosphocholine cytidylyltransferase alpha) transcript expression. All groups are shown relative to uninfected FBS-cultured control cells at 24 h and normalized to the average of β ACTIN and HSP90. The hashed line in (B) represents the level of uninfected FBS-cultured control cells at 24 h. Data are mean ± SEM and represent three independent experiments. Statistical significance was determined by two-way ANOVA with a Tukey post-hoc analysis (within each transcript), such that ** represents p < 0.01 compared to uninfected control within serum culture condition and # represents p < 0.05 compared between FBS- and HS-cultured conditions. Full article ">Figure 3
Choline transporter protein expression at 24 h and 96 h post-HCV infection. FBS- and HS-cultured Huh7.5 cells were infected at an MOI of 1 for (A) 24 h or (B) 96 h before cells were washed and protein isolated to measure choline transporter (CTL1; choline transporter-like 1 and CTL2; choline transporter-like 2) total protein expression. Samples were run on two individual gels, but transferred to a common membrane. Densitometry analyses depicts the density of CTL1 and CTL2 normalized to GAPDH and shown relative to uninfected FBS-cultured control cells at 24 h. The hashed line in (B) represents the level of uninfected FBS-cultured control cells at 24 h. Data are mean ± SEM and represent three independent experiments. Statistical significance was determined by two-way ANOVA with a Tukey post-hoc analysis (within each protein), such that * represents p < 0.05 compared to uninfected control within serum culture condition, and # and ## represents p < 0.05 and p < 0.01 compared between FBS- and HS-cultured conditions, respectively. Full article ">Figure 4
Choline uptake and incorporation into PC 24 h post-HCV infection. FBS-cultured Huh7.5 cells were infected at an MOI of 1 for 24 h. (A) [3H]-choline uptake was determined over the course of 30 min; (B) [3H]-choline uptake saturation kinetics were determined in the presence of increasing concentration of non-radiolabeled choline and fit to a Michaelis-Menten curve; and (C) [3H]-choline incorporation into PC was then determined. Data are mean ± SEM, are representative of three independent experiments and normalized to total protein content, where * and ** represent statistical significance compared to uninfected control cells at p < 0.05 and p < 0.01, respectively. Full article ">Figure 5
Inhibition of choline uptake augments HCV replication after 24 h. FBS- and HS-cultured Huh7.5 cells were infected at an MOI of 1 for (A) 24 h in the presence or absence of 20 or 200 μM hemicholinium-3 (HC-3) to inhibit choline uptake and (B) 96 h in the presence or absence of 20 or 200 μM HC-3 for the last 24 h of infection. Cells were washed and RNA was isolated to assess HCV RNA expression as an indication of viral replication. All treatments are shown relative to infected vehicle FBS-control cells and normalized to the average of β ACTIN and HSP90. Data are mean±SEM and are represent of 3-4 independent experiments. The hashed line in (B) represents the level of infected FBS-cultured control cells at 24 h. Statistical significance was determined within each culture system and HCV group, such that 0, 20 and 200 μM HC-3 from FBS- and HS-cultured cells were each determined by one-way ANOVA where * and *** represent p < 0.05 and p < 0.001, respectively (determined by a Tukey posthoc test) compared to vehicle control (0 μM HC-3). Full article ">Figure 6
Inhibition of choline uptake decreases HCV infectivity. FBS- and HS-cultured Huh7.5 cells were infected at an MOI of 1 for 96 h in the presence or absence of 20 or 200 μM hemicholinium-3 (HC-3) to inhibit choline uptake for the final 24 h of infection. Media was removed to assess TCID50 as a measure of HCV infectivity. Data are mean ± SEM and are represent of 5–7 independent experiments. Statistical significance was determined by two-way ANOVA with a Tukey post-hoc analysis, such that **** represents p < 0.0001 compared to vehicle control within serum culture condition and #### represents p < 0.0001 compared between FBS- and HS-cultured conditions. Full article ">

22 pages, 1707 KiB

Open AccessReview

Virus Metagenomics in Farm Animals: A Systematic Review

by Kirsty T. T. Kwok, David F. Nieuwenhuijse, My V. T. Phan and Marion P. G. Koopmans

Viruses 2020, 12(1), 107; https://doi.org/10.3390/v12010107 - 16 Jan 2020

Cited by 49 | Viewed by 9747

Abstract

A majority of emerging infectious diseases are of zoonotic origin. Metagenomic Next-Generation Sequencing (mNGS) has been employed to identify uncommon and novel infectious etiologies and characterize virus diversity in human, animal, and environmental samples. Here, we systematically reviewed studies that performed viral mNGS [...] Read more.

A majority of emerging infectious diseases are of zoonotic origin. Metagenomic Next-Generation Sequencing (mNGS) has been employed to identify uncommon and novel infectious etiologies and characterize virus diversity in human, animal, and environmental samples. Here, we systematically reviewed studies that performed viral mNGS in common livestock (cattle, small ruminants, poultry, and pigs). We identified 2481 records and 120 records were ultimately included after a first and second screening. Pigs were the most frequently studied livestock and the virus diversity found in samples from poultry was the highest. Known animal viruses, zoonotic viruses, and novel viruses were reported in available literature, demonstrating the capacity of mNGS to identify both known and novel viruses. However, the coverage of metagenomic studies was patchy, with few data on the virome of small ruminants and respiratory virome of studied livestock. Essential metadata such as age of livestock and farm types were rarely mentioned in available literature, and only 10.8% of the datasets were publicly available. Developing a deeper understanding of livestock virome is crucial for detection of potential zoonotic and animal pathogens and One Health preparedness. Metagenomic studies can provide this background but only when combined with essential metadata and following the “FAIR” (Findable, Accessible, Interoperable, and Reusable) data principles. Full article

(This article belongs to the Special Issue Viromics: Approaches, Advances, and Applications)

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Flow chart of systematic review. The procedures were adapted from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Full article ">Figure 2
An overview of study design and data (and metadata) availability of 120 included publications. Each column represents one paper, and numbers refer to the list of references (<a href="#app1-viruses-12-00107" class="html-app">Supplementary Materials</a>). Rows describe data fields extracted from the papers as follows (from top to bottom): study rationale, sequencing platforms, sample sizes, availability of sequencing data in public repository, availability of animal age description, availability of farm type description, indication of whether filtration was performed, indication of whether nuclease treatment was performed during sample preparation, nucleic acid extraction strategy, type of nucleic acid extracted, indication of whether nucleic acid amplification was performed, indication of whether sample pooling was performed in same/different farms, and mention of technical controls for validating sequencing results. Categories and color codes for each data field are indicated in the figure legend. Gray boxes indicate that the technique was not performed, or the information was not specified. mNGS = metagenomic Next-Generation Sequencing. SRA = Sequence Read Archive. ENA = European Nucleotide Archive. NA = nucleic acid. Full article ">Figure 3
(A) Geographical origin of included studies stratified by World Health Organization (WHO) regions. (B) An overview of sequencing platforms. (C) An overview of sample sizes. Full article ">Figure 4
Types of specimens tested in different farm animals by number of papers, stratified by reported health conditions. Categories per variable are color-coded as shown in the legend. GI = gastrointestinal. A version of Y-axis as frequency (%) can be found in <a href="#app1-viruses-12-00107" class="html-app">Supplementary Materials Figure S2</a>. Full article ">Figure 5
An overview of virus families found in different farm animals in included studies, stratified by reported health conditions. The bars are color-coded by host range of the viruses as shown in the figure legend. GI = gastrointestinal. Y axis shows proportion of studies that found the viruses specified. Full article ">Figure 6
Frequencies of virus genera of top three most abundant virus families stratified by health conditions. (A) Picornaviridae. (B) Parvoviridae. (C) Astroviridae. Color code is based on host range and adapted from <a href="#viruses-12-00107-f005" class="html-fig">Figure 5</a>. Full article ">

8 pages, 1548 KiB

Open AccessArticle

Modeling Challenges of Ebola Virus–Host Dynamics during Infection and Treatment

by Daniel S. Chertow, Louis Shekhtman, Yoav Lurie, Richard T. Davey, Theo Heller and Harel Dahari

Viruses 2020, 12(1), 106; https://doi.org/10.3390/v12010106 - 16 Jan 2020

Cited by 6 | Viewed by 3792

Abstract

Mathematical modeling of Ebola virus (EBOV)–host dynamics during infection and treatment in vivo is in its infancy due to few studies with frequent viral kinetic data, lack of approved antiviral therapies, and limited insight into the timing of EBOV infection of cells and [...] Read more.

Mathematical modeling of Ebola virus (EBOV)–host dynamics during infection and treatment in vivo is in its infancy due to few studies with frequent viral kinetic data, lack of approved antiviral therapies, and limited insight into the timing of EBOV infection of cells and tissues throughout the body. Current in-host mathematical models simplify EBOV infection by assuming a single homogeneous compartment of infection. In particular, a recent modeling study assumed the liver as the largest solid organ targeted by EBOV infection and predicted that nearly all cells become refractory to infection within seven days of initial infection without antiviral treatment. We compared our observations of EBOV kinetics in multiple anatomic compartments and hepatocellular injury in a critically ill patient with Ebola virus disease (EVD) with this model’s predictions. We also explored the model’s predictions, with and without antiviral therapy, by recapitulating the model using published inputs and assumptions. Our findings highlight the challenges of modeling EBOV–host dynamics and therapeutic efficacy and emphasize the need for iterative interdisciplinary efforts to refine mathematical models that might advance understanding of EVD pathogenesis and treatment. Full article

(This article belongs to the Special Issue Medical Advances in Viral Hemorrhagic Fever Research)

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13 pages, 2216 KiB

Open AccessArticle

Detection and Molecular Characterization of Picobirnaviruses (PBVs) in the Mongoose: Identification of a Novel PBV Using an Alternative Genetic Code

by Alyssa Kleymann, Anne A. M. J. Becker, Yashpal S. Malik, Nobumichi Kobayashi and Souvik Ghosh

Viruses 2020, 12(1), 99; https://doi.org/10.3390/v12010099 - 15 Jan 2020

Cited by 28 | Viewed by 3748

Abstract

We report high rates of detection (35.36%, 29/82) of genogroup-I (GI) picobirnaviruses (PBVs) in non-diarrheic fecal samples from the small Indian mongoose (Urva auropunctata). In addition, we identified a novel PBV-like RNA-dependent RNA polymerase (RdRp) gene sequence that uses an alternative [...] Read more.

We report high rates of detection (35.36%, 29/82) of genogroup-I (GI) picobirnaviruses (PBVs) in non-diarrheic fecal samples from the small Indian mongoose (Urva auropunctata). In addition, we identified a novel PBV-like RNA-dependent RNA polymerase (RdRp) gene sequence that uses an alternative mitochondrial genetic code (that of mold or invertebrate) for translation. The complete/nearly complete gene segment-2/RdRp gene sequences of seven mongoose PBV GI strains and the novel PBV-like strain were obtained by combining a modified non-specific primer-based amplification method with conventional RT-PCRs, facilitated by the inclusion of a new primer targeting the 3′-untranslated region (UTR) of PBV gene segment-2. The mongoose PBV and PBV-like strains retained the various features that are conserved in gene segment-2/RdRps of other PBVs. However, high genetic diversity was observed among the mongoose PBVs within and between host species. This is the first report on detection of PBVs in the mongoose. Molecular characterization of the PBV and PBV-like strains from a new animal species provided important insights into the various features and complex diversity of PBV gene segment-2/putative RdRps. The presence of the prokaryotic ribosomal binding site in the mongoose PBV genomes, and analysis of the novel PBV-like RdRp gene sequence that uses an alternative mitochondrial genetic code (especially that of mold) for translation corroborated recent speculations that PBVs may actually infect prokaryotic or fungal host cells. Full article

(This article belongs to the Section Animal Viruses)

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The small Indian mongoose (Urva auropunctata) on the Caribbean island of St. Kitts. Full article ">Figure 2
Map of island of St. Kitts showing the mongoose trapping sites. The trapping sites in wild and urban habitats are highlighted with blue and red, respectively. Full article ">Figure 3
Alignment of the 5′- region of the mongoose PBV and PBV-like RNA-dependent RNA polymerase (RdRp) gene sequences with that of prototype PBV GI strain PBV/Human/CHN/1-CHN-97/1997 (indicated with italic type). The 5′- terminal sequence (highlighted with yellow) and the bacterial ribosomal binding site (RBS) sequence (highlighted with blue) that are conserved in gene segment-2 of PBVs were retained in the mongoose PBV and PBV-like RdRp sequences. The putative start codon (ATG) for RdRp in gene segment-2 of mongoose PBV GI strains has been highlighted with green, whilst the putative alternative initiation codon (GTG) for RdRp in the novel PBV-like sequence (M17A) is shown with gray. A ‘*’denotes an identical nucleotide (nt) residue, whilst ‘-’ indicates absence of nt residue. Numbers to the right indicate the positions of the nt for respective PBV strains. Alignment of the complete/nearly complete mongoose PBV and PBV-like RdRp gene sequences with that of the prototype PBV GI strain is shown in <a href="#app1-viruses-12-00099" class="html-app">supplementary material S4</a>. Full article ">Figure 4
Alignment of the complete deduced amino acid (aa) sequences of putative RNA-dependent RNA polymerases (RdRps) of picobirnavirus (PBV) genogroup-I (GI) strains detected in the small Indian mongoose on the Caribbean island of St. Kitts with those of PBV strains from other host species using the ClustalW program (<a href="http://clustalw.ddbj.nig.ac.jp/" target="_blank">http://clustalw.ddbj.nig.ac.jp/</a>, accessed 15 October 2019). The PBV strains from mongoose are highlighted with underlines, whilst the prototype GI strain PBV/Human/CHN/1-CHN-97/1997 is indicated by italic type. The three conserved domains in the putative RdRps are highlighted with gray, whilst the seven polymerase motifs (G, F, A, B, C, D, and E) are shown with boxes [<a href="#B27-viruses-12-00099" class="html-bibr">27</a>]. Conserved cysteine and proline residues are shown by ‘#’ and ‘+’, respectively. A ‘*’ denotes an identical aa residue, whilst ‘-’ indicates absence of an aa residue. Positions of aa residues correspond to those of PBV strain PBV/Mongoose/KNA/M17B/2017. Full article ">Figure 5
Phylogenetic analysis of the putative RNA-dependent RNA polymerases of the mongoose picobirnavirus (PBV) genogroup-I (GI) strains and the novel mongoose PBV-like virus with those of PBVs that use the standard genetic code and PBV-like viruses that use an alternative mitochondrial genetic code. GenBank accession numbers are shown in parentheses. Bootstrap values < 70% are not shown. Scale bar, 0.5 substitutions per amino acid. Black circles: mongoose PBV GI strains; Black square: the novel mongoose PBV-like virus that uses an alternative mold mitochondrial genetic code for translation; pink triangle: prototype PBV GI strain; green triangle: prototype PBV GII strain; blue squares: PBV-like viruses that use alternative genetic code and cluster separately from PBVs using the standard genetic code; red squares: PBV-like strains that use alternative genetic code, yet cluster within PBVs using standard genetic code. Full article ">

20 pages, 4256 KiB

Open AccessArticle

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins

by Hao Chang, Lowela Siarot, Ryosuke Matsuura, Chieh-Wen Lo, Hirotaka Sato, Hiroyuki Otsuki and Yoko Aida

Viruses 2020, 12(1), 98; https://doi.org/10.3390/v12010098 - 15 Jan 2020

Cited by 6 | Viewed by 3465

Abstract

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of [...] Read more.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G₂/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10. Full article

(This article belongs to the Section Animal Viruses)

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27 pages, 6039 KiB

Open AccessArticle

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

by Aaron E. Lin, William E. Diehl, Yingyun Cai, Courtney L. Finch, Chidiebere Akusobi, Robert N. Kirchdoerfer, Laura Bollinger, Stephen F. Schaffner, Elizabeth A. Brown, Erica Ollmann Saphire, Kristian G. Andersen, Jens H. Kuhn, Jeremy Luban and Pardis C. Sabeti

Viruses 2020, 12(1), 105; https://doi.org/10.3390/v12010105 - 15 Jan 2020

Cited by 8 | Viewed by 5354

Abstract

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life [...] Read more.

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP’s roles in protein structure, virion budding, and transcription and replication. Full article

(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)

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22 pages, 13651 KiB

Open AccessArticle

An RNA Thermometer Activity of the West Nile Virus Genomic 3′-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching

by Alexandra Meyer, Marie Freier, Tobias Schmidt, Katja Rostowski, Juliane Zwoch, Hauke Lilie, Sven-Erik Behrens and Susann Friedrich

Viruses 2020, 12(1), 104; https://doi.org/10.3390/v12010104 - 15 Jan 2020

Cited by 19 | Viewed by 4172

Abstract

The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was [...] Read more.

The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was proposed to act as a replication silencer. The lower part of the 3′SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3′SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3′SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3′SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3′SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3′SL acts as an RNA thermometer that modulates flavivirus replication during host switching. Full article

(This article belongs to the Special Issue Flavivirus Replication and Pathogenesis)

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15 pages, 3737 KiB

Open AccessArticle

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

by Natasha D. Durham, Angela R. Howard, Ramesh Govindan, Fernando Senjobe, J. Maximilian Fels, William E. Diehl, Jeremy Luban, Kartik Chandran and James B. Munro

Viruses 2020, 12(1), 103; https://doi.org/10.3390/v12010103 - 15 Jan 2020

Cited by 15 | Viewed by 5651

Abstract

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential [...] Read more.

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization. Full article

(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)

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Figure 1
Site-specific fluorescent labelling of EBOV GP for smFRET. (a) Structural organization of GPΔTM. Adapted from Ref. [<a href="#B11-viruses-12-00103" class="html-bibr">11</a>]. FL, fusion loop; SP, signal peptide; TM, transmembrane helix. (b) EBOV GPΔTM and GPΔmuc were modified to contain peptide tags (bold) in GP1, between amino acid position 32 and 33, and in GP2, between amino acid positions 501 and 502, to facilitate enzymatic labelling with fluorophores at the serine residues highlighted in red. (c) Surface rendering of EBOV GPΔTM (derived from PDB accession 5JQ3 [<a href="#B11-viruses-12-00103" class="html-bibr">11</a>]) with fluorophores attached. MD simulation of fluorescently labeled GPΔTM 32-A1/501-A4 predicts an inter-fluorophore distance of approximately 30 Å. After minimization and equilibration, the time-averaged distance between the centers of mass of the fluorophores was determined through a 50-ns simulation (see <a href="#sec2-viruses-12-00103" class="html-sec">Section 2</a>). Full article ">Figure 2
Characterization of pseudovirions containing peptide tags for site-specific fluorophore attachment. (a) Infectivity of pseudovirions containing either GPΔmuc or GPΔmuc 32-A1/501-A4 using a recombinant vesicular stomatitis virus (VSV) core that encodes GFP. Viral infectivity was determined by counting eGFP-positive Vero cells 14–16 h post infection. Results are shown as the mean ± standard error (n = 3). (b) Western blot of retroviral pseudovirions containing GPΔmuc, GPΔmuc 32-A1/501-A4 or no GP after 1 h mock treatment (−) or treatment with thermolysin (+) to cleave the glycan cap and generate GPCL. The HIV structural protein p24 indicates the total amount of virus loaded. (c) Neutralization of retroviral pseudoparticles by monoclonal antibodies. Antibodies were tested at 15 µg/mL for neutralizing activity against retroviral pseudoparticles containing either GPΔmuc (black bars) or GPΔmuc 32-A1/501-A4 (red bars). Luciferase activity was detected 48 h post-infection and expressed as a percentage of infection with no antibody (No Ab). Results are shown as the mean ± standard error (n = 2). (d) ELISA for NPC1-C binding to retroviral pseudovirions containing only GPΔmuc or GPΔmuc 32-A1/501-A4, with or without thermolysin treatment to generate GPCL or GPCL 32-A1/501-A4. Results are shown as the mean ± standard error (n = 3). Full article ">Figure 3
Conformational dynamics of EBOV GPΔTM. (a) smFRET imaging of GPΔTM trimers containing a single fluorescently-labelled protomer within an otherwise wild-type GPΔTM trimer were surface-immobilized via biotin-NTA on a streptavidin-coated quartz microscope slide for smFRET imaging with TIRF microscopy. (b) Western blot of purified GPΔTM used in smFRET imaging using an anti-GP1 antibody (H3C8). (c) Representative fluorescence trace (top) showing fluorescence intensity over time for a donor (LD550; green) and acceptor (LD650; red) fluorophore pair on a single GPΔTM trimer, and the corresponding FRET trajectory (bottom). Overlaid on the FRET trajectory is the idealization (red) resulting from HMM analysis. (d) (Left) Population FRET histogram for GPΔTM trimers composed of FRET trajectories summed over the observation window. Overlaid on the histograms are Gaussian distributions with means 0.3, 0.5 and 0.7 that reflect the results of HMM analysis. N indicates the number of trimers in the population histogram. (Right) Transition density plot (TDP) displaying the relative frequencies of observed transitions generated from idealizations of individual FRET trajectories. N indicates the total number of transitions within the population of trimers analyzed. Full article ">Figure 4
Proteolytic priming and NPC1 binding to EBOV GPΔmuc pseudovirions promote a pre-existing conformation. (a) Pseudovirions containing a single fluorescently labelled protomer within an otherwise native GPΔmuc trimer were surface-immobilized on streptavidin-coated quartz slides for smFRET imaging with TIRF microscopy. (b) (Left) Structure of EBOV GPΔmuc, with subdomains labelled as in <a href="#viruses-12-00103-f001" class="html-fig">Figure 1</a>c. (Left/center) Representative fluorescence trace (top) showing fluorescence intensities over time for a donor (LD550; green) and acceptor (LD650; red) fluorophore pair on a single GPΔmuc on the surface of a pseudovirion. The corresponding FRET trajectory (bottom) is shown in blue. Overlaid on the FRET trajectory is the idealization (red) resulting from HMM analysis. (Right/center) Population FRET histogram for GPΔmuc trimers on pseudovirions composed of FRET trajectories summed over time. Overlaid on the histograms are Gaussian distributions with means 0.3, 0.5, and 0.7 that reflect the results of HMM analysis. N indicates the number of trimers in the population histogram. (Right) TDPs display the relative frequencies of observed transitions, generated from idealizations of individual FRET trajectories for the GPΔmuc population. N indicates the total number of transitions within the population of trimers analyzed. (c) Structural model and smFRET data, displayed as in (b), for GPΔmuc treated with thermolysin to remove the glycan cap (GPCL). (d) Structural models and smFRET data for GPCL bound to sNPC1-C. Structural models were adapted from PDB accessions 5JQ3 and 5F1B [<a href="#B11-viruses-12-00103" class="html-bibr">11</a>,<a href="#B14-viruses-12-00103" class="html-bibr">14</a>]. Approximate location of the viral membrane is shown. Full article ">Figure 5
Neutralizing antibodies stabilize distinct EBOV GPΔmuc conformations. Population FRET histograms and corresponding TDPs, displayed as in <a href="#viruses-12-00103-f003" class="html-fig">Figure 3</a> and <a href="#viruses-12-00103-f004" class="html-fig">Figure 4</a>, for EBOV GPΔmuc in the presence of 15 µg/mL (unless otherwise noted) of the following: (a) Base-binding antibodies KZ52, c2G4, c4G7, and ADI-15946; (b) base-binding antibodies that contact the GP2 fusion loop and adjacent GP1 protomer ADI-15878 and ADI-15742; (c) Glycan-cap-binding antibody ADI-15750; and (d) GP2 stalk-binding antibody ADI-16061. Full article ">

19 pages, 2418 KiB

Open AccessReview

Contribution of Dendritic Cells in Protective Immunity against Respiratory Syncytial Virus Infection

by Hi Eun Jung, Tae Hoon Kim and Heung Kyu Lee

Viruses 2020, 12(1), 102; https://doi.org/10.3390/v12010102 - 15 Jan 2020

Cited by 23 | Viewed by 13484

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. The socioeconomic burden of RSV infection is substantial because it leads to serious respiratory problems, subsequent hospitalization, and mortality. Despite its clinical significance, a safe and [...] Read more.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. The socioeconomic burden of RSV infection is substantial because it leads to serious respiratory problems, subsequent hospitalization, and mortality. Despite its clinical significance, a safe and effective vaccine is not yet available to prevent RSV infection. Upon RSV infection, lung dendritic cells (DCs) detecting pathogens migrate to the lymph nodes and activate the adaptive immune response. Therefore, RSV has evolved various immunomodulatory strategies to inhibit DC function. Due to the capacity of RSV to modulate defense mechanisms in hosts, RSV infection results in inappropriate activation of immune responses resulting in immunopathology and frequent reinfection throughout life. This review discusses how DCs recognize invading RSV and induce adaptive immune responses, as well as the regulatory mechanisms mediated by RSV to disrupt DC functions and ultimately avoid host defenses. Full article

(This article belongs to the Special Issue Dendritic Cells and Antiviral Defense)

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Figure 1
The structure of respiratory syncytial virus (RSV). The RSV genome is 15.2 kb of nonsegmented negative-sense RNA encoding 11 viral proteins. Viral envelope of RSV contains three transmembrane glycoproteins: attachment glycoprotein (G), fusion protein (F), and small hydrophobic protein (SH). Matrix proteins (M) are present on the inner side of the viral envelope. Viral RNA is tightly encapsidated by nucleoproteins (N) and the large proteins (L), phosphoproteins (P), and M2-1 proteins that mediate viral RNA transcription. M2-2 protein regulates viral RNA synthesis. Full article ">Figure 2
Innate sensors involved in RSV recognition, and immunomodulation strategies of RSV. Upon RSV infection, Toll-like receptors (TLR)2/6, TLR3, TLR4, TLR7, retinoic acid-inducible gene-I (RIG-I), and nucleotide-binding oligomerization domain (NOD2) are responsible for recognizing RSV pathogen-associated molecular patterns (PAMPs) in dendritic cells (DCs). The recognition of PAMPs by pattern recognition receptors (PRRs) activates downstream signaling pathways, which trigger DC activation and cytokine production. To avoid host immune responses, RSV has evolved various immunomodulatory strategies that inhibit DC functions. RSV proteins, specifically proteins G, NS1/NS2, and N, contribute to immunomodulation of RSV. Full article ">Figure 3
Lung dendritic cell subsets. Lung DCs are classified into conventional DC1s (cDC1s), cDC2s, and plasmacytoid DCs (pDCs). Each DC subset is widely distributed throughout the lungs and migrates to the lung-draining lymph node when they recognize RSV to initiate protective immune responses. cDC1s preferentially activate CD8+ T cells that mediate viral clearance, and cDC2s are responsible for Th2-mediated immune responses and RSV-mediated pulmonary diseases. pDCs are the main source of type I interferons (IFNs) and play an essential role in RSV-specific cytotoxic T lymphocyte (CTL) priming and regulation of disease severity. * Human-specific marker. Full article ">

11 pages, 1261 KiB

Open AccessArticle

Hepatitis C Virus Affects Tuberculosis-Specific T Cells in HIV-Negative Patients

by Mohamed Ahmed El-Mokhtar, Sherein G. Elgendy, Abeer Sharaf Eldin, Elham Ahmed Hassan, Ali Abdel Azeem Hasan, Muhamad R. Abdel Hameed, Douaa Sayed and Eman H. Salama

Viruses 2020, 12(1), 101; https://doi.org/10.3390/v12010101 - 15 Jan 2020

Cited by 9 | Viewed by 3142

Abstract

The occurrence of tuberculosis (TB) and hepatitis C virus (HCV) infections in the same patient presents a unique clinical challenge. The impact of HCV infection on the immune response to TB remains poorly investigated in TB⁺/HCV⁺ patients. This study was [...] Read more.

The occurrence of tuberculosis (TB) and hepatitis C virus (HCV) infections in the same patient presents a unique clinical challenge. The impact of HCV infection on the immune response to TB remains poorly investigated in TB⁺/HCV⁺ patients. This study was conducted to evaluate the impact of HCV on the T-cell-mediated immune response to TB in coinfected patients. Sixty-four patients with active TB infections were screened for coinfection with HCV. The expression of immune activation markers IFN-γ, CD38, and HLA-DR on TB-specific CD4⁺ T cells was evaluated by flow cytometry in TB-monoinfected patients, TB/HCV-coinfected patients, and healthy controls. IL-2, IL-4, IFN-γ, TNF-α, and IL-10 levels were measured using ELISA. The end-of-treatment response to anti-TB therapy was recorded for both patient groups. Significantly lower levels of CD4⁺IFN-γ⁺CD38⁺ and CD4⁺IFN-γ⁺HLA-DR⁺ T cells were detected in TB/HCV-coinfected patients compared to TB monoinfected patients and controls. TB⁺/HCV⁺-coinfected patients showed higher serum levels of IL-10. The baseline frequencies of TB-specific activated T-cell subsets did not predict the response to antituberculous therapy in TB⁺/HCV⁺ patients. We concluded that different subsets of TB-specific CD4⁺ T cells in TB/HCV-infected individuals are partially impaired in early-stage HCV infection. This was combined with increased serum IL-10 level. Such immune modulations may represent a powerful risk factor for disease progression in patients with HCV/TB coinfection. Full article

(This article belongs to the Section Animal Viruses)

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14 pages, 3251 KiB

Open AccessArticle

Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry

by Tran Bac Le, Hye Kwon Kim, Woonsung Na, Van Phan Le, Min-Suk Song, Daesub Song, Dae Gwin Jeong and Sun-Woo Yoon

Viruses 2020, 12(1), 100; https://doi.org/10.3390/v12010100 - 15 Jan 2020

Cited by 10 | Viewed by 5347

Abstract

Since the initial detection of H5N1, a highly pathogenic avian influenza (HPAI) virus, in 1996 in China, numerous HPAI H5 lineages have been classified, and they continue to pose a threat to animal and human health. In this study, we developed a novel [...] Read more.

Since the initial detection of H5N1, a highly pathogenic avian influenza (HPAI) virus, in 1996 in China, numerous HPAI H5 lineages have been classified, and they continue to pose a threat to animal and human health. In this study, we developed a novel primer/probe set that can be employed to simultaneously detect pan-H5 HPAI and two clades, 2.3.2.1 and 2.3.4.4, of H5Nx viruses using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The sensitivity and specificity of these primer sets and probes were confirmed with a number of different subtypes of influenza virus and the H5-HA gene plasmid DNA. In particular, the multiplex RT-qPCR assay was successfully applied to the simultaneous detection of H5 HPAI and different virus clades in clinical field samples from a poultry farm. Therefore, this multiplex assay and a novel detection primer set and probes will be useful for the laboratory diagnosis and epidemiological field studies of different circulating H5 HPAI virus clades in poultry and migratory wild birds. Full article

(This article belongs to the Section Animal Viruses)

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Design of the primer/probe set for the one-step multiplex RT-qPCR assay. Alignment of nucleotide sequences within the hemagglutinin (HA) gene of H5Nx avian influenza viruses that are representative of highly pathogenic avian influenza (HPAI) clades and low pathogenic avian influenza (LPAI) virus. The sequences of primers and probes specific to each target are on black background, and the reverse primer and probe specific for clade 2.3.2.1 show reverse complement sequence (R = A/G, Y = C/T, V = A/C/G). Full article ">Figure 2
Amplification plots and standard curves of the singleplex and multiplex realtime-qPCR assays using target-specific plasmid DNA. Comparison of the singleplex (A,C,E) and multiplex (B,D,F) realtime-qPCR assays was performed using standard plasmids corresponding to H5 HPAI and clades 2.3.4.4 and 2.3.2.1; each plasmid solution was serially diluted down to 5 × 108 to 5 × 100 copies of plasmid DNA/microliter. The cycle threshold in amplification plots, correlation coefficient (R2) and slope of the standard curve for the assays were drawn automatically by Lightcycler 96 software. Each concentration had triple replicates. Full article ">Figure 3
The one-step multiplex RT-qPCR assay for the detection of pan-H5 HPAI (A), H5 HPAI 2.3.2.1 clade (B), and H5 HPAI 2.3.4.4 clade (C) in field-infected feces samples. The validity of this assay was confirmed using M-gene positive field samples, H5Nx HPAI viruses for positive control, and Newcastle Disease virus for negative control. The results were visualized using an intuitive heat-map (green color-positive result, red color-negative result). Each concentration had triple replicates. (D) Clade classification of HPAI H5 subtype viruses from field-infected feces samples. The HA1 partial sequences of the HA gene with multiple basic cleavage sites were obtained by conventional RT-PCR and all H5 HPAI-positive sequences were verified using the Influenza Research Database (<a href="http://www.fludb.org" target="_blank">www.fludb.org</a>). Full article ">Figure 3 Cont.
The one-step multiplex RT-qPCR assay for the detection of pan-H5 HPAI (A), H5 HPAI 2.3.2.1 clade (B), and H5 HPAI 2.3.4.4 clade (C) in field-infected feces samples. The validity of this assay was confirmed using M-gene positive field samples, H5Nx HPAI viruses for positive control, and Newcastle Disease virus for negative control. The results were visualized using an intuitive heat-map (green color-positive result, red color-negative result). Each concentration had triple replicates. (D) Clade classification of HPAI H5 subtype viruses from field-infected feces samples. The HA1 partial sequences of the HA gene with multiple basic cleavage sites were obtained by conventional RT-PCR and all H5 HPAI-positive sequences were verified using the Influenza Research Database (<a href="http://www.fludb.org" target="_blank">www.fludb.org</a>). Full article ">

11 pages, 546 KiB

Open AccessArticle

Chronic Hepatitis B Virus Infection Associated with Increased Colorectal Cancer Risk in Taiwanese Population

by Fu-Hsiung Su, Thi Nga Le, Chih-Hsin Muo, Sister Arlene Te, Fung-Chang Sung and Chih-Ching Yeh

Viruses 2020, 12(1), 97; https://doi.org/10.3390/v12010097 - 14 Jan 2020

Cited by 21 | Viewed by 3816

Abstract

Chronic hepatitis B virus (HBV) infections and colorectal cancer (CRC) are prevalent in Taiwan. We carried out a population-based case-control study to assess the association between HBV infection and CRC risk. Using the National Health Insurance Research Database of Taiwan, we identified 69,478 [...] Read more.

Chronic hepatitis B virus (HBV) infections and colorectal cancer (CRC) are prevalent in Taiwan. We carried out a population-based case-control study to assess the association between HBV infection and CRC risk. Using the National Health Insurance Research Database of Taiwan, we identified 69,478 newly diagnosed patients with CRC from 2005 to 2011. We further randomly selected 69,478 age- and gender-matched controls without CRC from the same database. Odds ratios (ORs) were calculated to evaluate the association between chronic HBV infection and CRC using a logistic regression analysis. HBV infection was found to be associated with the risk of CRC (OR = 1.27, 95% confidence interval (CI) = 1.20–1.33). This relationship was similar in men and women. Age-specific analysis revealed that the CRC risk associated with HBV decreased with age. The adjusted ORs for patients aged <55, 55–64, and 65–74 years were 1.63 (95% CI = 1.48–1.79), 1.24 (95% CI = 1.13–1.37), and 1.02 (95% = 0.92–1.13), respectively. In conclusion, this study suggests that chronic HBV infection is significantly associated with an increased risk of CRC. Monitoring the risk of CRC development in young patients with HBV infection is crucial. Full article

(This article belongs to the Section Animal Viruses)

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23 pages, 3398 KiB

Open AccessArticle

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016

by Paulina Rajko-Nenow, Vasiliki Christodoulou, William Thurston, Honorata M. Ropiak, Savvas Savva, Hannah Brown, Mehnaz Qureshi, Konstantinos Alvanitopoulos, Simon Gubbins, John Flannery and Carrie Batten

Viruses 2020, 12(1), 96; https://doi.org/10.3390/v12010096 - 14 Jan 2020

Cited by 10 | Viewed by 4643

Abstract

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the [...] Read more.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2–5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42–98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July–September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus. Full article

(This article belongs to the Section Animal Viruses)

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Phylogenetic trees were constructed for the coding regions of BTV: (a) VP2 protein (Segment 2); (b) VP4 protein (Segment 4); (c) VP7 protein (Segment 7); (d) VP6 protein (Segment 9); and (e) NS3 protein (Segment 10). Maximum likelihood trees were constructed using IQ-Tree software [<a href="#B25-viruses-12-00096" class="html-bibr">25</a>] and the reliability of each tree was estimated by ultrafast bootstrap [<a href="#B26-viruses-12-00096" class="html-bibr">26</a>] analysis of 1000 replicates (bootstrap values of < 95% are not displayed). Full article ">Figure 1 Cont.
Phylogenetic trees were constructed for the coding regions of BTV: (a) VP2 protein (Segment 2); (b) VP4 protein (Segment 4); (c) VP7 protein (Segment 7); (d) VP6 protein (Segment 9); and (e) NS3 protein (Segment 10). Maximum likelihood trees were constructed using IQ-Tree software [<a href="#B25-viruses-12-00096" class="html-bibr">25</a>] and the reliability of each tree was estimated by ultrafast bootstrap [<a href="#B26-viruses-12-00096" class="html-bibr">26</a>] analysis of 1000 replicates (bootstrap values of < 95% are not displayed). Full article ">Figure 1 Cont.
Phylogenetic trees were constructed for the coding regions of BTV: (a) VP2 protein (Segment 2); (b) VP4 protein (Segment 4); (c) VP7 protein (Segment 7); (d) VP6 protein (Segment 9); and (e) NS3 protein (Segment 10). Maximum likelihood trees were constructed using IQ-Tree software [<a href="#B25-viruses-12-00096" class="html-bibr">25</a>] and the reliability of each tree was estimated by ultrafast bootstrap [<a href="#B26-viruses-12-00096" class="html-bibr">26</a>] analysis of 1000 replicates (bootstrap values of < 95% are not displayed). Full article ">Figure 1 Cont.
Phylogenetic trees were constructed for the coding regions of BTV: (a) VP2 protein (Segment 2); (b) VP4 protein (Segment 4); (c) VP7 protein (Segment 7); (d) VP6 protein (Segment 9); and (e) NS3 protein (Segment 10). Maximum likelihood trees were constructed using IQ-Tree software [<a href="#B25-viruses-12-00096" class="html-bibr">25</a>] and the reliability of each tree was estimated by ultrafast bootstrap [<a href="#B26-viruses-12-00096" class="html-bibr">26</a>] analysis of 1000 replicates (bootstrap values of < 95% are not displayed). Full article ">Figure 1 Cont.
Phylogenetic trees were constructed for the coding regions of BTV: (a) VP2 protein (Segment 2); (b) VP4 protein (Segment 4); (c) VP7 protein (Segment 7); (d) VP6 protein (Segment 9); and (e) NS3 protein (Segment 10). Maximum likelihood trees were constructed using IQ-Tree software [<a href="#B25-viruses-12-00096" class="html-bibr">25</a>] and the reliability of each tree was estimated by ultrafast bootstrap [<a href="#B26-viruses-12-00096" class="html-bibr">26</a>] analysis of 1000 replicates (bootstrap values of < 95% are not displayed). Full article ">Figure 2
(a) The Neighbor-Net network was estimated from an alignment of 18,621 characters for each of 73 taxa, which sequence composed of the concatenated coding regions of BTV (VP1–VP7 and NS1–NS3); and (b) genogroup D of the Neighbor-Net network. Nomenclature used in-line with that proposed by the BTV-GLUE resource for the BTV Segment 2. Solid coloured lines were added manually to indicate the genogroup while the dashed blue lines were added manually to indicate the distinct differences between the western and eastern topotypes, and genogroup K which contains the atypical BTV strains. Full article ">Figure 2 Cont.
(a) The Neighbor-Net network was estimated from an alignment of 18,621 characters for each of 73 taxa, which sequence composed of the concatenated coding regions of BTV (VP1–VP7 and NS1–NS3); and (b) genogroup D of the Neighbor-Net network. Nomenclature used in-line with that proposed by the BTV-GLUE resource for the BTV Segment 2. Solid coloured lines were added manually to indicate the genogroup while the dashed blue lines were added manually to indicate the distinct differences between the western and eastern topotypes, and genogroup K which contains the atypical BTV strains. Full article ">Figure 3
Maps of the near-surface history of air passing over the Larnaca region, aggregated over the period 21 July to 18 September 2016. Air parcels with a lifetime of (a) 24 h and (b) 36 h are shown. Full article ">

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