Viruses

24 pages, 1121 KiB

Open AccessReview

Drug Repositioning: New Approaches and Future Prospects for Life-Debilitating Diseases and the COVID-19 Pandemic Outbreak

by Zheng Yao Low, Isra Ahmad Farouk and Sunil Kumar Lal

Viruses 2020, 12(9), 1058; https://doi.org/10.3390/v12091058 - 22 Sep 2020

Cited by 107 | Viewed by 9399

Abstract

Traditionally, drug discovery utilises a de novo design approach, which requires high cost and many years of drug development before it reaches the market. Novel drug development does not always account for orphan diseases, which have low demand and hence low-profit margins for [...] Read more.

Traditionally, drug discovery utilises a de novo design approach, which requires high cost and many years of drug development before it reaches the market. Novel drug development does not always account for orphan diseases, which have low demand and hence low-profit margins for drug developers. Recently, drug repositioning has gained recognition as an alternative approach that explores new avenues for pre-existing commercially approved or rejected drugs to treat diseases aside from the intended ones. Drug repositioning results in lower overall developmental expenses and risk assessments, as the efficacy and safety of the original drug have already been well accessed and approved by regulatory authorities. The greatest advantage of drug repositioning is that it breathes new life into the novel, rare, orphan, and resistant diseases, such as Cushing’s syndrome, HIV infection, and pandemic outbreaks such as COVID-19. Repositioning existing drugs such as Hydroxychloroquine, Remdesivir, Ivermectin and Baricitinib shows good potential for COVID-19 treatment. This can crucially aid in resolving outbreaks in urgent times of need. This review discusses the past success in drug repositioning, the current technological advancement in the field, drug repositioning for personalised medicine and the ongoing research on newly emerging drugs under consideration for the COVID-19 treatment. Full article

(This article belongs to the Special Issue Drug-Repositioning Opportunities for Antiviral Therapy)

► Show Figures

Figure 1

18 pages, 2210 KiB

Open AccessArticle

An Unconventional Flavivirus and Other RNA Viruses in the Sea Cucumber (Holothuroidea; Echinodermata) Virome

by Ian Hewson, Mitchell R. Johnson and Ian R. Tibbetts

Viruses 2020, 12(9), 1057; https://doi.org/10.3390/v12091057 - 22 Sep 2020

Cited by 14 | Viewed by 5312

Abstract

Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting eight species (representing four families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a [...] Read more.

Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting eight species (representing four families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a hitherto undocumented wasting disease. The RNA virome comprised genome fragments of both single-stranded positive sense and double stranded RNA viruses, including those assigned to the Picornavirales, Ghabrivirales, and Amarillovirales. We discovered an unconventional flavivirus genome fragment which was most similar to a shark virus. Ghabivirales-like genome fragments were most similar to fungal totiviruses in both genome architecture and homology and had likely infected mycobiome constituents. Picornavirales, which are commonly retrieved in host-associated viral metagenomes, were similar to invertebrate transcriptome-derived picorna-like viruses. The greatest number of viral genome fragments was recovered from the wasting A. californicus library compared to the asymptomatic A. californicus library. However, reads from the asymptomatic library recruited to nearly all recovered wasting genome fragments, suggesting that they were present but not well represented in the grossly normal specimen. These results expand the known host range of flaviviruses and suggest that fungi and their viruses may play a role in holothurian ecology. Full article

(This article belongs to the Collection Unconventional Viruses)

► Show Figures

Figure 1

Figure 1
Contig map of Apostichopus californicus flavivirus-like contig 91. The open reading frame matched a flavivirus polyprotein by BLASTx [<a href="#B37-viruses-12-01057" class="html-bibr">37</a>]. Methyltransferase, NS5 and helicase domains were identified by comparison against the conserved domain database (CDD) at NCBI [<a href="#B41-viruses-12-01057" class="html-bibr">41</a>]. The location of the envelope region was determined by protein folding comparison in Phyre [<a href="#B39-viruses-12-01057" class="html-bibr">39</a>]. The hairpin like structure preceding the Envelope region was determined by folding all sites between start (AUG) codons by mFold [<a href="#B40-viruses-12-01057" class="html-bibr">40</a>]. Full article ">Figure 2
Phylogenetic representation of Apostichopus californicus flavivirus-like contig 91. The tree was constructed by performing an alignment of overlapping regions with best BLASTx matches at NCBI using the CLC Sequence Viewer 8.0 native alignment algorithm. The tree is based on a ~420 amino acid alignment by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. The green branches indicate the emerging aquatic and invertebrate-associated flavivirus clade [<a href="#B52-viruses-12-01057" class="html-bibr">52</a>]. An additional phylogenetic representation based on maximum likelihood is provided in the <a href="#app1-viruses-12-01057" class="html-app">Supplementary Materials</a>. Full article ">Figure 3
Contig maps for Picornavirales-like genome fragments recovered from holothurians by viral metagenomics. The expect values of best matches by BLASTx against the non-redundant database at NCBI are indicated to the right of each contig. Solid lines indicate the total contig length, and arrows indicate open reading frames. Colored bars indicate shared homology between contigs based on reciprocal tBLASTx. Full article ">Figure 4
Phylogenetic representation of holothurian-associated Picornavirales-like genome fragments. The tree was constructed by performing an alignment of an overlapping region (~100 amino acid) of the rhv domain with best BLASTx matches in the non-redundant database at NCBI. The tree was constructed by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. An additional phylogenetic representation based on maximum likelihood is provided in the <a href="#app1-viruses-12-01057" class="html-app">Supplementary Materials</a>. Full article ">Figure 5
Contig maps for totivirus-like genome fragments recovered from holothurians. The expected values of best matches by BLASTx [<a href="#B37-viruses-12-01057" class="html-bibr">37</a>] to the non-redundant database at NCBI are indicated to the right of the contigs. Solid lines indicate the total contig length, and arrows indicate open reading frames. RdRp = RNA-dependent RNA polymerase, Cp = capsid protein. Full article ">Figure 6
Phylogenetic representations of holothurian-associated totivirus-like genome fragments. The trees were constructed by performing an alignment of an overlapping region of the RdRp (top) and Cp (bottom) domains with best BLASTx matches at NCBI. The trees are based on ~156 amino acid (for RdRp) and 87 amino acid (for Cp) alignments by neighbor joining and based on Jukes-Cantor distance. Values above nodes indicate bootstrap statistics (>50%) based on 1000 iterations. An additional phylogenetic representation based on maximum likelihood is provided in the <a href="#app1-viruses-12-01057" class="html-app">Supplementary Materials</a>. Full article ">

23 pages, 7699 KiB

Open AccessArticle

A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes

by Caitlin A. O’Brien, Cassandra L. Pegg, Amanda S. Nouwens, Helle Bielefeldt-Ohmann, Bixing Huang, David Warrilow, Jessica J. Harrison, John Haniotis, Benjamin L. Schulz, Devina Paramitha, Agathe M. G. Colmant, Natalee D. Newton, Stephen L. Doggett, Daniel Watterson, Jody Hobson-Peters and Roy A. Hall

Viruses 2020, 12(9), 1056; https://doi.org/10.3390/v12091056 - 22 Sep 2020

Cited by 9 | Viewed by 4234

Abstract

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. [...] Read more.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes. Full article

(This article belongs to the Special Issue Mosquito-Specific Viruses: Their Role in Nature and Potential Use for Vector Control or Control of Arboviral Pathogens)

► Show Figures

Figure 1

Figure 1
Isolation and prevalence of Aedes birnavirus (ABV). (a) Summary of screening for ABV and known insect-specific viruses (ISVs) in MAVRIC-positive mosquito pools collected in NSW between 2013 and 2014. (b) Anti-double-stranded RNA (dsRNA) immunolabeling in C6/36 cells inoculated with mosquito pools 177833 and 178287, or mock infected (media only) in immunofluorescence assay. Images were taken at 63× magnification. Green: dsRNA. Blue: nuclei. (c) C6/36 cells infected with ABV isolates 177833 and 178287, NDiV/LNV co-infected isolate 179853 and mock-infected cells. Gaps in the monolayer typical of NDiV/LNV infection are depicted by arrows for isolate 179853. Cell monolayer images were taken at 20x magnification. * Screened by RT-PCR with ABV-specific primers. The † LNV/NDiV-positive pool did not contain ABV. ‡ Ballina LNV/NDiV isolate originally reported in Prow et al. [<a href="#B28-viruses-12-01056" class="html-bibr">28</a>]. Full article ">Figure 2
Phylogenetic analysis of ABV VP1. Unrooted maximum likelihood trees constructed from (a) VP1 nucleotide coding sequences (CDS) aligned using the MUSCLE algorithm and (b) VP1 amino acid sequences aligned with MAFFT. The broad host-species classification for each virus is indicated by icons next to the virus name. Currently recognized genera are indicated in italics. Maximum likelihood analyses were performed in MEGA 7.0.26 using the general time-reversible (GTR) model with gamma distribution (5 categories ( + G, parameter = 1.8479)) and invariable variation for some sites ([ + I], 2.92% sites) for nucleotide sequences and the Jones–Taylor–Thornton (JTT) model with gamma substitutions and no deletions for amino acid sequences. Full article ">Figure 3
Phylogenetic analysis of ABV polyprotein. Unrooted maximum likelihood trees constructed from (a) nucleotide coding sequences (CDS) for birnavirus polyproteins aligned using the MUSCLE algorithm and (b) polyprotein amino acid sequences aligned with MAFFT. The broad host-species classification for each virus is by icons next to the virus name. Currently recognized genera are indicated in italics. Maximum likelihood analyses were performed in MEGA 7.0.26 using the general time-reversible (GTR) model with gamma distribution (5 categories ( + G, parameter = 1.8479)) and invariable variation for some sites ([ + I], 2.92% sites) for nucleotide sequences and the Jones–Taylor–Thornton (JTT) model with gamma substitutions and no deletions for amino acid sequences. Full article ">Figure 4
Protein analysis of ABV. (a) Transmission electron micrograph of purified ABV particles negatively stained with uranyl acetate and imaged at 200 K. Scale bar represents 100 nm. (b) Infectivity of two preparations of CsCl gradient-purified ABV particles in C6/36 cells as compared to ABV-infected cell supernatant used at a known MOI (0.1). Values graphed are averaged virus titers in supernatants taken from infected cells at 7 days post-infection (dpi). Error bars represent standard deviation in titers between 3 replicate wells. (c) Ruby stained image of SDS-PAGE analysis on purified ABV boiled and reduced with 1 M DTT, and results of mass spectrometry identification of excised protein bands as indicated by arrows. Protein ID refers to top ranking proteins identified in each band by mass spectrometry analysis. * No. peptides, number of peptides identified with equal to or greater than 50% confidence; † Coverage, percentage of protein covered by peptides identified with >50% confidence. (d) Ruby stained image of SDS-PAGE analysis of ABV further denatured in 3 M urea. Protein identities based on mass spectrometry are indicated next to each band. Kaleidoscope protein ladder was run alongside all samples. Full article ">Figure 5
Digestion of ABV proteins by trypsin and chymotrypsin. Schematic showing ABV polyprotein sequence identified by mass spectrometry following digestion with (a) trypsin or (b) chymotrypsin. Sequence detected by mass spectrometry is highlighted in grey and underlined. Colored bars above sequence denote predicted individual proteins: pVP2, magenta; VP4, green; VP3, black. Full article ">Figure 6
Clustal W alignment of amino acid polyprotein sequences for ABV, RBV (CAX33877), IBDV (ANY27027), BSNV (CAD30689), ESV (AEW87521), DXV (NP_690836) and IPNV (NP_047196). Non-conserved insertions of 61 and 85 amino acids in the ABV polyprotein are indicated by red lines. Vertical lines in magenta depict published polyprotein cleavage sites for these birnaviruses [<a href="#B41-viruses-12-01056" class="html-bibr">41</a>,<a href="#B42-viruses-12-01056" class="html-bibr">42</a>]. ★ Denotes predicted cleavage sites for ABV based on mass spectrometry data; Molecular weights predicted using ExPASy compute pI/MW tool are marked at the c-terminus of each resulting protein. Green box, pVP2. Purple box, VP4; brown box, VP3. Full article ">Figure 7
Antibody response to ABV. (a) Reactivity of anti-ABV mouse immune serum to ABV in Western blot. Lane 1, supernatant from ABV-infected C6/36 cells grown in minimal (< 1%) FBS; 2, ABV virus stock supernatant from C6/36 cells, supplemented with 10% FBS; 3, CsCl gradient-purified preparation one of ABV in PBS; 4, CsCl gradient-purified preparation two of ABV in PBS. U, unreduced; BR, boiled and reduced. (b) Summary of ABV-specific monoclonal antibody panel. † Most likely target based on neutralizing activity. * Highest reciprocal dilution at which virus infectivity is inhibited. –, OD450 nm <0.25 and less than two times the average OD of the negative control; +, OD450 nm is >0.5 and at least two times greater than the average OD of negative control; +/-, positive result but inconsistent between replicates. Full article ">Figure 8
ABV replication in mosquito cell lines. (a) IFA performed on mosquito cells infected with ABV or mock infected (media only) fixed with 4% formaldehyde (FA) and 0.5% Triton X-100 at 7 dpi and immunolabeled for dsRNA (green) and nuclei (blue). Main panel images taken at 40x and inset at 63x magnifications. (b) Mean titers in supernatants harvested from mosquito cells at 2 hpi (white) and 7 dpi (black). Error bars represent standard deviations between three replicates. Statistical significance was calculated by t tests using the Holm–Sidak method, with alpha = 0.05. Each row was analyzed individually, without assuming a consistent SD. ****, p < 0.0001; *, p < 0.05; ns, not significant. (c) Localization of dsRNA (red) in ABV-infected mosquito cells fixed with either 100% acetone, or 4% formaldehyde + 0.5% Triton X-100. DsRNA (red); nuclei (blue). Images taken at 63x magnification. (d) ABV or mock-infected mosquito cells stained with VP3- and VP2-specific mAbs. ABV protein (green); nuclei (blue). Images taken at 40x magnification. C6/36, RNAi-deficient Ae. albopictus; RML-12, RNAi-competent Ae. albopictus; Chao Ball, Culex tarsalis; Mos55, Anopheles gambiae. Full article ">

16 pages, 4373 KiB

Open AccessArticle

The Epidemiological Signature of Pathogen Populations That Vary in the Relationship between Free-Living Parasite Survival and Virulence

by Lourdes M. Gomez, Victor A. Meszaros, Wendy C. Turner and C. Brandon Ogbunugafor

Viruses 2020, 12(9), 1055; https://doi.org/10.3390/v12091055 - 22 Sep 2020

Cited by 6 | Viewed by 4042

Abstract

The relationship between parasite virulence and transmission is a pillar of evolutionary theory that has implications for public health. Part of this canon involves the idea that virulence and free-living survival (a key component of transmission) may have different relationships in different host–parasite [...] Read more.

The relationship between parasite virulence and transmission is a pillar of evolutionary theory that has implications for public health. Part of this canon involves the idea that virulence and free-living survival (a key component of transmission) may have different relationships in different host–parasite systems. Most examinations of the evolution of virulence-transmission relationships—Theoretical or empirical in nature—Tend to focus on the evolution of virulence, with transmission being a secondary consideration. Even within transmission studies, the focus on free-living survival is a smaller subset, though recent studies have examined its importance in the ecology of infectious diseases. Few studies have examined the epidemic-scale consequences of variation in survival across different virulence–survival relationships. In this study, we utilize a mathematical model motivated by aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) natural history to investigate how evolutionary changes in survival may influence several aspects of disease dynamics at the epidemiological scale. Across virulence–survival relationships (where these traits are either positively or negatively correlated), we found that small changes (5% above and below the nominal value) in survival can have a meaningful effect on certain outbreak features, including R₀, and on the size of the infectious peak in the population. These results highlight the importance of properly understanding the mechanistic relationship between virulence and parasite survival, as the evolution of increased survival across different relationships with virulence may have considerably different epidemiological signatures. Full article

(This article belongs to the Special Issue Virus Ecology and Evolution: Current Research and Future Directions)

► Show Figures

Figure 1

Figure 1
Compartmental diagram of the SEAIR-W (susceptible-exposed-asymptomatic-infected-recovered) version of a-WAIT (Waterborne Abiotic or other Indirect Transmission)) model: this is based on a previously developed mathematical model used to interrogate environmental transmission of SARS-CoV-2 (see [<a href="#B22-viruses-12-01055" class="html-bibr">22</a>]). Full article ">Figure 2
Tornado plot showing the sensitivity of epidemic properties to individual parameter changes: (A) the number of infected individuals (asymptomatic and symptomatic) at the epidemic peak; (B) the rate at which the epidemic peak is reached, tmax−1; (C) the total infected population after 30 days; and (D) the basic reproductive ratio (R0). Filled bars indicate the value of the epidemic feature when the associated parameter is increased by 5.0% from its nominal value. White bars indicate the value of a feature when the associated parameter is decreased by 5.0%. Blue coloring with checkered patterning indicates a parameter associated with survival, and orange coloring with lined patterning indicates a parameter associated with virulence. Full article ">Figure 3
Sample dynamics for the model system: (A) the dynamics for all host compartments within the model and (B) the fraction of environmental reservoirs in a setting that are contaminated with infectious virus. Full article ">Figure 4
Heatmap describing the impact of varying virus virulence and survival trait values assessed across four key epidemic metrics: these heatmaps express the change in (A) the number of infected individuals (asymptomatic and symptomatic) at the epidemic peak; (B) the rate at which the epidemic peak is reached, tmax−1; (C) the total infected population after 30 days; and (D) the basic reproductive ratio (R0) when virulence and survival are modulated by ±5% within the model. Contour lines are available for clarity. Full article ">Figure 5
The expected effect of increasing survival on virulence for the two correlation models considered: here, we present a schematic of how the different hypotheses for the relationship between virulence and survival manifest on a map with a structure similar to the heat maps shown in <a href="#viruses-12-01055-f004" class="html-fig">Figure 4</a>. The directions of the arrows depict how increasing survival would affect virulence under the two hypotheses: the blue arrow indicates the flow of an increasing positive correlation dynamic, while the direction of the orange arrow indicates an increasing negative correlation dynamic. Full article ">Figure 6
The percent change in SEAIR-W outbreak metrics as survival increases from −5% to +5% for different virulence–survival relationships (positive correlation and negative correlation). For each metric analyzed, we present the percent difference between the minimum and maximum survival values given the two hypotheses tested: (i) positive correlation between survival and virulence (comparing low virulence/low survival to high virulence/high survival) and (ii) negative correlation (high virulence/low survival to high survival/low virulence). The bars here correspond to the values (percent) in the third columns of <a href="#viruses-12-01055-t005" class="html-table">Table 5</a> and <a href="#viruses-12-01055-t006" class="html-table">Table 6</a>, which denote the differences between the minimum and maximum values. Full article ">Figure 7
Virus outbreak dynamics for the extreme values of virulence and free-living survival considered for the two different relationships (positive or negative) between virulence and survival traits: these plots are similar to the illustrative dynamics in <a href="#viruses-12-01055-f001" class="html-fig">Figure 1</a>. Here, we observe the dynamics of disease corresponding to the extreme values presented in <a href="#viruses-12-01055-t005" class="html-table">Table 5</a> and <a href="#viruses-12-01055-t006" class="html-table">Table 6</a>. Subfigures (A,C,E,G) depict disease dynamics, and (B,D,F,H) depict the dynamics of contaminated environments. (A–D) correspond to the parameter values considered for the positive correlation scenario, while (E–H) correspond to the negative correlation scenario. Full article ">

13 pages, 2477 KiB

Open AccessArticle

Identification of a Membrane Binding Peptide in the Envelope Protein of MHV Coronavirus

by Entedar A. J. Alsaadi, Benjamin W. Neuman and Ian M. Jones

Viruses 2020, 12(9), 1054; https://doi.org/10.3390/v12091054 - 22 Sep 2020

Cited by 9 | Viewed by 5405

Abstract

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in [...] Read more.

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding. Full article

(This article belongs to the Collection Coronaviruses)

► Show Figures

Figure 1

Figure 1
Multiple sequence alignment of coronavirus E protein. Alignment was done using Jalview 2.9.0b2 and the position of key features, TM, EPTM, Cys region and conserved prolines indicated. Four genera of coronavirus are represented as follows: α-CoV is represented by HCOV-229E, Human coronavirus 229E (NP_073554.1); M-BatCoV-HKU8, Miniopterus bat coronavirus Hong Kong University 8 (YP_001718614.1); TGEV-Purdue, transmissible gastroenteritis virus- Purdue (ABG89336.1); HCoV-NL63, Human coronavirus NL63 (YP_003769.1); PEDV, Porcine epidemic diarrhea virus (NP_598312.1); FCoV, Feline coronavirus (YP_004070197.1); β-CoV include HCoV-HKU1, Human coronavirus Hong Kong University 1(YP_173240.1); MHV-A59, Murine hepatitis virus-A59 (NP_068673.1); SARS-CoV, Severe acute respiratory syndrome coronavirus (NP_828854.1); MERS-CoV, Middle East respiratory syndrome coronavirus (YP_009047209.1); BCoV-HKU9, Bat coronavirus Hong Kong University 9 (YP_001039973.1); γ-CoV include IBV, Infectious bronchitis virus (ADP06512.1); SW1, sperm Whale coronavirus 1(YP_001876438.1); BdCoV-HKU22, Bottlenose dolphin coronavirus Hong Kong University 22 (AHB63482.1); δ-CoV consists of NHCoV-HKU19, Night-heron-coronavirus- Hong Kong University 19 (AFD29227.1); PD-CoV, Porcine coronavirus Hong Kong University 15 (YP_005352832.1); MCoV-HKU13, Munia coronavirus Hong Kong University 13-3514(YP_002308507.1). TM: transmembrane domain; highly conserved cysteine residues indicated; conserved proline indicated by stars. Blue represents hydrophobic amino acids (A, I, L, M, F, W, V); Red represents positive charge amino acids (K, R); Magenta represents negative charge amino acids (E, D); Green represents polar amino acids (N, Q, S, T); Pink represents cysteines (C); Orange represents glycines (G); Yellow represents prolines (P); Cyan represents aromatic amino acids (H, Y); White represents any unconserved/gap. The SARS-CoV-2 E protein is 95% identical to SARS E, so was not included as a distinct entry. Full article ">Figure 2
Effect of MHV E protein-derived peptides on size and shape of GUVs. (A) Fluorescent images of electroformed GUVs treated with peptides MHV-EPTM, MHV-ETM and M2-Infleunza and imaged at 0, 1, 2 and 5 min postaddition. (B) GUV relative size estimated by Ramanujan’s first approximation. Then standard deviation for both long and short measurements for a vesicle was also measured and averaged for each GUV for three separate experiments of 40 GUVs each. (C) GUV shape, measured as the ratio between the longest and shortest radii and averaged for each GUV for three separate experiments of 40 GUVs each. The scale bar indicates 20 µm. Error bars shown are mean ± SEM. The stars *** indicate significance (p ≤ 0.001; with respect to the corresponding buffer; Linear Mixed). Each coloured group of four columns represents the data for a single peptide test at each time point. Left to right, 0, 1, 2 and 5 min. Some error bars are too small to be observed. Full article ">Figure 3
Immunofluorescent staining of MHV E protein expression in Lenti-X 293T cells. (A) Cells were transfected with pTriEx1.1 vectors encoding WT MHV-CoV E or various alanine mutants, fixed and permeabilized and detected with anti-His Ab conjugated to Alexa Flour 488 (green). Nuclei were counterstained with DAPI (blue). Punctate staining is indicated. (B) Enlarged images of the WT and L52A panels with punctate staining indicated. In both panels the scale bar is 20 M. Full article ">Figure 4
Western blot analysis of WT MHV-E protein expression and mutants following expression in insect cells. (A) Western blot using anti-His antibody. Lane M - See Blue™ Plus2 Pre-Stained Protein Standard (Invitrogen). Lanes 1-11 are samples as follows: 1: WT E, 2: L50A, 3: V51A, 4: L52A, 5: P54A, 6: Y57A, 7: Y59A, 8: all mutants, 9: deleted EPTM, 10: GFP baculovirus (positive control), 11: mock. (B) Western blot by anti baculovirus surface glycoprotein gp64 following stripping of the membrane used for panel A Key molecular mass markers are indicated to the left of each panel. Full article ">Figure 5
Western blot Analysis of WT MHV-E and mutants following membrane partition by differential centrifugation. (A) Detection of E protein by anti-His antibody in the low- and high-speed membrane fractions (marked). Lane M - See Blue™ Plus2 Pre-Stained Protein Standard (Invitrogen) with 14KDa marker identified. Lanes 1-9 are samples as follows: 1: WT E, 2: L50A, 3: V51A, 4: L52A, 5: P54A, 6: Y57A, 7: Y59A, 8: all mutants, 9: deleted EPTM. The single panel to the right of panel A, marked C, is the LSP and HSP fractions blotted with an antibody to calnexin. (B) The altered distribution of E, dependent on its sequence, is evident by the relative band intensity and was measured by ImageJ densitometry. The filled bars are the LSP samples, the open bars the HSP. Full article ">Figure 6
Predicted topological models of E and the EPTM peptide. Above: Two of the suggested topologies of E are shown with TM, N- and C-termini indicated. Middle: The EPTM peptide is shown within the C-terminal region. Mutated residues are enlarged and coloured according to the level of membrane repartitioning observed. Below: A helical wheel depiction of the EPTM peptide with a suggested interpretation of sidedness with respect to membrane attachment. Full article ">

18 pages, 4696 KiB

Open AccessReview

Structural Biology of Influenza Hemagglutinin: An Amaranthine Adventure

by Nicholas C. Wu and Ian A. Wilson

Viruses 2020, 12(9), 1053; https://doi.org/10.3390/v12091053 - 22 Sep 2020

Cited by 39 | Viewed by 7656

Abstract

Hemagglutinin (HA) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. Over the past four decades, X-ray crystallography has [...] Read more.

Hemagglutinin (HA) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. Over the past four decades, X-ray crystallography has greatly facilitated our understanding of HA receptor binding, membrane fusion, and antigenicity. The recent advances in cryo-EM have further deepened our comprehension of HA biology. Since influenza HA constantly evolves in natural circulating strains, there are always new questions to be answered. The incessant accumulation of knowledge on the structural biology of HA over several decades has also facilitated the design and development of novel therapeutics and vaccines. This review describes the current status of the field of HA structural biology, how we got here, and what the next steps might be. Full article

(This article belongs to the Special Issue In Memory of Michael Rossmann)

► Show Figures

Figure 1

16 pages, 3728 KiB

Open AccessArticle

Characterization of PlyB221 and PlyP32, Two Novel Endolysins Encoded by Phages Preying on the Bacillus cereus Group

by Audrey Leprince, Manon Nuytten, Annika Gillis and Jacques Mahillon

Viruses 2020, 12(9), 1052; https://doi.org/10.3390/v12091052 - 21 Sep 2020

Cited by 14 | Viewed by 3800

Abstract

Endolysins are phage-encoded enzymes implicated in the breaching of the bacterial cell wall at the end of the viral cycle. This study focuses on the endolysins of Deep-Blue (PlyB221) and Deep-Purple (PlyP32), two phages preying on the Bacillus cereus group. Both enzymes exhibit [...] Read more.

Endolysins are phage-encoded enzymes implicated in the breaching of the bacterial cell wall at the end of the viral cycle. This study focuses on the endolysins of Deep-Blue (PlyB221) and Deep-Purple (PlyP32), two phages preying on the Bacillus cereus group. Both enzymes exhibit a typical modular organization with an enzymatically active domain (EAD) located in the N-terminal and a cell wall binding domain (CBD) in the C-terminal part of the protein. In silico analysis indicated that the EAD domains of PlyB221 and PlyP32 are endowed with peptidase and muramidase activities, respectively, whereas in both proteins SH3 domains are involved in the CBD. To evaluate their antimicrobial properties and binding specificity, both endolysins were expressed and purified. PlyB221 and PlyP32 efficiently recognized and lysed all the tested strains from the B. cereus group. Biochemical characterization showed that PlyB221 activity was stable under a wide range of pHs (5–9), NaCl concentrations (up to 200 mM), and temperature treatments (up to 50 °C). Although PlyP32 activity was less stable than that of PlyB221, the endolysin displayed high activity at pH 6–7, NaCl concentration up to 100 mM and the temperature treatment up to 45 °C. Overall, PlyB221 and PlyP32 display suitable characteristics for the development of biocontrol and detection tools. Full article

(This article belongs to the Section Bacterial Viruses)

► Show Figures

Graphical abstract

23 pages, 704 KiB

Open AccessReview

Immune Checkpoints in Viral Infections

by Huiming Cai, Ge Liu, Jianfeng Zhong, Kai Zheng, Haitao Xiao, Chenyang Li, Xun Song, Ying Li, Chenshu Xu, Haiqiang Wu, Zhendan He and Qinchang Zhu

Viruses 2020, 12(9), 1051; https://doi.org/10.3390/v12091051 - 21 Sep 2020

Cited by 45 | Viewed by 4803

Abstract

As evidence has mounted that virus-infected cells, such as cancer cells, negatively regulate the function of T-cells via immune checkpoints, it has become increasingly clear that viral infections similarly exploit immune checkpoints as an immune system escape mechanism. Although immune checkpoint therapy has [...] Read more.

As evidence has mounted that virus-infected cells, such as cancer cells, negatively regulate the function of T-cells via immune checkpoints, it has become increasingly clear that viral infections similarly exploit immune checkpoints as an immune system escape mechanism. Although immune checkpoint therapy has been successfully used in cancer treatment, numerous studies have suggested that such therapy may also be highly relevant for treating viral infection, especially chronic viral infections. However, it has not yet been applied in this manner. Here, we reviewed recent findings regarding immune checkpoints in viral infections, including COVID-19, and discussed the role of immune checkpoints in different viral infections, as well as the potential for applying immune checkpoint blockades as antiviral therapy. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Figure 1

15 pages, 3376 KiB

Open AccessArticle

Metagenomic Insights into the Sewage RNA Virosphere of a Large City

by Sergio Guajardo-Leiva, Jonás Chnaiderman, Aldo Gaggero and Beatriz Díez

Viruses 2020, 12(9), 1050; https://doi.org/10.3390/v12091050 - 21 Sep 2020

Cited by 25 | Viewed by 5084

Abstract

Sewage-associated viruses can cause several human and animal diseases, such as gastroenteritis, hepatitis, and respiratory infections. Therefore, their detection in wastewater can reflect current infections within the source population. To date, no viral study has been performed using the sewage of any large [...] Read more.

Sewage-associated viruses can cause several human and animal diseases, such as gastroenteritis, hepatitis, and respiratory infections. Therefore, their detection in wastewater can reflect current infections within the source population. To date, no viral study has been performed using the sewage of any large South American city. In this study, we used viral metagenomics to obtain a single sample snapshot of the RNA virosphere in the wastewater from Santiago de Chile, the seventh largest city in the Americas. Despite the overrepresentation of dsRNA viruses, our results show that Santiago’s sewage RNA virosphere was composed mostly of unknown sequences (88%), while known viral sequences were dominated by viruses that infect bacteria (60%), invertebrates (37%) and humans (2.4%). Interestingly, we discovered three novel genogroups within the Picobirnaviridae family that can fill major gaps in this taxa’s evolutionary history. We also demonstrated the dominance of emerging Rotavirus genotypes, such as G8 and G6, that have displaced other classical genotypes, which is consistent with recent clinical reports. This study supports the usefulness of sewage viral metagenomics for public health surveillance. Moreover, it demonstrates the need to monitor the viral component during the wastewater treatment and recycling process, where this virome can constitute a reservoir of human pathogens. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Map of el Trebal Wastewater Treatment Plant location in Santiago de Chile. El Trebal wastewater treatment plant (WWTP) is indicated by a white-star red baloon. Full article ">Figure 2
Relative abundances of predicted protein sequences in Trebal viral RNA metagenome, classified by LCA algorithm trough local alignment to NCBI nr database. (A) Domain level, and (B) Family level for sequences classified as Virus in A. (C) Putative host for sequences classified as Virus in A. Sequences were normalized by protein length. Full article ">Figure 3
Hierarchical clustering analysis of 2266 RNA-dependent RNA polymerase (RdRP) predicted protein sequences from Trebal and NCBI RefSeq database based on Bray–Curtis amino acid distance (k = 2). Dendrogram was divided in 12 main cluster based on the “unrooted” dendrogram. Pie charts represent the frequency of sequences in each cluster classified by the putative host trough LCA algorithm. Bar charts represent the source (NCBI or Trebal) from which sequences were retrieved inside each cluster. Full article ">Figure 4
Maximum Likelihood phylogenetic reconstruction of 31 RNA-dependent RNA polymerase (RdRP) predicted proteins from Picobirnaviridae family. Node numbers indicate ultra-fast bootstrap values. RdRP sequence of White clover cryptic virus 1 was used as an outgroup and appeared in grey letters. The sequences characterized in the present study are reported in red letters. Scale bar: 0.5 aminoacid substitution per site. Full article ">Figure 5
Relative abundances of Human Rotavirus species and genotypes inferred from Pfam annotated VP4, VP6, and VP7 genes and classified by local alignment (BLASTn) to NCBI nt database. Sequences were normalized by gene length. (A) Relative abundance of Human Rotavirus species based on VP6. (B) Relative abundance of Human Rotavirus G types based on VP7. (C) Relative abundance of Human Rotavirus P types based on VP4. Full article ">

3 pages, 140 KiB

Open AccessEditorial

Special Issue: “Plant Virus Pathogenesis and Disease Control”

by Bryce W. Falk and Shahideh Nouri

Viruses 2020, 12(9), 1049; https://doi.org/10.3390/v12091049 - 21 Sep 2020

Cited by 3 | Viewed by 4190

Abstract

Plant viruses are emerging and re-emerging to cause important diseases in many plants that humans grow for food and/or fiber, and sustainable, effective strategies for controlling many plant virus diseases remain unavailable [...] Full article

(This article belongs to the Special Issue Plant Virus Pathogenesis and Disease Control)

13 pages, 10351 KiB

Open AccessBrief Report

The Emergence of a vv + MDV Can Break through the Protections Provided by the Current Vaccines

by Meng-ya Shi, Min Li, Wei-wei Wang, Qiao-mu Deng, Qiu-hong Li, Yan-li Gao, Pei-kun Wang, Teng Huang and Ping Wei

Viruses 2020, 12(9), 1048; https://doi.org/10.3390/v12091048 - 20 Sep 2020

Cited by 21 | Viewed by 3909

Abstract

Marek’s disease (MD) is an infectious malignant T-cell lymphoma proliferative disease caused by Marek’s disease virus (MDV). In recent years, the emergence of very virulent (vv) and/or very virulent plus (vv +) strains of MDV in the field has been suggested as one [...] Read more.

Marek’s disease (MD) is an infectious malignant T-cell lymphoma proliferative disease caused by Marek’s disease virus (MDV). In recent years, the emergence of very virulent (vv) and/or very virulent plus (vv +) strains of MDV in the field has been suggested as one of the causes of vaccination failure. The pathogenicity of the MDV strain GX18NNM4, isolated from a clinical outbreak in a broiler breeder flock that was vaccinated with CVI988/Rispens, was investigated. In the vaccination-challenge test, GX18NNM4 was able to break through the protections provided by the vaccines CVI988 and 814. It also significantly reduced body weight gain and caused marked gross lesions and a large area of infiltration of neoplastic lymphocyte cells in the heart, liver, pancreas, etc. of the infected birds. In addition, the expressions of programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), in the spleens and cecal tonsils (CTs) of the unvaccinated challenged birds were significantly increased compared to those in the vaccinated challenged birds, indicating that the PD-1/PD-L1 pathway is related to immune evasion mechanisms. The results showed that the GX18NNM4 strain could cause severe immunosuppression and significantly decrease the protections provided by the current commercial vaccines, thus showing GX18NNM4 to be a vv + MDV strain. Full article

(This article belongs to the Special Issue Animal Herpesviruses Pathogenesis and Immunity)

► Show Figures

Figure 1

Figure 1
Statistics of body weight of experimental birds (X ± SD). Means not sharing a common letter are significantly different (p < 0.05). Full article ">Figure 2
Dynamics of immune organ indices of the experimental birds. (A) spleen; (B) thymus; (C) bursa. Means not sharing a common letter are significantly different (p < 0.05). Full article ">Figure 3
The viral load of MDV in the peripheral blood lymphocytes (PBLs) of the experimental birds. Means not sharing a common letter are significantly different (p < 0.05). Full article ">Figure 4
The mRNA expressions of PD-1 and PD-L1 in the tissues from birds experimentally infected with MDV. (A) PD-1 in the spleen; (B) PD-1 in the cecal tonsils; (C) PD-L1 in the spleen; (D) PD-L1 in the cecal tonsils. Determined by real-time PCR. Means not sharing a common letter are significantly different (p < 0.05). Full article ">Figure 5
Survival patterns showing the overall effects of the various treatments. Full article ">Figure 6
Anatomical and histological lesions: (A,L) white solid nodules in the lungs; (E,P) numerous proliferating tumor cells concentrated in the lung tissue; (B,D,J) tumor or tumor-like lesions in the heart; (F,H,N) numerous infiltrations of diffuse lymphocytosis in myocardial fibers; (C) pancreas hemorrhage with tumor; (G) numerous lymphocytosis proliferations in pancreas; (I) white tumor nodules, approximately 1–3 mm in diameter, on the liver surface; (M) numerous proliferating tumor cells were concentrated in the hepatic tissue; (K) white tumor nodule on the surface of spleen; (O) large number of lymphocytes infiltrated in the spleen tissue. Full article ">

15 pages, 897 KiB

Open AccessArticle

Renal Allograft Biopsies with Polyomavirus BK Nephropathy: Turin Transplant Center, 2015–19

by Elisa Zanotto, Anna Allesina, Antonella Barreca, Francesca Sidoti, Ester Gallo, Paolo Bottino, Marco Iannaccone, Gabriele Bianco, Luigi Biancone, Rossana Cavallo and Cristina Costa

Viruses 2020, 12(9), 1047; https://doi.org/10.3390/v12091047 - 20 Sep 2020

Cited by 6 | Viewed by 3143

Abstract

Background: In kidney transplant patients, polyomavirus-associated nephropathy (PVAN) represents a serious complication; the key factor for the development of PVAN is immunosuppression level and modulation of anti-rejection treatment represents the first line of intervention. Allograft biopsy and histology remain the criterion standard for [...] Read more.

Background: In kidney transplant patients, polyomavirus-associated nephropathy (PVAN) represents a serious complication; the key factor for the development of PVAN is immunosuppression level and modulation of anti-rejection treatment represents the first line of intervention. Allograft biopsy and histology remain the criterion standard for diagnosing PVAN. Methods: All consecutive renal biopsies with the diagnosis of PVAN carried out at the University Hospital City of Health and Science of Turin over a five-years period were studied. Renal allograft biopsy was performed due to renal function alterations associated to medium-high polyomavirus BK (BKV)-DNA levels on plasma specimen. Results: A total of 21 patients underwent a first biopsy to diagnose a possible BKV nephropathy, in 18, a second biopsy was made, in eight, a third biopsy, and finally, three underwent the fourth renal biopsy; following the results of each biopsies, immunosuppressant agents dosages were modified in order to reduce the effect of PVAN. Conclusions: In this study, the clinical and histological features of 21 kidney transplant recipients with BKV reactivation and development of PVAN are described. To date, the only treatment for PVAN consists in the reduction of immunosuppressive agents, constantly monitoring viral load. Full article

(This article belongs to the Special Issue BK Virus and Transplantation)

► Show Figures

Figure 1

19 pages, 2736 KiB

Open AccessArticle

PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems

by Ahmed Mostafa, Sara H. Mahmoud, Mahmoud Shehata, Christin Müller, Ahmed Kandeil, Rabeh El-Shesheny, Hanaa Z. Nooh, Ghazi Kayali, Mohamed A. Ali and Stephan Pleschka

Viruses 2020, 12(9), 1046; https://doi.org/10.3390/v12091046 - 20 Sep 2020

Cited by 14 | Viewed by 4530

Abstract

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility [...] Read more.

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1_EGY and H9N2_EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2_EGY, expressed individually and in combination in the genetic background of H5N1_EGY, were genetically compatible with the other H5N1_EGY segments. At 37 °C the replication efficiencies of H5N1_EGY reassortants expressing the H9N2_EGY polymerase subunits PB2 and PA (H5N1_PB2-H9N2EGY, H5N1_PA-H9N2EGY) were higher than the wild-type H5N1_EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1_PB2-H9N2EGY, but reduced for H5N1_PA-H9N2EGY. At 33 °C and 39 °C, H5N1_PB2-H9N2EGY and H5N1_PA-H9N2EGY replicated to higher levels than the wild-type H5N1_EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1_EGY. Genetic analysis of the polymerase-encoding segments revealed that the PA_H9N2EGY and PB2_H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1_EGY enabled the mutant virus H5N1_PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1_PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures. Full article

(This article belongs to the Special Issue Evolution and Pathogenesis of Avian and Animal Influenza Viruses)

► Show Figures

Figure 1

9 pages, 2016 KiB

Open AccessArticle

RNAemia Corresponds to Disease Severity and Antibody Response in Hospitalized COVID-19 Patients

by Kirsten Alexandra Eberhardt, Charlotte Meyer-Schwickerath, Eva Heger, Elena Knops, Clara Lehmann, Jan Rybniker, Philipp Schommers, Dennis A. Eichenauer, Florian Kurth, Michael Ramharter, Rolf Kaiser, Udo Holtick, Florian Klein, Norma Jung and Veronica Di Cristanziano

Viruses 2020, 12(9), 1045; https://doi.org/10.3390/v12091045 - 18 Sep 2020

Cited by 48 | Viewed by 5091

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global health emergency. To improve the understanding of the systemic component of SARS-CoV-2, we investigated if viral load dynamics in plasma and respiratory samples are associated with antibody response and severity of coronavirus disease [...] Read more.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global health emergency. To improve the understanding of the systemic component of SARS-CoV-2, we investigated if viral load dynamics in plasma and respiratory samples are associated with antibody response and severity of coronavirus disease 2019 (COVID-19). SARS-CoV-2 RNA was found in plasma samples from 14 (44%) out of 32 patients. RNAemia was detected in 5 out of 6 fatal cases. Peak IgG values were significantly lower in mild/moderate than in severe (0.6 (interquartile range, IQR, 0.4–3.2) vs. 11.8 (IQR, 9.9–13.0), adjusted p = 0.003) or critical cases (11.29 (IQR, 8.3–12.0), adjusted p = 0.042). IgG titers were significantly associated with virus Ct (Cycle threshold) value in plasma and respiratory specimens ((ß = 0.4, 95% CI (confidence interval, 0.2; 0.5), p < 0.001 and ß = 0.5, 95% CI (0.2; 0.6), p = 0.002). A classification as severe or a critical case was additionally inversely associated with Ct values in plasma in comparison to mild/moderate cases (ß = −3.3, 95% CI (−5.8; 0.8), p = 0.024 and ß = −4.4, 95% CI (−7.2; 1.6), p = 0.007, respectively). Based on the present data, our hypothesis is that the early stage of SARS-CoV-2 infection is characterized by a primary RNAemia, as a potential manifestation of a systemic infection. Additionally, the viral load in plasma seems to be associated with a worse disease outcome. Full article

(This article belongs to the Collection Coronaviruses)

► Show Figures

Figure 1

21 pages, 3726 KiB

Open AccessArticle

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line

by Hossein M. Elbadawy, Mohi I. Mohammed Abdul, Naif Aljuhani, Adriana Vitiello, Francesco Ciccarese, Mohamed A. Shaker, Heba M. Eltahir, Giorgio Palù, Veronica Di Antonio, Hanieh Ghassabian, Claudia Del Vecchio, Cristiano Salata, Elisa Franchin, Eleonora Ponterio, Saleh Bahashwan, Khaled Thabet, Mekky M. Abouzied, Ahmed M. Shehata, Cristina Parolin, Arianna Calistri and Gualtiero Alvisi Show full author list Hide full author list

Viruses 2020, 12(9), 1044; https://doi.org/10.3390/v12091044 - 18 Sep 2020

Cited by 6 | Viewed by 4112

Abstract

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon [...] Read more.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection. Full article

(This article belongs to the Special Issue RNA Interference (RNAi) for Antiviral Therapy)

► Show Figures

Figure 1

18 pages, 5160 KiB

Open AccessArticle

Merkel Cell Polyomavirus Large T Antigen Unique Domain Regulates Its Own Protein Stability and Cell Growth

by Nnenna Nwogu, Luz E. Ortiz and Hyun Jin Kwun

Viruses 2020, 12(9), 1043; https://doi.org/10.3390/v12091043 - 18 Sep 2020

Cited by 9 | Viewed by 4402

Abstract

Merkel cell polyomavirus (MCV) is the only known human oncogenic virus in the polyomaviridae family and the etiological agent of most Merkel cell carcinomas (MCC). MCC is an aggressive and highly metastatic skin cancer with a propensity for recurrence and poor prognosis. Large [...] Read more.

Merkel cell polyomavirus (MCV) is the only known human oncogenic virus in the polyomaviridae family and the etiological agent of most Merkel cell carcinomas (MCC). MCC is an aggressive and highly metastatic skin cancer with a propensity for recurrence and poor prognosis. Large tumor antigen (LT), is an essential oncoprotein for MCV transcription, viral replication, and cancer cell proliferation. MCV LT is a short-lived protein that encodes a unique domain: MCV LT unique regions (MURs). These domains consist of phosphorylation sites that interact with multiple E3 ligases, thus limiting LT expression and consequently, viral replication. In this study, we show that MURs are necessary for regulating LT stability via multiple E3 ligase interactions, resulting in cell growth arrest. While expression of wild-type MCV LT induced a decrease in cellular proliferation, deletion of the MUR domains resulted in increased LT stability and cell proliferation. Conversely, addition of MURs to SV40 LT propagated E3 ligase interactions, which in turn, reduced SV40 LT stability and decreased cell growth activity. Our results demonstrate that compared to other human polyomaviruses (HPyVs), MCV LT has evolved to acquire the MUR domains that are essential for MCV LT autoregulation, potentially leading to viral latency and MCC. Full article

(This article belongs to the Special Issue Polyomaviruses)

► Show Figures

Figure 1

Figure 1
Merkel cell polyomavirus large tumor antigen (MCV LT) similarity and disorder plots. (A) LT amino acid similarity of 14 human polyomaviruses (HPyVs) and disordered region of MCV LT protein. Sliding window plot (6 aa window size) analysis. LT amino acid similarity was compared using PLOTCON (EMBOSS). For disordered region prediction, IUPred2 and ANCHOR2 were utilized to identify disordered protein regions and disordered binding regions in MCV LT, respectively. MCV LT contains a unique disordered region (MUR) divided in two fragments by the conserved LXCXE motif [<a href="#B23-viruses-12-01043" class="html-bibr">23</a>,<a href="#B24-viruses-12-01043" class="html-bibr">24</a>]. (B) Diagram of MCV LT and SV40 LT domain structures. Compared with SV40 LT, MCV LT has an extended structure, MUR, that serves as an interacting domain with multiple cellular factors. The MUR domain also consists of phosphorylated serines for SCF E3 ligase recognition (phospho-degron motifs) [<a href="#B15-viruses-12-01043" class="html-bibr">15</a>]. Full article ">Figure 2
MCV LT unique region (MUR) regulates LT stability. (A) Diagram of MCV LT coding regions and sites of deletion mutations. MCV LT MUR domains: MUR1 (102–207 aa) and MUR2 (220–258 aa) were simultaneously deleted to evaluate LT stability. (B) LT MUR destabilizes LT. LT protein turnover was measured by a cycloheximide (CHX) chase assay using quantitative immunoblot analysis. Cells transfected with LTWT or LTdMUR constructs (0.3 µg and 0.9 µg respectively) were treated with CHX (0.1 mg/mL) 24 h after transfection and harvested at each time point indicated. (C) Protein expression was quantified using a LI-COR IR imaging system. Deletion of the MUR extended the half-life of LT from ~3–4 h up to >8 h. Error bars represent SEM and were calculated using GraphPad Prism software. Data were analyzed using three biological replicates per experiment, n = 3. (D) Diagram of SV40 LT coding regions and sites of insertion mutations. SV40 LT amino acids (99–123) were deleted, and the MCV LT complete MUR domain (102–258 aa) was inserted. (E) MCV LT MUR destabilizes SV40 LT. SV40 LT protein turnover was assessed by a CHX chase assay using quantitative immunoblot analysis. 293 cells transfected with either wild-type SV40 LT (SV40 LTWT) (0.3 µg) or SV40 LT+MUR (0.6 µg) were treated with CHX (0.1 mg/mL) 24 h after transfection and harvested at each time point indicated. (F) Protein expression was quantified using the laser-scanning Odyssey CLX (LI-COR) infrared (IR) imaging system. MCV MUR insertion into SV40 LT reduced SV40 LT turnover to ~6 h. Error bars represent standard errors of the mean (SEM) and were calculated using GraphPad Prism software. Data were analyzed using three biological replicates per experiment, n = 3. Full article ">Figure 3
MCV LT maintains origin binding and replication capacity independent of the MUR domain. (A) Schematic of MCV LT coding regions and sites of deletion mutations. MCV LT MUR1 (102–207 aa) (LTdM1), MUR2 (220-258 aa) (LTdM2) or MUR1+MUR2 (LTdMUR) (102-258aa) were deleted to evaluate LT mediated viral replication. (B) Diagram of MCV transcription reporter. The MCV promoter region (nt 4928–195; GenBank accession no. EU375804) was cloned into a bidirectional dual Firefly (early) and Renilla (late) luciferase reporter [<a href="#B15-viruses-12-01043" class="html-bibr">15</a>]. (C) Early gene transcription activity was measured by luciferase activity using co-transfection of the reporter (0.5 µg) with either wild-type LT or MUR deletion mutants (0.5 µg) into 293 cells. Relative luciferase activity was normalized to empty vector control (mean ± SEM, n = 3). Deletion of either MUR1 or MUR2 impaired repression of MCV early gene transcription while deletion of both MUR1 and MUR2 (dMUR) successfully downregulated MCV early gene transcription compared to wild-type LT. There was no significant increased late gene transcription by either LTwt or mutant LTs because no robust amplification of origin replication occurred as shown in <a href="#viruses-12-01043-f003" class="html-fig">Figure 3</a>D. Similar results are observed using the replication-deficient reporter [<a href="#B15-viruses-12-01043" class="html-bibr">15</a>]. (D) MCV origin replication assay. Either deletion of MUR1 or MUR2 impaired MCV origin replication. Deletion of both MUR1 and MUR2 (LTdMUR) retained its ability to replicate MCV origin as compared to wild-type LT. Error bars represent SEM and were calculated using GraphPad Prism software. Data were analyzed using three biological replicates per experiment, n = 3. Differences between means (*, p value ≤ 0.05) were analyzed using a t-test with GraphPad Prism software. Full article ">Figure 4
MCV LT MUR regulates cell growth inhibition activity of LT. (A) Diagram of MCV LT and SV40 LT lentiviral constructs used for cell proliferation analysis. Enhanced GFP (eGFP) was tagged to the N-terminus. P2A self-cleaving peptide was fused between eGFP and LT sequences to allow non-tagged LT expression. (B) Immunoblot analysis of eGFP expression in BJ-hTERT fibroblasts. eGFP can effectively be used as a marker of LT expression. (C) Validation of the P2A lentivirus system by immunofluorescence staining. Cells were fixed, and GFP fluorescence was analyzed by direct visualization, whereas LT antigen expression was identified by indirect immunofluorescence using specific antibodies. Nuclear counterstain (DAPI-Blue), GFP for T antigen expression (Green), and various T antigen antibodies (Pab416, 2T2, CM2B4) (Red). Scale bar = 10 µm. (D) MCV LT MUR regulates serum-independent human BJ-hTERT cell proliferation. LT proteins were transduced by lentiviral vector into immortalized BJ-hTERT cells, and cell proliferation was determined in 0.1% FBS using a CCK-8 colorimetric cell proliferation assay. Wild-type LT mediated cell growth inhibition, as previously reported [<a href="#B7-viruses-12-01043" class="html-bibr">7</a>,<a href="#B8-viruses-12-01043" class="html-bibr">8</a>]. In the absence of MUR, LT accelerated BJ-hTERT cell growth (normalized by mean OD values on day 1 for two independent experiments performed in triplicate). In contrast, wild-type SV40 LT promoted cell proliferation while the insertion of MCV MUR decreased cell proliferation potential in the absence of serum. Full article ">Figure 5
MCV LT MUR is LT-FBW7 and Skp2 interaction domain. (A) MCV LT MUR interacts with SCF E3 ligase complexes. HA-tagged F-box proteins (FBW7 and Skp2) (6 µg) were co-transfected with LTs (6 µg) into 293 cells, and proximity ligation assay (PLA)-flow cytometric analysis was performed with anti-HA and either 2T2 (MCV LT) or pAb416 (SV40 LT) antibodies. c-Myc/FBW7 interaction was included as a positive control for PLA-flow cytometry. Wild-type LT mediated E3 ligase interaction through the known serine phosphorylation sites within the MUR domain, as previously reported [<a href="#B15-viruses-12-01043" class="html-bibr">15</a>]. Insertion of the MCV LT MUR into SV40 LT (SV40 LT+MUR) increased Skp-Cullin-F-box (SCF) protein interactions. (B) Calculated fold difference of mean fluorescence intensity of PLA analyzed by Flow Jo software. (C) Protein expression was evaluated by immunoblot analysis. Full article ">Figure 6
Serines 142 and 147 are the LT phosphorylation sites for β-TrCP recruitment. (A) β-TrCP binds to the DpSGXX(X)pS dual-site phosphorylation motif in its substrates [<a href="#B38-viruses-12-01043" class="html-bibr">38</a>]. (B) MCV LT MUR interacts with β-TrCP. β-TrCP (6 µg) was co-transfected with LTs (6 µg) into 293 cells, and PLA-flow cytometric analysis was performed with anti-HA and either 2T2 (MCV LT) or pAb416 (SV40 LT) antibodies. c-Myc/FBW7 interaction was included as a positive control for PLA-flow cytometry. (C) Calculated fold difference of mean fluorescence intensity of PLA analyzed by Flow Jo software. (D) Protein expression was evaluated by immunoblot analysis. (E) LT alanine (Ala, A) substitution mutants at S142 and S147 potential β-TrCP binding residues were tested for stability by a CHX chase assay using quantitative immunoblot analysis. Cell transfected with LTs (0.5 µg) were treated with CHX (0.1 mg/mL) 24 h after transfection and harvested at each time point indicated. (F) Protein expression was quantified in triplicate using an LI-COR IR imaging system. Error bars represent SEM; n = 3. Both S147 and S142 to Ala mutations along with the double mutant increased LT stability. The double mutant is the most stable of all three while S147A mutant is more stable than S142A. Full article ">

14 pages, 3035 KiB

Open AccessArticle

Divergent Influenza-Like Viruses of Amphibians and Fish Support an Ancient Evolutionary Association

by Rhys Parry, Michelle Wille, Olivia M. H. Turnbull, Jemma L. Geoghegan and Edward C. Holmes

Viruses 2020, 12(9), 1042; https://doi.org/10.3390/v12091042 - 18 Sep 2020

Cited by 24 | Viewed by 8771

Abstract

Influenza viruses (family Orthomyxoviridae) infect a variety of vertebrates, including birds, humans, and other mammals. Recent metatranscriptomic studies have uncovered divergent influenza viruses in amphibians, fish and jawless vertebrates, suggesting that these viruses may be widely distributed. We sought to identify additional [...] Read more.

Influenza viruses (family Orthomyxoviridae) infect a variety of vertebrates, including birds, humans, and other mammals. Recent metatranscriptomic studies have uncovered divergent influenza viruses in amphibians, fish and jawless vertebrates, suggesting that these viruses may be widely distributed. We sought to identify additional vertebrate influenza-like viruses through the analysis of publicly available RNA sequencing data. Accordingly, by data mining, we identified the complete coding segments of five divergent vertebrate influenza-like viruses. Three fell as sister lineages to influenza B virus: salamander influenza-like virus in Mexican walking fish (Ambystoma mexicanum) and plateau tiger salamander (Ambystoma velasci), Siamese algae-eater influenza-like virus in Siamese algae-eater fish (Gyrinocheilus aymonieri) and chum salmon influenza-like virus in chum salmon (Oncorhynchus keta). Similarly, we identified two influenza-like viruses of amphibians that fell as sister lineages to influenza D virus: cane toad influenza-like virus and the ornate chorus frog influenza-like virus, in the cane toad (Rhinella marina) and ornate chorus frog (Microhyla fissipes), respectively. Despite their divergent phylogenetic positions, these viruses retained segment conservation and splicing consistent with transcriptional regulation in influenza B and influenza D viruses, and were detected in respiratory tissues. These data suggest that influenza viruses have been associated with vertebrates for their entire evolutionary history. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Genome architecture of the novel influenza-like viruses identified here. Genome architecture of influenza A virus (IAV)/influenza B virus (IBV) and influenza C virus (ICV)/influenza D virus (IDV) is shown on the left. For each novel virus, we provide the segment name and size. PB1, RNA-dependent RNA polymerase basic subunit 1; PB2, RNA-dependent RNA polymerase basic subunit 2; PA, RNA-dependent RNA polymerase acidic subunit; P3, Polymerase protein 3; NP, nucleoprotein; HA, hemagglutinin; NB, glycoprotein NB; HEF, hemagglutinin esterase; NA, neuraminidase; M, matrix; CM1, viral matrix protein; NS, non-structural protein; NEP nuclear export protein. Detailed annotations for each virus are presented in <a href="#app1-viruses-12-01042" class="html-app">Supplementary Tables S1–S6 and Figures S1–S5</a>. Full article ">Figure 2
Phylogenetic relationships of vertebrate influenza-like viruses. Maximum likelihood tree of the PB1 segment, which encodes the RNA-dependent RNA polymerase, of various influenza-like viruses. Lineages corresponding to IAV, IBV, ICV, IDV are coloured blue, green, red and orange, respectively. Viruses identified in this study are denoted by a black circle and pictogram of the host species of the library. Wenling hagfish influenza virus is set as the outgroup as per Shi et al. [<a href="#B3-viruses-12-01042" class="html-bibr">3</a>]. The scale bar indicates the number of amino acid substitutions per site. Full article ">Figure 3
Phylogenetic trees for segments in cases where complete coding genomes could be recovered. Maximum likelihood trees for each segment ordered by (segment) size. Lineages are coloured by influenza virus, in which IAV, IBV, ICV, IDV are coloured blue, green, red and orange, respectively. Viruses identified in this study are denoted by a black circle and pictogram of the host species of the library. The phylogenetic position of each virus is traced across the trees with grey dashed lines. ICV, IDV, cane toad and chorus frog influenza-like viruses do not have an NA segment. The scale bar for each tree indicates the number of amino acid substitutions per site. Where possible, the trees are rooted using the hagfish influenza virus (PB2, PB1, PA/P3, NP). The HA/HEF, M and NS trees were midpoint rooted. Full article ">Figure 4
Heat map of the abundance of salamander influenza-like virus reads in the multi-tissue transcriptome library of developmental stages of plateau tiger salamander [<a href="#B44-viruses-12-01042" class="html-bibr">44</a>]. Read abundance for this virus was highest in the gills of late metamorphosis salamanders, and lungs of post metamorphosis salamander, strongly suggesting tropism for respiratory epithelium. Full article ">

24 pages, 3980 KiB

Open AccessArticle

Identification of Inhibitors of ZIKV Replication

by Desarey Morales Vasquez, Jun-Gyu Park, Ginés Ávila-Pérez, Aitor Nogales, Juan Carlos de la Torre, Fernando Almazan and Luis Martinez-Sobrido

Viruses 2020, 12(9), 1041; https://doi.org/10.3390/v12091041 - 18 Sep 2020

Cited by 17 | Viewed by 4402

Abstract

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no [...] Read more.

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Figure 1

22 pages, 1773 KiB

Open AccessArticle

CBF-1 Promotes the Establishment and Maintenance of HIV Latency by Recruiting Polycomb Repressive Complexes, PRC1 and PRC2, at HIV LTR

by Adhikarimayum Lakhikumar Sharma, Joseph Hokello, Shilpa Sonti, Sonia Zicari, Lin Sun, Aseel Alqatawni, Michael Bukrinsky, Gary Simon, Ashok Chauhan, Rene Daniel and Mudit Tyagi

Viruses 2020, 12(9), 1040; https://doi.org/10.3390/v12091040 - 18 Sep 2020

Cited by 21 | Viewed by 4098

Abstract

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in [...] Read more.

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting polycomb group (PcG/PRC) corepressor complexes or polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, the treatment of latently infected primary CD4+ T cells with the PRC2/EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation. Full article

(This article belongs to the Special Issue HIV-1 Transcription Regulation)

► Show Figures

Figure 1

Figure 1
Knockdown of C-promoter binding factor-1 (CBF-1) in primary CD4+ T cells reactivates latent human immunodeficiency virus (HIV) proviruses. (a) Structure of lentiviral vector (pHR’-PNL-Luc), which carries reporter luciferase gene under HIV LTR promoter. (b) Western blot demonstrating CBF-1 knockdown in cells expressing shRNAs against CBF-1, cells expressing scrambled shRNA and control unstimulated cells. (c) Densitometric analyses of immunoblot bands using ImageJ software, and represented graphically after normalization to actin. (d) Luciferase assay showing proviral reactivation in primary cells with pHR’-PNL-Luc that are superinfected with different amounts of lentiviral vectors expressing either shRNAs against CBF-1, scrambled shRNA and control unstimulated cells. Error bars represent the Mean ± SD of three independent and separate experiments. The p value of statistical significance was set as; p < 0.05 (*), 0.01 (**) or 0.001 (***). Full article ">Figure 2
Knockdown of polycomb group (PcG) complex led to proviral reactivation. Some of the core PcG complex components were knocked down individually by transfecting latently infected Jurkat-pHR’-PNL-Luc cells with four specific siRNAs. HIV-1 reactivation of latent provirus was quantified through luciferase assays performed after 52 h either post siRNA transfection or 48 h post DZNep treatment. (a) Western blot showing the efficiency of siRNA to knockdown indicated subunits of PRCs. The densitometry analyses were then represented graphically after normalization to actin. Quantitative luciferase assays marking proviral reactivation either after (b) knockdown of individual subunits belonging to PRCs or (c) upon DZNep treatment (from 2 µM to 32 µM) of cells. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was done using Microsoft Excel and GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set as: p < 0.01 (**). Full article ">Figure 3
CBF-1 restricts HIV transcription by inducing multiple types of repressive epigenetic modifications at HIV LTR. Chromatin immunoprecipitation (ChIP) analyses were performed using latently infected Jurkat T cells to evaluate the turnover of different epigenetic modifications at HIV LTR in the absence or presence of knockdown of endogenous CBF-1, using the indicated antibodies. Primer sets directed to the (a) Promoter region (−116 to +4) with respect to transcription start site; (b) Nucleosome 1 (+30 to +134) with respect to transcription start site of HIV-1 LTR. The depicted ChIP assay results were reproduced 5 times. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was calculated with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set at either; p < 0.05 (*) or 0.01 (**). Full article ">Figure 4
CBF-1 knockdown resulted in dissociation of different factors belonging to both PRCs (PRC1 and PRC2). ChIP analyses were performed using latently infected Jurkat T cells in the absence or presence of CBF-1 knockdown. CBF-1 knockdown leads to the dissociation of various core components of both PRCs, showing the role of CBF-1 in their recruitment at HIV LTR. (a) Promoter region (−116 to +4); (b) Nucleosome 1 (+30 to +134). Error bars represent the SEM of three independent experiments and three separate qPCR measurements from each experiment. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was calculated with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set at either; p < 0.05 (*) or 0.01 (**). Full article ">Figure 5
Cell activation leads to fluctuation in the levels of different chromatin-associated factors that belong to PRC1 and PRC2. ChIP analyses were performed before and after activation of latently infected primary CD4+ T cells with α-CD3/-CD28 antibodies, in the presence of IL-2 for 30 min. (a) Structure of lentiviral vectors. mCherry was used as reporter depicted in this diagram. ChIP results in latency systems harboring proviruses with the vector pHR’-PNL-H13LTat-mCherry (b,c), and pHR’-PNL-wild-typeTat-mCherry (d,e). Error bars represent the SEM of two independent experiments and three separate qPCR measurements from each analysis. Graphs represent the average and standard deviation from three independent and replicate samples. Statistical analysis was calculated with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The p value of statistical significance was set at either; p < 0.05 (*) or 0.01 (**). Full article ">Figure 6
Model of CBF-1 functioning. Based on our findings, we propose the following model for the regulation of HIV latency by CBF-1. The higher levels of CBF-1 and lack of transcription factors such as NF-kB and NFAT in quiescent cells facilitates the binding of CBF-1 at HIV LTR. CBF-1 after binding to LTR recruits PRCs. PRCs subsequently promote heterochromatin environment at HIV LTR and inhibit the free flow of transcription machinery, thus facilitating the establishment and maintenance of HIV latency. Following cellular activation, the levels of CBF-1 drop, but the levels of NF-kB and NFAT rise in the nucleus, which displaces CBF-1 and corepressor complexes from their binding sites. Eventually, these factors recruit coactivator complexes at HIV LTR, which then establishes the euchromatin environment at HIV LTR that facilitate the access of transcription machinery at the LTR promoter, and thus leads to the reactivation of latent proviruses. Full article ">

19 pages, 1184 KiB

Open AccessReview

Induction of the Antiviral Immune Response and Its Circumvention by Coronaviruses

by Ping Liu, Yan Hong, Bincai Yang, Prasha Shrestha, Nelam Sajjad and Ji-Long Chen

Viruses 2020, 12(9), 1039; https://doi.org/10.3390/v12091039 - 18 Sep 2020

Cited by 10 | Viewed by 5245

Abstract

Some coronaviruses are zoonotic viruses of human and veterinary medical importance. The novel coronavirus, severe acute respiratory symptoms coronavirus 2 (SARS-CoV-2), associated with the current global pandemic, is characterized by pneumonia, lymphopenia, and a cytokine storm in humans that has caused catastrophic impacts [...] Read more.

Some coronaviruses are zoonotic viruses of human and veterinary medical importance. The novel coronavirus, severe acute respiratory symptoms coronavirus 2 (SARS-CoV-2), associated with the current global pandemic, is characterized by pneumonia, lymphopenia, and a cytokine storm in humans that has caused catastrophic impacts on public health worldwide. Coronaviruses are known for their ability to evade innate immune surveillance exerted by the host during the early phase of infection. It is important to comprehensively investigate the interaction between highly pathogenic coronaviruses and their hosts. In this review, we summarize the existing knowledge about coronaviruses with a focus on antiviral immune responses in the respiratory and intestinal tracts to infection with severe coronaviruses that have caused epidemic diseases in humans and domestic animals. We emphasize, in particular, the strategies used by these coronaviruses to circumvent host immune surveillance, mainly including the hijack of antigen-presenting cells, shielding RNA intermediates in replication organelles, 2′-O-methylation modification for the evasion of RNA sensors, and blocking of interferon signaling cascades. We also provide information about the potential development of coronavirus vaccines and antiviral drugs. Full article

(This article belongs to the Special Issue Emerging Viruses 2020: Surveillance, Prevention, Evolution and Control)

► Show Figures

Figure 1

Figure 1
Architecture of the mucosal epithelial barrier in the respiratory and intestinal tracts that guards against viral invasion. (A). The airway epithelium is composed of ciliated cells, goblet cells, basal cells, and Clara cells. The mucus on the epithelial surface is the first barrier against human infection by coronaviruses, such as SARS-CoV, MERS-CoV, and the novel emerging coronavirus, SARS-CoV-2. The mucins secreted by goblet cells on the epithelial surface include two layers, a viscous layer on top and a periciliary layer below. The innate immune cells in the submucosal layer such as dendritic cells and macrophages are involved in controlling viral infection. (B) Enteric coronaviruses, such as PEDV and PDCoV, principally infect swine by causing histopathological lesions in the intestinal tract. In spite of their similar histological structures, there are substantial differences in the functional purposes and internal environments of the gut and respiratory tract. The mucus of the intestinal tract mainly consists of MUC2 mucin, antimicrobial peptides, and secreted IgA (sIgA) produced by goblet cells, Paneth cells, and plasma cells, respectively. In particular, the commensal bacterial communities resident in the mucus of the gut are involved in various physiological processes that modulate the homeostasis of mucosal immunity. In addition, the intraepithelial lymphocytes (IELs) are located between intestinal epithelial cells, and these cells constitute a large and highly conserved T cell compartment. Intestinal microfold cells (M cells) are only found in the gut-associated lymphoid tissues (GALT) of Peyer’s patches in the intestinal tract, and they are unique antigen-presenting cells that are important for the initiation of mucosal immune responses. The diverse immune cells reside in the lamina propria and mainly include B cells, T cells, dendritic cells, and macrophages. These immune cells interact with the epithelium to detect invading pathogens. Full article ">Figure 2
Schematic diagram of the antiviral immune response and evasion mechanism of coronaviruses. Coronaviruses are internalized into susceptible target cells by the fusion of viral and cellular membranes with unique receptors, such as ACE2, DPP4, and APN, and the RNA of the viral genome is released into the cytosol. SARS-CoV and SARS-CoV-2 exploit the serine protease TMPRSS2 for spike protein priming. The virion and the pathogen-associated molecular patterns (PAMPs) of coronavirus can be recognized by immune sensors called pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs) and cytoplasm retinoic acid-inducible gene (RIG) type I like receptors (RLRs) (RIG-I/MDA5). The extracellular membrane of TLRs (TLR2/4) and endosome TLRs (TLR3/7/8) are widely expressed in epithelial cells and dendritic cells. The PAMPs of coronaviruses induce the interferon (IFN) signaling pathway for antiviral innate immune responses. RIG-I/MDA5 conveys signals through mitochondrial antiviral-signaling protein (MAVS), while TLRs signal through TIR-containing adapter protein inducing IFN-β/myeloid differentiation factor 88 (TRIF/MyD88). TNF receptor-associated factor 3 (TRAF3) activates tank-binding kinase 1/IκB kinase epsilon (TBK1/IKKε), while TRAF6 signal transduction requires activation of the IKK complex. Activated transcription factors are translocated into the nucleus to promote Type I and III IFN expression. IFNs are secreted into the extracellular space and bound to their cognate receptors IFNAR and IFNLR (IFNLR1 and IL10Rβ) to activate downstream the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, followed by nuclear localization of the interferon-stimulated gene factor 3 (ISGF3) complex and expression of numerous interferon stimulating genes (ISGs), leading to the establishment of an antiviral state. Suppression of IFN signal pathways by coronaviruses and their antagonists is shown in red boxes. S, severe acute respiratory symptom coronavirus (SARS-CoV); M, Middle East respiratory syndrome coronavirus (MERS-CoV); P, porcine epidemic diarrhea virus (PEDV); PD, porcine Deltacoronavirus (PDCoV); ACE2, angiotensin-converting enzyme 2; DPP4, dipeptidyl peptidase-4; APN, aminopeptidase N; TMPRSS2, transmembrane serine protease 2. Full article ">

16 pages, 3618 KiB

Open AccessArticle

The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission

by Jennifer H. Kopanke, Justin S. Lee, Mark D. Stenglein and Christie E. Mayo

Viruses 2020, 12(9), 1038; https://doi.org/10.3390/v12091038 - 18 Sep 2020

Cited by 9 | Viewed by 3421

Abstract

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of [...] Read more.

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV. Full article

(This article belongs to the Section Invertebrate Viruses)

► Show Figures

Figure 1

Figure 1
Experimental Set-up. A field isolate of bluetongue virus (BTV)-17 (BTV17-INPUT) was passaged under three different cell culture conditions: serial passages in bovine cells (BTV17-BPAEC); serial passages in Culicoides sonorensis cells (BTV17-CUVA); and alternating passages in bovine and C. sonorensis cells (BTV17-ALT) for 10 consecutive passages. Full article ">Figure 2
BTV Genetic Distance by Segment across Cell Culture Conditions. Genetic distances (the sum of single nucleotide variant (SNV) frequencies per segment) were normalized by each segments’ coding sequence length. Segments 1–10 are represented along the x-axis (s1, s2, s3, s4, s5, s6, s7, s8, s9, and s10). For BTV17-CUVA, -ALT, and -BPAEC, mean distance (and standard deviation) for each segment across passages 3, 6, and 9 is shown. Full article ">Figure 3
BTV Genetic Richness across Cell Culture Conditions. (a) Richness of each segment was calculated as the sum of single nucleotide variants (SNV) sites normalized by the number of BTV reads (i.e., variant sites per 10,000 BTV reads), and collective data across all segments are shown by box-and-whisker plots (median, interquartile range, and minimum/maximum are depicted; *** = p < 0.005). Box-and-whisker plots for BTV17-CUVA, -ALT, and -BPAEC were constructed using the richness of all segments across passages 3, 6, and 9. (b) Mean richness and standard deviation for each segment are shown. Segments 1–10 are represented along the x-axis (s1, s2, s3, s4, s5, s6, s7, s8, s9, and s10). Bars depicting BTV17-CUVA, -ALT, and -BPAEC represent collective data from passages 3, 6, and 9. Full article ">Figure 4
BTV Genetic Complexity across Cell Culture Conditions. Shannon entropy was calculated as a measure of population complexity across viral coding sequences. Shannon entropy was calculated for each segment and cumulative data from all segments are shown by box-and-whisker plots (median, interquartile range, and minimum/maximum are depicted; *** = p < 0.005). Box-and-whisker plots for BTV17-CUVA, -ALT, and -BPAEC were constructed using the mean Shannon entropy of all segments across passages 3, 6, and 9. Full article ">Figure 5
BTV Genetic Complexity by Segment across Cell Culture Conditions. Shannon entropy was calculated for each segment as a measure of viral population complexity. Segments 1–10 are represented along the x-axis (s1, s2, s3, s4, s5, s6, s7, s8, s9, and s10). For BTV17-CUVA, -ALT, and -BPAEC, mean Shannon entropy (and standard deviation) for each segment across passages 3, 6, and 9 are shown. Full article ">Figure 6
SNVs and Indels by Passage and Cell Culture Condition. The total number of novel SNVs or indels was calculated for each sample and normalized by the nucleotide length of the coding sequence (CDS) of each segment. The mean number of normalized novel sites per segment is plotted according to passage and cell culture condition. Segments 1–10 are represented in each bar graph (s1, s2, s3, s4, s5, s6, a7, s8, s9, s10). Viruses harvested from each condition at passage 3 (CuVa p3, Alternate (Alt) p3, and BPAEC p3), passage 6 (CuVa p6, Alt p6, BPAEC p6) and passage 9 (CuVa p9, Alt p9, BPAEC p9) are depicted. Full article ">Figure 7
Genetic Selection by Segment and Cell Culture Condition. The proportion of nonsynonymous (dN) to synonymous (dS) changes was used as an estimate of selection. (a) dN/dS for each sample was calculated across the entire BTV coding sequence (CDS; inclusive of all ten segments), *** = p < 0.0005. (b) dN/dS from all passages and replicates are shown. Error bars depict mean and standard deviation of each segment according to cell culture condition. BTV17-INPUT is shown by black dots and the dashed line. Segments 1–10 are represented along the x-axis (s1, s2, s3, s4, s5, s6, s7, s8, s9, and s10). Full article ">

15 pages, 1266 KiB

Open AccessReview

Functional Characterization of Pepper Vein Banding Virus-Encoded Proteins and Their Interactions: Implications in Potyvirus Infection

by Pallavi Sabharwal and Handanahal S. Savithri

Viruses 2020, 12(9), 1037; https://doi.org/10.3390/v12091037 - 17 Sep 2020

Cited by 4 | Viewed by 3707

Abstract

Pepper vein banding virus (PVBV) is a distinct species in the Potyvirus genus which infects economically important plants in several parts of India. Like other potyviruses, PVBV encodes multifunctional proteins, with several interaction partners, having implications at different stages of the potyviral infection. [...] Read more.

Pepper vein banding virus (PVBV) is a distinct species in the Potyvirus genus which infects economically important plants in several parts of India. Like other potyviruses, PVBV encodes multifunctional proteins, with several interaction partners, having implications at different stages of the potyviral infection. In this review, we summarize the functional characterization of different PVBV-encoded proteins with an emphasis on their interaction partners governing the multifunctionality of potyviral proteins. Intrinsically disordered domains/regions of these proteins play an important role in their interactions with other proteins. Deciphering the function of PVBV-encoded proteins and their interactions with cognitive partners will help in understanding the putative mechanisms by which the potyviral proteins are regulated at different stages of the viral life-cycle. This review also discusses PVBV virus-like particles (VLPs) and their potential applications in nanotechnology. Further, virus-like nanoparticle-cell interactions and intracellular fate of PVBV VLPs are also discussed. Full article

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

► Show Figures

Figure 1

Figure 1
Genome organization of viruses belonging to Potyvirus genus, indicating the transcriptional slippage at the slippery sequences and polyprotein processing by the viral-encoded proteases to form mature proteins. Pink arrow at the P1 and orange arrow at the HC-Pro cleavage sites indicate self-cleavage of the two proteases. Red arrows indicate trans cleavage by VPg-Pro and black arrows indicate the cis cleavage by VPg-Pro. Full article ">Figure 2
Effect of ionic strength and pH in the disassembly of pepper vein-banding virus (PVBV) virus-like particles (VLPs). At pH < 6.5 and a low ionic strength, VLPs are the dominant species which get dissociated into 16 S ring and further into coat protein (CP) subunits (monomer/dimer) as the ionic strength keeps on increasing. As the pH increases, even a low ionic strength is sufficient to disassemble the VLPs into ring intermediate. At higher ionic strength (>0.9 M) and high pH (>10), VLPs are completely dissociated into individual CP subunits. Full article ">Figure 3
Schematic representation of the generation of chimeric VLPs and their potential applications (a) IgG-binding B-domain is cloned at the N-terminus of CP gene which assembles to form PVBV chimeric VLPs with each subunit expressing the B-domain at its N-terminus (b) Incubating the PVBV chimeric VLPs with anti-α tubulin antibody led to the formation of chimeric VLPs + antibody complex (c) PVBV chimeric VLPs, protein A as well as the monomer BCP (PVBV CP N-terminal fusion with B-domain) subunit + antibody complex were subjected to DAC ELISA (direct antigen coating-enzyme linked immunosorbent assay) where an antibody unrelated to CP was used. The dissociation constant (Kd) for each of the complexes is depicted, indicating an approximately 500-fold higher antibody-binding affinity of chimeric VLPs when compared to protein A (d) chimeric VLPs + anti-α tubulin antibody complex could internalize into mammalian cells and deliver the functional tubulin antibodies, which caused the disruption of the tubulin network ultimately causing cell death (e) whereas the BCP monomer (Ni-NTA purified BCP after size exclusion chromatography) + antibody complex fail to enter the mammalian cells. Full article ">

15 pages, 1991 KiB

Open AccessArticle

Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1

by Ajani Athukorala, Jade K. Forwood, David N. Phalen and Subir Sarker

Viruses 2020, 12(9), 1036; https://doi.org/10.3390/v12091036 - 17 Sep 2020

Cited by 15 | Viewed by 4515

Abstract

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine [...] Read more.

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine adenovirus 1 (PaAdV-1). The PaAdV-1 genome is 39,664 bp in length, which was the longest atadenovirus to be sequenced, to the best of our knowledge, and contained 42 putative genes. Its genome organisation was characteristic of the members of genus Atadenovirus; however, the novel PaAdV-1 genome was highly divergent and showed the highest sequence similarity with psittacine adenovirus-3 (55.58%). Importantly, PaAdV-1 complete genome was deemed to contain 17 predicted novel genes that were not present in any other adenoviruses sequenced to date, with several of these predicted novel genes encoding proteins that harbour transmembrane helices. Subsequent analysis of the novel PaAdV-1 genome positioned phylogenetically to a distinct sub-clade with all others sequenced atadenoviruses and did not show any obvious close evolutionary relationship. This study concluded that the PaAdV-1 complete genome described here is not closely related to any other adenovirus isolated from avian or other natural host species and that it should be considered a separate species. Full article

(This article belongs to the Special Issue Animal and Wildlife Viruses)

► Show Figures

Figure 1

Figure 1
Schematic illustration of the avian atadenoviruses. Schematic map of the passerine adenovirus 1 (PaAdV-1, GenBank accession no. MT674683), in comparison with duck adenovirus A (DAdV-A, GenBank accession no. AC_000004) and psittacine adenovirus 3 (PsAdV-3, GenBank accession no. KJ675568), using CLC Genomic Workbench (version 9.5.4, CLC bio, a QIAGEN Company, Prismet, Aarhus C, Denmark). The arrows symbolize adenovirus genes and open reading frames (ORFs) predicted to code for proteins, indicating their direction of transcription. Each gene or ORF is colour coded, as indicated by the colour key in the legend. The bottom graph represents the sequence conservation between the aligned PaAdV-1, DAdV-A, and PsAdV-3 sequences at a given coordinate at each position in the alignment. The gradient of the colour reflects the conservation of that particular position is in the alignment. Red presents 100% conservation across all three viruses, black 50% conserved regions, and blue less than 50% conserved regions. Full article ">Figure 2
Predicted structure of the unique PaAdV1-ORF10. (A) prediction of transmembrane helices (TMHs) in unique PaAdV1-ORF10 gene using EMBOSS 6.5.7 tool in Geneious (version 10.2.2) (A), TMHMM (B), and TMpred (C). All the programs consistently predicted two TMHs. (A) TMHs detected by EMBOSS also showed the presence of alpha-helices within TMHs predicted region that has been dominated by highly hydrophobic residue (red colour). (B,C) The x-axis represents the position of residue, whereas y-axis represents the posterior probability (B), and scores (above 500 are considered significant) (C) for the predicted TMHs. (C) Solid and dashed black lines indicate protein orientation as inside to outside, and outside to inside, respectively. Full article ">Figure 3
Predicted structure of the unique PaAdV1-ORF18. (A) prediction of transmembrane helices (TMHs) in unique PaAdV1-ORF18 gene, using EMBOSS 6.5.7 tool in Geneious (version 10.2.2) (A), TMHMM (B), and TMpred (C). All the programs consistently predicted two TMHs. (A) TMHs detected by EMBOSS also showed the presence of alpha-helices within TMHs predicted region that has been dominated by highly hydrophobic residue (red colour). (B,C) The x-axis represents the position of residue, whereas the y-axis represents the posterior probability (B) and scores (above 500 were considered significant) (C) for the predicted TMHs. (C) Solid and dashed black lines indicate protein orientation as inside to outside, and outside to inside, respectively. Full article ">Figure 4
Phylogenetic tree shows the possible evolutionary relationship of novel passerine adenovirus 1 with other selected AdVs. Maximum likelihood (ML) tree was constructed by using concatenated amino acid sequences of the complete DNA-dependent DNA polymerase, pTP, penton, and hexon genes. Concatenated protein sequences were aligned with MAFTT (version 7.450) [<a href="#B41-viruses-12-01036" class="html-bibr">41</a>] in Geneious (version 10.2.2, Biomatters, Ltd., Auckland, New Zealand), under the BLOSUM62 scoring matrix and gap open penalty = 1.53. The gap >20 residues deleted from the alignments. The unrooted ML tree was constructed with PhyML [<a href="#B42-viruses-12-01036" class="html-bibr">42</a>] under the LG substitution model, and 1000 bootstrap re-samplings were chosen to generate ML trees, using tools available in Geneious (version 10.2.2, Biomatters, Ltd., Auckland, New Zealand). The numbers on the left show bootstrap values as percentages, and the labels at branch tips refer to original AdVs species name, followed by GenBank accession number in parentheses. The final tree is visualised with FigTree (version 1.4.4) [<a href="#B43-viruses-12-01036" class="html-bibr">43</a>]. The five official genera are highlighted as different background colours, and novel passerine adenovirus 1 is shown in pink colour. Full article ">

13 pages, 1561 KiB

Open AccessArticle

Evolutionary Study of the Crassphage Virus at Gene Level

by Alessandro Rossi, Laura Treu, Stefano Toppo, Henrike Zschach, Stefano Campanaro and Bas E. Dutilh

Viruses 2020, 12(9), 1035; https://doi.org/10.3390/v12091035 - 17 Sep 2020

Cited by 9 | Viewed by 4538

Abstract

crAss-like viruses are a putative family of bacteriophages recently discovered. The eponym of the clade, crAssphage, is an enteric bacteriophage estimated to be present in at least half of the human population and it constitutes up to 90% of the sequences in some [...] Read more.

crAss-like viruses are a putative family of bacteriophages recently discovered. The eponym of the clade, crAssphage, is an enteric bacteriophage estimated to be present in at least half of the human population and it constitutes up to 90% of the sequences in some human fecal viral metagenomic datasets. We focused on the evolutionary dynamics of the genes encoded on the crAssphage genome. By investigating the conservation of the genes, a consistent variation in the evolutionary rates across the different functional groups was found. Gene duplications in crAss-like genomes were detected. By exploring the differences among the functional categories of the genes, we confirmed that the genes encoding capsid proteins were the most ubiquitous, despite their overall low sequence conservation. It was possible to identify a core of proteins whose evolutionary trees strongly correlate with each other, suggesting their genetic interaction. This group includes the capsid proteins, which are thus established as extremely suitable for rebuilding the phylogenetic tree of this viral clade. A negative correlation between the ubiquity and the conservation of viral protein sequences was shown. Together, this study provides an in-depth picture of the evolution of different genes in crAss-like viruses. Full article

(This article belongs to the Special Issue Bioinformatics and Computational Approaches in Viral Genomics and Evolution)

► Show Figures

Figure 1

Figure 1
Number of sequences in each group of protein homologs (cluster size) according to functional categories. The three color bars refer to the different similarity thresholds applied to filter alignments (50%, 80%, and 95% respectively). The capsid proteins are the most frequently present in the crAss-like contigs. See <a href="#app1-viruses-12-01035" class="html-app">Table S2</a> for the names of all homologous groups of proteins and values for all statistics. Full article ">Figure 2
Correlation between the logarithm of the number of sequences in a protein family and the Shannon information content of the positions in the protein sequence alignment. Inverse correlations were obtained both for crAss-like viruses and pVOGs. For proteins in crAss-like viruses, only functional classes having more than four genes are reported. Full article ">Figure 3
Mirrortree algorithm applied to the homologous groups of proteins. Histogram representing the distribution of the pairwise Pearson’s r coefficients. A great number of genes appear to be coevolving. Heatmap of the Pearson correlation coefficient of each protein with any other. Along the x and y axis the 92 ORFs identified in the reference crAssphage genome are represented. The interactions between clusters sharing less than five sequences were colored in white, in order to avoid confusion due to a low size. Histogram representing the average correlation coefficient of every protein represented in their position on the reference genome. The different colors represent the six functional groups. Full article ">

20 pages, 3415 KiB

Open AccessReview

Regulation of KSHV Latency and Lytic Reactivation

by Grant Broussard and Blossom Damania

Viruses 2020, 12(9), 1034; https://doi.org/10.3390/v12091034 - 17 Sep 2020

Cited by 77 | Viewed by 8467

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with three malignancies— Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and [...] Read more.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with three malignancies— Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Central to the pathogenesis of these diseases is the KSHV viral life cycle, which is composed of a quiescent latent phase and a replicative lytic phase. While the establishment of latency enables persistent KSHV infection and evasion of the host immune system, lytic replication is essential for the dissemination of the virus between hosts and within the host itself. The transition between these phases, known as lytic reactivation, is controlled by a complex set of environmental, host, and viral factors. The effects of these various factors converge on the regulation of two KSHV proteins whose functions facilitate each phase of the viral life cycle—latency-associated nuclear antigen (LANA) and the master switch of KSHV reactivation, replication and transcription activator (RTA). This review presents the current understanding of how the transition between the phases of the KSHV life cycle is regulated, how the various phases contribute to KSHV pathogenesis, and how the viral life cycle can be exploited as a therapeutic target. Full article

(This article belongs to the Special Issue New Advances in Kaposi's Sarcoma-Associated Herpesvirus Research)

► Show Figures

Figure 1

Figure 1
The Kaposi’s sarcoma-associated herpesvirus (KSHV) genome enters a latent state after de novo infection. In some cells, early expression of lytic genes such as replication and transcription activator (RTA) triggers expression of the master organizer of latency, latency-associated nuclear antigen (LANA). LANA recruits many components of the host epigenetic machinery to promote the formation of latent KSHV episomes. A pattern of transcriptionally-permissive histone modifications across the KSHV genome gives way to a generally-repressive chromatin state, sparing robust latent gene expression. Lytic gene expression becomes minimal but poised for upregulation upon reactivation. Full article ">Figure 2
KSHV modulates host signaling pathways, chromatin structure, and gene expression to maintain latency. Viral proteins and miRNAs stimulate latency-promoting signaling pathways and chromatin modifiers. The expression and function of RTA is restricted by a variety of proteins and miRNAs from the virus and the host. LANA promotes repressive histone modifications and regulatory loops. Lytic gene expression is minimal but poised for reactivation upon shifts in the relative activities of RTA and LANA. Full article ">Figure 3
Environmental stimuli alter regulation of RTA to promote KSHV reactivation. Host signaling pathways integrate information about the cellular microenvironment that affects the expression and activity of RTA. Lytic viral proteins promote KSHV reactivation in a feed-forward loop. Various isoforms and posttranslational modifications of LANA switch it from an organizer of latency to a facilitator of the lytic cycle. The cell-intrinsic immune system is antagonized by host and viral factors to augment viral reactivation. Full article ">Figure 4
The latent and lytic stages of the KSHV life cycle drive pathogenesis. Latency maintains a population of infected cells by segregating KSHV episomes to daughter cells after mitosis. Lytic reactivation produces infectious virions that expand the pool of infected cells. Both latent and lytic factors promote tumor growth through viral oncogenes or paracrine factors. Lytic replication is sensitive to chemical inhibition, while latent episomes are difficult to target. Therapeutic interventions can force the virus out of latency, promoting cell death. Full article ">

19 pages, 5398 KiB

Open AccessReview

Progress in the Development of Universal Influenza Vaccines

by Wenqiang Sun, Tingrong Luo, Wenjun Liu and Jing Li

Viruses 2020, 12(9), 1033; https://doi.org/10.3390/v12091033 - 17 Sep 2020

Cited by 33 | Viewed by 5735

Abstract

Influenza viruses pose a significant threat to human health. They are responsible for a large number of deaths annually and have a serious impact on the global economy. There are numerous influenza virus subtypes, antigenic variations occur continuously, and epidemic trends are difficult [...] Read more.

Influenza viruses pose a significant threat to human health. They are responsible for a large number of deaths annually and have a serious impact on the global economy. There are numerous influenza virus subtypes, antigenic variations occur continuously, and epidemic trends are difficult to predict—all of which lead to poor outcomes of routine vaccination against targeted strain subtypes. Therefore, the development of universal influenza vaccines still constitutes the ideal strategy for controlling influenza. This article reviews the progress in development of universal vaccines directed against the conserved regions of hemagglutinin (HA), neuraminidase (NA), and other structural proteins of influenza viruses using new technologies and strategies with the goals of enhancing our understanding of universal influenza vaccines and providing a reference for research into the exploitation of natural immunity against influenza viruses. Full article

(This article belongs to the Special Issue Influenza Virus Vaccines)

► Show Figures

Figure 1

Figure 1
Mutation frequency of different antigenic regions and surface amino acids in the hemagglutinin (HA) protein of influenza viruses. H1 represents the 3D structure of A/Puerto Rico/8/34 (H1N1) HA protein (PDB ID: 1RU7) on which the location and distribution of different antigenic regions (Ca1, Ca2, Sa, Sb, Cb, and H1C) are indicated. H3 represents the 3D structure of A/X-31 H3 subtype HA protein (PBD ID: 2VIU) illustrating the location and distribution of different antigenic regions (A, B, C, D, and E). B represents the 3D structure of B/Lee/40 B subtype HA protein (PDB ID: 1RFT) highlighting the location and distribution of different antigenic regions (A, B, C, D, and E). H1 abs, H3 abs, and B abs illustrate the mutation frequency of surface amino acids on the respective HA proteins, with their color representing the intensity of mutation frequency based on H1N1 (n = 531, isolated between 1918–2008), H3N2 (n = 968, 1968–2005), and flu B (n = 209, 1940–2007). Full article ">Figure 2
Phylogenetic tree of HAs of different subtypes (H1–H16) of influenza viruses. The phylogenetic tree was constructed using the neighbor-joining (NJ) method within MEGA software (version 7.0). The colors of the trees are edited using Adobe Illustrator software. The scale bar indicates the average number of amino acid substitutions per site. Full article ">Figure 3
Schematic diagram of immune responses activated by different types of potential universal influenza vaccines. Universal influenza vaccines developed using different strategies involving different target proteins are administered by subcutaneous, intranasal, and intramuscular routes. The antigen is phagocytosed and processed by macrophages and other APC cells. Subsequently, B cell epitopes form a complex with MHC-II and are presented to the cell surface. Under the combined action of CD4 cells, B cells are activated to differentiate into plasma cells and secrete antibodies—e.g., anti-HA, anti-NA, anti-NP, anti-M2e, and anti-HA stem–to neutralize the virus. T cell epitopes—mainly, NP, M1, and HA stem—form a complex with MHC-I and are presented to the cell surface, under the action of CD8 cells and activate T cells to differentiate into CTLs to kill virus-infected cells. Full article ">

16 pages, 5904 KiB

Open AccessArticle

Abrogating ALIX Interactions Results in Stuttering of the ESCRT Machinery

by Shilpa Gupta, Mourad Bendjennat and Saveez Saffarian

Viruses 2020, 12(9), 1032; https://doi.org/10.3390/v12091032 - 16 Sep 2020

Cited by 9 | Viewed by 3727

Abstract

Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently [...] Read more.

Endosomal sorting complexes required for transport (ESCRT) proteins assemble on budding cellular membranes and catalyze their fission. Using live imaging of HIV virions budding from cells, we followed recruitment of ESCRT proteins ALIX, CHMP4B and VPS4. We report that the ESCRT proteins transiently co-localize with virions after completion of virion assembly for durations of 45 ± 30 s. We show that mutagenizing the YP domain of Gag which is the primary ALIX binding site or depleting ALIX from cells results in multiple recruitments of the full ESCRT machinery on the same virion (referred to as stuttering where the number of recruitments to the same virion >3). The stuttering recruitments are approximately 4 ± 3 min apart and have the same stoichiometry of ESCRTs and same residence time (45 ± 30 s) as the single recruitments in wild type interactions. Our observations suggest a role for ALIX during fission and question the linear model of ESCRT recruitment, suggesting instead a more complex co-assembly model. Full article

(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)

► Show Figures

Figure 1

Figure 1
Single versus multiple transient recruitments of ALIX into HIV Gag VLPs versus HIV Gag (YP−) VLPs. HeLa cells stably expressing ALIX–h30–eGFP were transfected with 1500 ng of Gag–mCherry (A,B) or Gag (YP−)–mCherry (C,D) and imaged 4 h after transfection. Assemblies of individual representative VLPs are shown with intensity plots (as gray dots and fitted with gray line) and cropped TIRF images of the Gag (top, gray) and ALIX (bottom, Blue) for (A) Gag-mChery VLPs and (C) Gag (YP−)–mCherry VLPs. Histograms of the first time (dark blue) and later recruitments (light blue) of ALIX are shown for (B) Gag-mChery VLPs and (C) Gag (YP−)–mCherry VLPs. The majority of the first ALIX recruitment took place within 1–10 min after the VLP assembly completing during both YP− and WT assembly. Full article ">Figure 2
Single versus multiple transient recruitments of CHMP4 into HIV Gag VLPs versus HIV Gag (YP-) VLPs. HeLa cells were transfected with 900 ng of ΔCMV–eGFP–flex–CHMP4b and 600 ng (total 1500 ng plasmids) of Gag–mCherry (A,B) or Gag (YP-)–mCherry (C,D) and imaged 7 h after transfection. Assemblies of individual representative VLPs are shown with intensity plots (as gray dots and fitted with gray line) and cropped TIRF images of the Gag (top, gray) and CHMP4b (bottom, green) for (A) Gag–mCherry VLPs and (C) Gag (YP-)–mCherry VLPs. Histograms of the first time (dark green) and later recruitments (light Green) of CHMP4b are shown for (B) Gag–mCherry VLPs and (C) Gag (YP-)–mCherry VLPs. The majority of the first CHMP4b recruitment was within 1–10 min after the VLP assembly completed during both YP- and WT assembly. Full article ">Figure 3
Single versus multiple transient recruitments of VPS4 into HIV Gag VLPs versus HIV Gag (YP-) VLPs.HeLa cells were transfected with 1200 ng of ΔCMV-VPS4-h37-mcherry and 300 ng (total 1500 ng of plasmids) of Gag–mCherry (A,B) or Gag (YP-)–mCherry (C,D) and imaged 7 h after transfection. Assemblies of individual representative VLPs are shown with intensity plots (as gray dots and fitted with gray line) and cropped TIRF images of the Gag (top, gray) and VPS4 (bottom, red) for (A) Gag–mCherry VLPs and (C) Gag (YP-)–mCherry VLPs. Histograms of the first time (dark red) and later recruitments (light red) of VPS4 are shown for (B) Gag-mChery VLPs and (C) Gag (YP-)–mCherry VLPs. The majority of the first VPS4 recruitment was within 1–10 min after the VLP assembly completed during both YP- and WT assembly. Full article ">Figure 4
Multiple transient recruitments of VPS4 into HIV Gag WT and Gag (YP-) VLPs under ALIX depletion. HeLa cells were treated with two rounds of siRNA against ALIX. (A–C) HeLa ALIX–h37–eGFP cell line treated with siRNA against ALIX and then transfected with 1500 ng of Gag–mCherry WT. An individual multinucleated cell was chosen (A) and a representative HIV Gag–mCherry VLP assembly in this cell is shown in (B) with intensity plots (as gray dots and fitted with gray line) and cropped TIRF images of the Gag (top, gray) and ALIX (bottom, Blue). In 40 VLPs analyzed, there was no recruitment of ALIX, as shown in (C). HeLa cells were treated with siRNA against ALIX and then transfected with 1200 ng of ΔCMV–VPS4–h37–mCherry and 300 ng of HIV Gag–eGFP (D–F) and Gag (YP-)–eGFP (G–I). (D,G) Multinucleated cells chosen for experiments with (E&H) showing a representative HIV Gag–mCherry (E) or HIV Gag (YP-)–mCherry (H) VLP assembly with intensity plots and cropped TIRF images of the Gag (top, gray) and VPS4 (bottom, Red). (F,I) Histograms of the number and timing of the first (dark red) and later recruitment (light red) of VPS4 in (F) HIV Gag–mCherry VLPs and (I) HIV Gag (YP−)–mCherry VLPs. Full article ">Figure 5
Delayed transient recruitment of ALIX into HIV Gag (YP− + PTAP−) VLPs or HIV Gag (PTAP−) VLPs. HeLa cells stably expressing ALIX–h30–eGFP were transfected with 1500 ng of Gag (PTAP−)–mCherry (A,B) or Gag (PTAP− + YP−)–mCherry (C,D) and imaged 5 h after transfection. Intensity plots (as gray dots and fitted with gray line) of individual fully assembled representative VLPs are shown and cropped TIRF images of the Gag (top, gray) and AIX (bottom, blue) for (A) Gag(PTAP−)–mCherry VLPs and (C) Gag (PTAP− + YP−)–mCherry VLPs. Histograms of the first time (dark blue) and later recruitments (light blue) of ALIX are shown for (B) Gag (PTAP−)–mCherry VLPs and (C) Gag (PTAP− + YP−)–mCherry VLPs. Alix was recruited within 2 h after the start of the actual experiment. Full article ">

13 pages, 1874 KiB

Open AccessCommunication

Avian Influenza Virus Prevalence and Subtype Diversity in Wild Birds in Shanghai, China, 2016–2018

by Ling Tang, Wangjun Tang, Xiaofang Li, Chuanxia Hu, Di Wu, Tianhou Wang and Guimei He

Viruses 2020, 12(9), 1031; https://doi.org/10.3390/v12091031 - 16 Sep 2020

Cited by 11 | Viewed by 3970

Abstract

From 2016 to 2018, surveillance of influenza A viruses in wild birds was conducted in Shanghai, located at the East Asian–Australian flyway, China. A total of 5112 samples from 51 species of wild birds were collected from three different wetlands. The total three-year [...] Read more.

From 2016 to 2018, surveillance of influenza A viruses in wild birds was conducted in Shanghai, located at the East Asian–Australian flyway, China. A total of 5112 samples from 51 species of wild birds were collected from three different wetlands. The total three-year prevalence of influenza A viruses among them was 8.8%, as assessed using real-time polymerase chain reaction (PCR) methods, and the total prevalence was higher in Anseriformes (26.3%) than in the Charadriiformes (2.3%) and the other orders (2.4%) in the Chongmin wetlands. Anseriformes should be the key monitoring group in future surveillance efforts. The peak prevalence of influenza A viruses in Charadriiformes were in April and September, and in other bird orders, the peaks were in November and December. Twelve subtypes of haemagglutinin (HA; H1–H12) and eight subtypes of neuraminidase (NA; N1, N2, N4–N9) were identified in 21 different combinations. The greatest subtype diversity could be found in common teal, suggesting that this species of the bird might play an important role in the ecology and epidemiology of influenza A viruses in Shanghai. These results will increase our understanding of the ecology and epidemiology of influenza A viruses in wild bird hosts in eastern China, and provide references for subsequent surveillance of influenza A virus in wild birds in this area. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Figure 1

16 pages, 3573 KiB

Open AccessArticle

Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson’s-Fixed Paraffin-Embedded Shrimp Tissue

by Lauren Marie Ochoa, Roberto Cruz-Flores and Arun K. Dhar

Viruses 2020, 12(9), 1030; https://doi.org/10.3390/v12091030 - 16 Sep 2020

Cited by 11 | Viewed by 3889

Abstract

Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection [...] Read more.

Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection and phylogenetic analyses. Total RNA was isolated from known TSV-infected DFPE tissues using three commercially available kits and the purity and ability to detect TSV in the isolated RNA were compared. TSV was successfully detected through RT-qPCR in all the tested samples. Among the TSV-specific primers screened through RT-PCR, primer pair TSV-20 for the RNA-dependent RNA polymerase (RdRp), primers TSV-15 and TSV-16 for the capsid protein gene VP2 and primers TSV-5 for the capsid protein gene VP1 amplified the highest number of samples. To assess the phylogenetic relation among different TSV isolates, the VP1 gene was amplified and sequenced in overlapping segments. Concatenated sequences from smaller fragments were taken for phylogenetic analyses. The results showed that the TSV isolates from this study generally clustered with homologous isolates from the corresponding geographical regions indicating RNA derived from DFPE tissues can be used for pathogen detection and retrospective analyses. The ability to perform genomic characterization from archived tissue will expedite pathogen discovery, development of diagnostic tools and prevent disease spread in shrimp and potentially other aquaculture species worldwide. Full article

(This article belongs to the Section Animal Viruses)

► Show Figures

Figure 1

Figure 1
Taura syndrome virus (TSV) infection in Litopenaeus vannamei. (A) Focal acute-phase infection (large arrow) in the cuticular epithelium characterized by spherical intracytoplasmic inclusions bodies, pyknosis and karyorrhexis. Scale bar, 50 μM. (B) High magnification of the same section; the basophilic intracytoplasmic inclusions bodies (black arrow) and pyknotic nuclei (red arrow) are shown. Scale bar, 20 μM. Full article ">Figure 2
Quantity and quality assessment of total RNA isolated from archived TSV-infected Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissues. Each test was run in triplicate and a mean value for each data set was obtained. The figure in (A) shows a comparison of the mean nucleic acid concentrations for 29 samples from each extraction kit. The figure in (B) shows a comparison of the mean 260/230 ratio values for 29 samples from each extraction kit. The figure in (C) shows a comparison of the mean 260/280 ratio values for the 29 samples from each extraction kit. The horizontal line in each group represents a geometric mean of the data in the corresponding vertical data set. Full article ">Figure 2 Cont.
Quantity and quality assessment of total RNA isolated from archived TSV-infected Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissues. Each test was run in triplicate and a mean value for each data set was obtained. The figure in (A) shows a comparison of the mean nucleic acid concentrations for 29 samples from each extraction kit. The figure in (B) shows a comparison of the mean 260/230 ratio values for 29 samples from each extraction kit. The figure in (C) shows a comparison of the mean 260/280 ratio values for the 29 samples from each extraction kit. The horizontal line in each group represents a geometric mean of the data in the corresponding vertical data set. Full article ">Figure 3
Analysis of the Ct values of TSV amplicons generated by RT-qPCR. Three commercially available extraction kits were utilized for total RNA extraction and total RNA isolated from archived TSV-infected DFPE shrimp tissues was used to generate a 72 bp amplicon of the TSV genome. The horizontal line in each group represents a geometric mean of the Ct values of the corresponding vertical data set. Asterisks represent statistical significance (p < 0.05). Full article ">Figure 4
Representative agarose gel electrophoresis images for the detection of TSV genes and the internal control gene EF-1α (elongation factor 1-α). (A) TSV VP1 gene (primer pairs TSV-5, amplicon size 122 bp). (B) Shrimp EF-1α gene. SPF = Specific Pathogen Free negative control, NC = no template control, PC = TSV positive control. The first lane in each gel represents a molecular weight marker (Invitrogen 1 kb Plus DNA Ladder). Full article ">Figure 5
A pictograph of the RT-PCR amplification of TSV capsid protein VP1 and VP2 genes, RNA-dependent RNA polymerase (RdRp) and shrimp internal control gene EF-1α. The primer pairs (TSV-1 to TSV-21) used to amplify different TSV genes are shown on the top row of the map. The case numbers are indicated in the far-left column (excluding Sample No. 25, as mentioned previously) and the total number of primer pairs (out of 21) that were successful in amplifying each sample can be seen in the far-right column. Green boxes indicate amplification and red boxes indicate no amplification for the corresponding primers. Full article ">Figure 6
Phylogenetic analysis of TSV isolates. The evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 0.87629497 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis included 12 samples from the present study (denoted by ** on the tree) and 29 GenBank accessions. Full article ">

21 pages, 2563 KiB

Open AccessArticle

Identification and Distribution of Novel Cressdnaviruses and Circular Molecules in Four Penguin Species in South Georgia and the Antarctic Peninsula

by Hila Levy, Rafaela S. Fontenele, Ciara Harding, Crystal Suazo, Simona Kraberger, Kara Schmidlin, Anni Djurhuus, Caitlin E. Black, Tom Hart, Adrian L. Smith and Arvind Varsani

Viruses 2020, 12(9), 1029; https://doi.org/10.3390/v12091029 - 16 Sep 2020

Cited by 11 | Viewed by 4531

Abstract

There is growing interest in uncovering the viral diversity present in wild animal species. The remote Antarctic region is home to a wealth of uncovered microbial diversity, some of which is associated with its megafauna, including penguin species, the dominant avian biota. Penguins [...] Read more.

There is growing interest in uncovering the viral diversity present in wild animal species. The remote Antarctic region is home to a wealth of uncovered microbial diversity, some of which is associated with its megafauna, including penguin species, the dominant avian biota. Penguins interface with a number of other biota in their roles as marine mesopredators and several species overlap in their ranges and habitats. To characterize the circular single-stranded viruses related to those in the phylum Cressdnaviricota from these environmental sentinel species, cloacal swabs (n = 95) were obtained from King Penguins in South Georgia, and congeneric Adélie Penguins, Chinstrap Penguins, and Gentoo Penguins across the South Shetland Islands and Antarctic Peninsula. Using a combination of high-throughput sequencing, abutting primers-based PCR recovery of circular genomic elements, cloning, and Sanger sequencing, we detected 97 novel sequences comprising 40 ssDNA viral genomes and 57 viral-like circular molecules from 45 individual penguins. We present their detection patterns, with Chinstrap Penguins harboring the highest number of new sequences. The novel Antarctic viruses identified appear to be host-specific, while one circular molecule was shared between sympatric Chinstrap and Gentoo Penguins. We also report viral genotype sharing between three adult-chick pairs, one in each Pygoscelid species. Sequence similarity network approaches coupled with Maximum likelihood phylogenies of the clusters indicate the 40 novel viral genomes do not fall within any known viral families and likely fall within the recently established phylum Cressdnaviricota based on their replication-associated protein sequences. Similarly, 83 capsid protein sequences encoded by the viruses or viral-like circular molecules identified in this study do not cluster with any of those encoded by classified viral groups. Further research is warranted to expand knowledge of the Antarctic virome and would help elucidate the importance of viral-like molecules in vertebrate host evolution. Full article

(This article belongs to the Special Issue Viromics)

► Show Figures

Figure 1

Figure 1
Sampling sites of the four-penguin species based on their breeding colonies in South Georgia and the Antarctic Peninsula. The numbers in the pie chart indicate the number of individual samples per species. Full article ">Figure 2
Heat map illustrating the dataset-wide detection scaled to sample size, of virus and circular molecule genotypes with the linearized genome organization shown on the right. The detection is based on the recovery of complete sequences by PCR, cloning, and Sanger sequencing. For the purpose of this study, the viruses and circular molecules have been classified as unique species based on a cutoff threshold of 80% genome-wide pairwise identity and genotypes based on 98% genome-wide pairwise identity. The penguin colony key is as follows: (first two letters) AP = Adélie Penguin, CP = Chinstrap Penguin, GP = Gentoo Penguin, and KP = King Penguin, followed by locations BOOT = Booth Island (Western Antarctic Peninsula), KINN = Kinnes Cove (Northern Antarctic Peninsula), BAIL = Baily Head (Deception Island), GEOR = Georges Point (Western Antarctic Peninsula), HALF = Half Moon Island (South Shetland Islands), MOOT = Moot Point (Western Antarctic Peninsula), YANK = Yankee Harbor (South Shetland Islands), and STA = St Andrews Bay (South Georgi). Full article ">Figure 3
Sequence similarity network (SSN) analysis and Maximum likelihood (ML) phylogenetic tree of the Rep amino acid sequences identified in this study compared with those of known viruses in classified families and plasmids identified by Kazlauskas et al. [<a href="#B35-viruses-12-01029" class="html-bibr">35</a>]. The ML phylogenetic tree based on the SSN is mid-point rooted, and branches with aLRT support < 0.8 have been collapsed. The Reps identified in this study are highlighted in green and relevant clades of the tree are shown in the expanded view. Sequences with taxa names CruV-203, -227, -319, and -320 are from metagenomic derived sequences reported in de la Higuera et al. [<a href="#B64-viruses-12-01029" class="html-bibr">64</a>] and do not have accession #s assigned. The level of branch support (>0.8) is depicted as different sized shaded circles. Full article ">Figure 4
Sequence similarity network (SSN) analysis and Maximum likelihood (ML) phylogenetic tree of the CP amino acid sequences that fall within specific networks that encompass sequences identified in this study (highlighted in purple). The ML phylogenetic trees based on the SSNs are mid-point rooted, and branches with aLRT support < 0.8 have been collapsed. The level of branch support (>0.8) is depicted as different sized shaded circles. Full article ">Figure 5
Summary of the recombination detected by RDP4. The methods used to detect recombination are RDP (R) GENCONV (G), BOOTSCAN (B), MAXCHI (M), CHIMERA (C), SISCAN (S), and 3SEQ (T). The method with the highest p-value for each recombination event is bolded. For each genome, a graphic on the left indicates the recombination event with its breakpoint location within the genome. Roman numerals in parenthesis after accession numbers indicate genotype. Full article ">

Journal Menu

Journal Browser

Viruses, Volume 12, Issue 9 (September 2020) – 154 articles

Further Information

Guidelines

MDPI Initiatives

Follow MDPI