Viruses

18 pages, 4010 KiB

Open AccessArticle

How HIV-1 Integrase Associates with Human Mitochondrial Lysyl-tRNA Synthetase

by Xaysongkhame Phongsavanh, Noha Al-Qatabi, Mohammed Samer Shaban, Fawzi Khoder-Agha, Merwan El Asri, Martine Comisso, Raphaël Guérois and Marc Mirande

Viruses 2020, 12(10), 1202; https://doi.org/10.3390/v12101202 - 21 Oct 2020

Cited by 3 | Viewed by 3341

Abstract

Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNA^Lys,3 from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNA^Lys, an essential [...] Read more.

Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNA^Lys,3 from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNA^Lys, an essential step to initiate reverse transcription in the virions. The C-terminal integrase moiety of GagPol is essential for its association with mLysRS. We show that integrases from HIV-1 and HIV-2 bind mLysRS with the same efficiency. In this work, we have undertaken to probe the three-dimensional (3D) architecture of the complex of integrase with mLysRS. We first established that the C-terminal domain (CTD) of integrase is the major interacting domain with mLysRS. Using the pBpa-photo crosslinking approach, inter-protein cross-links were observed involving amino acid residues located at the surface of the catalytic domain of mLysRS and of the CTD of integrase. In parallel, using molecular docking simulation, a single structural model of complex was found to outscore other alternative conformations. Consistent with crosslinking experiments, this structural model was further probed experimentally. Five compensatory mutations in the two partners were successfully designed which supports the validity of the model. The complex highlights that binding of integrase could stabilize the tRNA^Lys:mLysRS interaction. Full article

(This article belongs to the Special Issue Function and Structure of Viral Ribonucleoproteins Complexes)

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6 pages, 234 KiB

Open AccessCase Report

Six Cases of Zika/Dengue Coinfection in a Brazilian Cohort, 2015–2019

by Claudio Siqueira, Valéria Féres, Livia Coutinho, Isabela Junqueira, Luziane Bento, Larissa Montes and João Bosco Siqueira, Jr.

Viruses 2020, 12(10), 1201; https://doi.org/10.3390/v12101201 - 21 Oct 2020

Cited by 6 | Viewed by 2519

Abstract

Brazil is one of the countries which has been most affected by dengue epidemics. This scenario became more challenging with the emergence of Zika virus after 2014. The cocirculation of dengue and Zika viruses makes their diagnosis and treatment a challenge for health [...] Read more.

Brazil is one of the countries which has been most affected by dengue epidemics. This scenario became more challenging with the emergence of Zika virus after 2014. The cocirculation of dengue and Zika viruses makes their diagnosis and treatment a challenge for health professionals, especially due to their similar clinical outcomes. From 2015 to 2019, we followed a cohort of 2017 participants in Goiania, Goias, Central Brazil. Febrile cases were monitored weekly, and after identification of fever, the physician performed a home visit for clinical evaluation and collection of blood/urine for diagnosis of acute dengue/Zika infection in suspected cases. Dengue acute infection was investigated by NS1 antigen and real time RT-PCR and seroconversion of anti-dengue IgM. ZIKV infection was confirmed by real time RT-PCR. Six cases of Zika/dengue coinfection among participants were reported. The clinical outcomes were suggestive for both DENV and ZIKV infection. No coinfected patient had neurological clinical manifestation, warning signs or need for hospitalization. A continuous specific laboratory confirmation for both dengue and Zika viruses should be enforced as part of the surveillance systems even in the presence of very suggestive cases of dengue fever, minimizing the risk of a late detection of ZIKV circulation. Full article

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

27 pages, 1875 KiB

Open AccessReview

Extracellular Vesicles in the Pathogenesis of Viral Infections in Humans

by Allen Caobi, Madhavan Nair and Andrea D. Raymond

Viruses 2020, 12(10), 1200; https://doi.org/10.3390/v12101200 - 21 Oct 2020

Cited by 61 | Viewed by 6134

Abstract

Most cells can release extracellular vesicles (EVs), membrane vesicles containing various proteins, nucleic acids, enzymes, and signaling molecules. The exchange of EVs between cells facilitates intercellular communication, amplification of cellular responses, immune response modulation, and perhaps alterations in viral pathogenicity. EVs serve a [...] Read more.

Most cells can release extracellular vesicles (EVs), membrane vesicles containing various proteins, nucleic acids, enzymes, and signaling molecules. The exchange of EVs between cells facilitates intercellular communication, amplification of cellular responses, immune response modulation, and perhaps alterations in viral pathogenicity. EVs serve a dual role in inhibiting or enhancing viral infection and pathogenesis. This review examines the current literature on EVs to explore the complex role of EVs in the enhancement, inhibition, and potential use as a nanotherapeutic against clinically relevant viruses, focusing on neurotropic viruses: Zika virus (ZIKV) and human immunodeficiency virus (HIV). Overall, this review’s scope will elaborate on EV-based mechanisms, which impact viral pathogenicity, facilitate viral spread, and modulate antiviral immune responses. Full article

(This article belongs to the Section Animal Viruses)

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15 pages, 2669 KiB

Open AccessArticle

Oncolytic H-1 Parvovirus Enters Cancer Cells through Clathrin-Mediated Endocytosis

by Tiago Ferreira, Amit Kulkarni, Clemens Bretscher, Karsten Richter, Marcelo Ehrlich and Antonio Marchini

Viruses 2020, 12(10), 1199; https://doi.org/10.3390/v12101199 - 21 Oct 2020

Cited by 9 | Viewed by 4504

Abstract

H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma [...] Read more.

H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV. Full article

(This article belongs to the Special Issue Advances in Parvovirus Research 2020)

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14 pages, 272 KiB

Open AccessArticle

Antibody Response to Canine Adenovirus-2 Virus Vaccination in Healthy Adult Dogs

by Michèle Bergmann, Monika Freisl, Yury Zablotski, Stephanie Speck, Uwe Truyen and Katrin Hartmann

Viruses 2020, 12(10), 1198; https://doi.org/10.3390/v12101198 - 21 Oct 2020

Cited by 6 | Viewed by 2961

Abstract

Background: Re-vaccination against canine adenovirus (CAV) is performed in ≤3-year-intervals but their necessity is unknown. The study determined anti-CAV antibodies within 28 days of re-vaccination and factors associated with the absence of antibodies and vaccination response. Methods: Ninety-seven healthy adult dogs (last vaccination [...] Read more.

Background: Re-vaccination against canine adenovirus (CAV) is performed in ≤3-year-intervals but their necessity is unknown. The study determined anti-CAV antibodies within 28 days of re-vaccination and factors associated with the absence of antibodies and vaccination response. Methods: Ninety-seven healthy adult dogs (last vaccination ≥12 months) were re-vaccinated with a modified live CAV-2 vaccine. Anti-CAV antibodies were measured before vaccination (day 0), and after re-vaccination (day 7, 28) by virus neutralization. A ≥4-fold titer increase was defined as vaccination response. Fisher’s exact test and multivariate regression analysis were performed to determine factors associated with the absence of antibodies and vaccination response. Results: Totally, 87% of dogs (90/97; 95% CI: 85.61–96.70) had anti-CAV antibodies (≥10) before re-vaccination. Vaccination response was observed in 6% of dogs (6/97; 95% CI: 2.60–13.11). Time since last vaccination (>3–5 years, OR = 9.375, p = 0.020; >5 years, OR= 25.000, p = 0.006) was associated with a lack of antibodies. Dogs from urban areas were more likely to respond to vaccination (p = 0.037). Conclusion: Many dogs had anti-CAV pre-vaccination antibodies, even those with an incomplete vaccination series. Most dogs did not respond to re-vaccination. Based on this study, dogs should be re-vaccinated every 3 years or antibodies should be determined. Full article

(This article belongs to the Special Issue Viral Infections in Companion Animals)

16 pages, 3283 KiB

Open AccessArticle

Maturation of Pseudo-Nucleus Compartment in P. aeruginosa, Infected with Giant phiKZ Phage

by Yana A. Danilova, Viktoriia V. Belousova, Andrey V. Moiseenko, Innokentii E. Vishnyakov, Maria V. Yakunina and Olga S. Sokolova

Viruses 2020, 12(10), 1197; https://doi.org/10.3390/v12101197 - 21 Oct 2020

Cited by 15 | Viewed by 5313

Abstract

The giant phiKZ phage infection induces the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used RT-PCR, fluorescent in situ hybridization (FISH), electron tomography, and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the [...] Read more.

The giant phiKZ phage infection induces the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used RT-PCR, fluorescent in situ hybridization (FISH), electron tomography, and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the distribution of phiKZ and bacterial DNA in infected Pseudomonas aeruginosa cells. The maturation of the pseudo-nucleus was traced in short intervals for 40 min after infection and revealed the continuous spatial separation of the phage and host DNA. Immediately after ejection, phage DNA was located inside the newly-identified round compartments; at a later infection stage, it was replicated inside the pseudo-nucleus; in the mature pseudo-nucleus, a saturated internal network of filaments was observed. This network consisted of DNA bundles in complex with DNA-binding proteins. On the other hand, the bacterial nucleoid underwent significant rearrangements during phage infection, yet the host DNA did not completely degrade until at least 40 min after phage application. Energy dispersive x-ray spectroscopy (EDX) analysis revealed that, during the infection, the sulfur content in the bacterial cytoplasm increased, which suggests an increase of methionine-rich DNA-binding protein synthesis, whose role is to protect the bacterial DNA from stress caused by infection. Full article

(This article belongs to the Special Issue Giant or Jumbo Phages)

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24 pages, 1408 KiB

Open AccessReview

System-Based Approaches to Delineate the Antiviral Innate Immune Landscape

by Karsten Krey, Aleksandra W. Babnis and Andreas Pichlmair

Viruses 2020, 12(10), 1196; https://doi.org/10.3390/v12101196 - 21 Oct 2020

Cited by 6 | Viewed by 5406

Abstract

Viruses pose substantial challenges for society, economy, healthcare systems, and research. Their distinctive pathologies are based on specific interactions with cellular factors. In order to develop new antiviral treatments, it is of central importance to understand how viruses interact with their host and [...] Read more.

Viruses pose substantial challenges for society, economy, healthcare systems, and research. Their distinctive pathologies are based on specific interactions with cellular factors. In order to develop new antiviral treatments, it is of central importance to understand how viruses interact with their host and how infected cells react to the virus on a molecular level. Invading viruses are commonly sensed by components of the innate immune system, which is composed of a highly effective yet complex network of proteins that, in most cases, mediate efficient virus inhibition. Central to this process is the activity of interferons and other cytokines that coordinate the antiviral response. So far, numerous methods have been used to identify how viruses interact with cellular processes and revealed that the innate immune response is highly complex and involves interferon-stimulated genes and their binding partners as functional factors. Novel approaches and careful experimental design, combined with large-scale, high-throughput methods and cutting-edge analysis pipelines, have to be utilized to delineate the antiviral innate immune landscape at a global level. In this review, we describe different currently used screening approaches, how they contributed to our knowledge on virus–host interactions, and essential considerations that have to be taken into account when planning such experiments. Full article

(This article belongs to the Special Issue Innate Immune Sensing of Viruses and Viral Evasion)

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13 pages, 3269 KiB

Open AccessArticle

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants

by Przemysław Wieczorek, Marta Budziszewska, Patryk Frąckowiak and Aleksandra Obrępalska-Stęplowska

Viruses 2020, 12(10), 1195; https://doi.org/10.3390/v12101195 - 20 Oct 2020

Cited by 10 | Viewed by 4624

Abstract

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTV_pJL-Kra) that could [...] Read more.

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTV_pJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTV_pJL-Kra_GFP)—was engineered as well. RNA2 of ToTV_pJL-Kra_GFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTV_pJL-Kra_GFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology. Full article

(This article belongs to the Special Issue Plant Virus Emergence)

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15 pages, 4212 KiB

Open AccessArticle

Adeno-Associated Virus (AAV-DJ)—Cryo-EM Structure at 1.56 Å Resolution

by Qing Xie, Craig K. Yoshioka and Michael S. Chapman

Viruses 2020, 12(10), 1194; https://doi.org/10.3390/v12101194 - 20 Oct 2020

Cited by 21 | Viewed by 6511

Abstract

Adeno-associated virus is the leading viral vector for gene therapy. AAV-DJ is a recombinant variant developed for tropism to the liver. The AAV-DJ structure has been determined to 1.56 Å resolution through cryo-electron microscopy (cryo-EM). Only apoferritin is reported in preprints at 1.6 [...] Read more.

Adeno-associated virus is the leading viral vector for gene therapy. AAV-DJ is a recombinant variant developed for tropism to the liver. The AAV-DJ structure has been determined to 1.56 Å resolution through cryo-electron microscopy (cryo-EM). Only apoferritin is reported in preprints at 1.6 Å or higher resolution, and AAV-DJ nearly matches the highest resolutions ever attained through X-ray diffraction of virus crystals. However, cryo-EM has the advantage that most of the hydrogens are clear, improving the accuracy of atomic refinement, and removing ambiguity in hydrogen bond identification. Outside of secondary structures where hydrogen bonding was predictable a priori, the networks of hydrogen bonds coming from direct observation of hydrogens and acceptor atoms are quite different from those inferred even at 2.8 Å resolution. The implications for understanding viral assembly mean that cryo-EM will likely become the favored approach for high resolution structural virology. Full article

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

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12 pages, 3099 KiB

Open AccessCommunication

Evaluation in Swine of a Recombinant African Swine Fever Virus Lacking the MGF-360-1L Gene

by Elizabeth Ramirez-Medina, Elizabeth A. Vuono, Ayushi Rai, Sarah Pruitt, Ediane Silva, Lauro Velazquez-Salinas, James Zhu, Douglas P. Gladue and Manuel V. Borca

Viruses 2020, 12(10), 1193; https://doi.org/10.3390/v12101193 - 20 Oct 2020

Cited by 23 | Viewed by 3882

Abstract

The African swine fever (ASF) pandemic is currently affecting pigs throughout Eurasia, resulting in significant swine production losses. The causative agent, ASF virus (ASFV), is a large, structurally complex virus with a genome encoding more than 160 genes. The function of most of [...] Read more.

The African swine fever (ASF) pandemic is currently affecting pigs throughout Eurasia, resulting in significant swine production losses. The causative agent, ASF virus (ASFV), is a large, structurally complex virus with a genome encoding more than 160 genes. The function of most of those genes remains unknown. Here, we presented the previously uncharacterized ASFV gene MGF360-1L, the first gene in the genome. The kinetic studies of virus RNA transcription demonstrated that the MGF360-1L gene was transcribed as a late virus protein. The essentiality of MGF360-1L to virus replication was evaluated by developing a recombinant ASFV lacking the gene (ASFV-G-ΔMGF360-1L). In primary swine macrophage cell cultures, ASFV-G-ΔMGF360-1L showed similar replication kinetics as the parental highly virulent field isolate Georgia2007 (ASFV-G). Domestic pigs experimentally infected with ASFV-G-ΔMGF360-1L presented with a clinical disease indistinguishable from that caused by ASFV-G, demonstrating that MGF360-1L was not involved in virulence in swine, the natural host of ASFV. Full article

(This article belongs to the Special Issue Endemic and Emerging Swine Viruses)

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33 pages, 4178 KiB

Open AccessArticle

A Comprehensive Proteomics Analysis of the JC Virus (JCV) Large and Small Tumor Antigen Interacting Proteins: Large T Primarily Targets the Host Protein Complexes with V-ATPase and Ubiquitin Ligase Activities While Small t Mostly Associates with Those Having Phosphatase and Chromatin-Remodeling Functions

by Sami Saribas and Mahmut Safak

Viruses 2020, 12(10), 1192; https://doi.org/10.3390/v12101192 - 20 Oct 2020

Cited by 9 | Viewed by 3578

Abstract

The oncogenic potential of both the polyomavirus large (LT-Ag) and small (Sm t-Ag) tumor antigens has been previously demonstrated in both tissue culture and animal models. Even the contribution of the MCPyV tumor antigens to the development of an aggressive human skin cancer, [...] Read more.

The oncogenic potential of both the polyomavirus large (LT-Ag) and small (Sm t-Ag) tumor antigens has been previously demonstrated in both tissue culture and animal models. Even the contribution of the MCPyV tumor antigens to the development of an aggressive human skin cancer, Merkel cell carcinoma, has been recently established. To date, the known primary targets of these tumor antigens include several tumor suppressors such as pRb, p53, and PP2A. However, a comprehensive list of the host proteins targeted by these proteins remains largely unknown. Here, we report the first interactome of JCV LT-Ag and Sm t-Ag by employing two independent “affinity purification/mass spectroscopy” (AP/MS) assays. The proteomics data identified novel targets for both tumor antigens while confirming some of the previously reported interactions. LT-Ag was found to primarily target the protein complexes with ATPase (v-ATPase and Smc5/6 complex), phosphatase (PP4 and PP1), and ligase (E3-ubiquitin) activities. In contrast, the major targets of Sm t-Ag were identified as Smarca1/6, AIFM1, SdhA/B, PP2A, and p53. The interactions between “LT-Ag and SdhB”, “Sm t-Ag and Smarca5”, and “Sm t-Ag and SDH” were further validated by biochemical assays. Interestingly, perturbations in some of the LT-Ag and Sm t-Ag targets identified in this study were previously shown to be associated with oncogenesis, suggesting new roles for both tumor antigens in novel oncogenic pathways. This comprehensive data establishes new foundations to further unravel the new roles for JCV tumor antigens in oncogenesis and the viral life cycle. Full article

(This article belongs to the Special Issue Polyomaviruses)

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17 pages, 25575 KiB

Open AccessReview

Poxviral Targeting of Interferon Regulatory Factor Activation

by Clara Lawler and Gareth Brady

Viruses 2020, 12(10), 1191; https://doi.org/10.3390/v12101191 - 20 Oct 2020

Cited by 10 | Viewed by 4025

Abstract

As viruses have a capacity to rapidly evolve and continually alter the coding of their protein repertoires, host cells have evolved pathways to sense viruses through the one invariable feature common to all these pathogens—their nucleic acids. These genomic and transcriptional pathogen-associated molecular [...] Read more.

As viruses have a capacity to rapidly evolve and continually alter the coding of their protein repertoires, host cells have evolved pathways to sense viruses through the one invariable feature common to all these pathogens—their nucleic acids. These genomic and transcriptional pathogen-associated molecular patterns (PAMPs) trigger the activation of germline-encoded anti-viral pattern recognition receptors (PRRs) that can distinguish viral nucleic acids from host forms by their localization and subtle differences in their chemistry. A wide range of transmembrane and cytosolic PRRs continually probe the intracellular environment for these viral PAMPs, activating pathways leading to the activation of anti-viral gene expression. The activation of Nuclear Factor Kappa B (NFκB) and Interferon (IFN) Regulatory Factor (IRF) family transcription factors are of central importance in driving pro-inflammatory and type-I interferon (TI-IFN) gene expression required to effectively restrict spread and trigger adaptive responses leading to clearance. Poxviruses evolve complex arrays of inhibitors which target these pathways at a variety of levels. This review will focus on how poxviruses target and inhibit PRR pathways leading to the activation of IRF family transcription factors. Full article

(This article belongs to the Special Issue Innate Immune Sensing of Viruses and Viral Evasion)

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16 pages, 1879 KiB

Open AccessArticle

Phosphoinositide 3′-Kinase γ Facilitates Polyomavirus Infection

by Paul Clark, Gretchen V. Gee, Brandon S. Albright, Benedetta Assetta, Ying Han, Walter J. Atwood and Daniel DiMaio

Viruses 2020, 12(10), 1190; https://doi.org/10.3390/v12101190 - 20 Oct 2020

Cited by 9 | Viewed by 3314

Abstract

Polyomaviruses are small, non-enveloped DNA tumor viruses that cause serious disease in immunosuppressed people, including progressive multifocal leukoencephalopathy (PML) in patients infected with JC polyomavirus, but the molecular events mediating polyomavirus entry are poorly understood. Through genetic knockdown approaches, we identified phosphoinositide 3′-kinase [...] Read more.

Polyomaviruses are small, non-enveloped DNA tumor viruses that cause serious disease in immunosuppressed people, including progressive multifocal leukoencephalopathy (PML) in patients infected with JC polyomavirus, but the molecular events mediating polyomavirus entry are poorly understood. Through genetic knockdown approaches, we identified phosphoinositide 3′-kinase γ (PI3Kγ) and its regulatory subunit PIK3R5 as cellular proteins that facilitate infection of human SVG-A glial cells by JCPyV. PI3Kα appears less important for polyomavirus infection than PI3Kγ. CRISPR/Cas9-mediated knockout of PIK3R5 or PI3Kγ inhibited infection by authentic JCPyV and by JC pseudovirus. PI3Kγ knockout also inhibited infection by BK and Merkel Cell pseudoviruses, other pathogenic human polyomaviruses, and SV40, an important model polyomavirus. Reintroduction of the wild-type PI3Kγ gene into the PI3Kγ knock-out SVG-A cells rescued the JCPyV infection defect. Disruption of the PI3Kγ pathway did not block binding of JCPyV to cells or virus internalization, implying that PI3Kγ facilitates some intracellular step(s) of infection. These results imply that agents that inhibit PI3Kγ signaling may have a role in managing polyomavirus infections. Full article

(This article belongs to the Special Issue Polyomaviruses)

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6 pages, 195 KiB

Open AccessEditorial

Are Viral Vectors Any Good for RNAi Antiviral Therapy?

by Kenneth Lundstrom

Viruses 2020, 12(10), 1189; https://doi.org/10.3390/v12101189 - 20 Oct 2020

Cited by 11 | Viewed by 3456

Abstract

RNA interference (RNAi) represents a novel approach for alternative antiviral therapy. However, issues related to RNA delivery and stability have presented serious obstacles for obtaining good therapeutic efficacy. Viral vectors are capable of efficient delivery of RNAi as short interfering RNA (siRNA), short [...] Read more.

RNA interference (RNAi) represents a novel approach for alternative antiviral therapy. However, issues related to RNA delivery and stability have presented serious obstacles for obtaining good therapeutic efficacy. Viral vectors are capable of efficient delivery of RNAi as short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA). Efficacy in gene silencing for therapeutic applications against viral diseases has been demonstrated in various animal models. Rotavirus (RV) miR-7 can inhibit rotavirus replication by targeting the RV nonstructural protein 5. Viral gene silencing by targeting the RNAi pathway showed efficient suppression of hepatitis B virus replication by adeno-associated virus (AAV)-based delivery of RNAi hepatitis B virus (HBV) cassettes. Hepatitis C virus replication has been targeted by short hairpin RNA molecules expressed from lentivirus vectors. Potentially, RNAi-based approaches could be suitable for antiviral drugs against COVID-19. Full article

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

9 pages, 1172 KiB

Open AccessReview

Budding of a Retrovirus: Some Assemblies Required

by Kevin M. Rose, Vanessa M. Hirsch and Fadila Bouamr

Viruses 2020, 12(10), 1188; https://doi.org/10.3390/v12101188 - 20 Oct 2020

Cited by 24 | Viewed by 3586

Abstract

One of the most important steps in any viral lifecycle is the production of progeny virions. For retroviruses as well as other viruses, this step is a highly organized process that occurs with exquisite spatial and temporal specificity on the cellular plasma membrane. [...] Read more.

One of the most important steps in any viral lifecycle is the production of progeny virions. For retroviruses as well as other viruses, this step is a highly organized process that occurs with exquisite spatial and temporal specificity on the cellular plasma membrane. To facilitate this process, retroviruses encode short peptide motifs, or L domains, that hijack host factors to ensure completion of this critical step. One such cellular machinery targeted by viruses is known as the Endosomal Sorting Complex Required for Transport (ESCRTs). Typically responsible for vesicular trafficking within the cell, ESCRTs are co-opted by the retroviral Gag polyprotein to assist in viral particle assembly and release of infectious virions. This review in the Viruses Special Issue “The 11th International Retroviral Nucleocapsid and Assembly Symposium”, details recent findings that shed light on the molecular details of how ESCRTs and the ESCRT adaptor protein ALIX, facilitate retroviral dissemination at sites of viral assembly. Full article

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

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20 pages, 2545 KiB

Open AccessArticle

A Systematic Evaluation of High-Throughput Sequencing Approaches to Identify Low-Frequency Single Nucleotide Variants in Viral Populations

by David J. King, Graham Freimanis, Lidia Lasecka-Dykes, Amin Asfor, Paolo Ribeca, Ryan Waters, Donald P. King and Emma Laing

Viruses 2020, 12(10), 1187; https://doi.org/10.3390/v12101187 - 20 Oct 2020

Cited by 9 | Viewed by 3875

Abstract

High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have [...] Read more.

High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have taken a systematic approach to evaluate laboratory and bioinformatic pipelines to accurately identify low-frequency SNVs in viral populations. Artificial DNA and RNA “populations” were created by introducing known SNVs at predetermined frequencies into template nucleic acid before being sequenced on an Illumina MiSeq platform. These were used to assess the effects of abundance and starting input material type, technical replicates, read length and quality, short-read aligner, and percentage frequency thresholds on the ability to accurately call variants. Analyses revealed that the abundance and type of input nucleic acid had the greatest impact on the accuracy of SNV calling as measured by a micro-averaged Matthews correlation coefficient score, with DNA and high RNA inputs (10⁷ copies) allowing for variants to be called at a 0.2% frequency. Reduced input RNA (10⁵ copies) required more technical replicates to maintain accuracy, while low RNA inputs (10³ copies) suffered from consensus-level errors. Base errors identified at specific motifs identified in all technical replicates were also identified which can be excluded to further increase SNV calling accuracy. These findings indicate that samples with low RNA inputs should be excluded for SNV calling and reinforce the importance of optimising the technical and bioinformatics steps in pipelines that are used to accurately identify sequence variants. Full article

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

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22 pages, 2371 KiB

Open AccessReview

A Decade in Review: A Systematic Review of Universal Influenza Vaccines in Clinical Trials during the 2010 Decade

by Brigette N. Corder, Brianna L. Bullard, Gregory A. Poland and Eric A. Weaver

Viruses 2020, 12(10), 1186; https://doi.org/10.3390/v12101186 - 20 Oct 2020

Cited by 26 | Viewed by 5317

Abstract

On average, there are 3–5 million severe cases of influenza virus infections globally each year. Seasonal influenza vaccines provide limited protection against divergent influenza strains. Therefore, the development of a universal influenza vaccine is a top priority for the NIH. Here, we report [...] Read more.

On average, there are 3–5 million severe cases of influenza virus infections globally each year. Seasonal influenza vaccines provide limited protection against divergent influenza strains. Therefore, the development of a universal influenza vaccine is a top priority for the NIH. Here, we report a comprehensive summary of all universal influenza vaccines that were tested in clinical trials during the 2010–2019 decade. Of the 1597 studies found, 69 eligible clinical trials, which investigated 27 vaccines, were included in this review. Information from each trial was compiled for vaccine target, vaccine platform, adjuvant inclusion, clinical trial phase, and results. As we look forward, there are currently three vaccines in phase III clinical trials which could provide significant improvement over seasonal influenza vaccines. This systematic review of universal influenza vaccine clinical trials during the 2010–2019 decade provides an update on the progress towards an improved influenza vaccine. Full article

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

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13 pages, 2257 KiB

Open AccessArticle

Deletion of CD2-Like (CD2v) and C-Type Lectin-Like (EP153R) Genes from African Swine Fever Virus Georgia-∆9GL Abrogates Its Effectiveness as an Experimental Vaccine

by Douglas P. Gladue, Vivian O’Donnell, Elizabeth Ramirez-Medina, Ayushi Rai, Sarah Pruitt, Elizabeth A. Vuono, Ediane Silva, Lauro Velazquez-Salinas and Manuel V. Borca

Viruses 2020, 12(10), 1185; https://doi.org/10.3390/v12101185 - 20 Oct 2020

Cited by 52 | Viewed by 4713

Abstract

African swine fever virus (ASFV) is currently the most dreaded infectious disease affecting the global swine production industry. There is no commercial vaccine available, making the culling of infected animals the current solution to control outbreaks. Effective experimental vaccines have been developed by [...] Read more.

African swine fever virus (ASFV) is currently the most dreaded infectious disease affecting the global swine production industry. There is no commercial vaccine available, making the culling of infected animals the current solution to control outbreaks. Effective experimental vaccines have been developed by deleting virus genes associated with virulence. Deletion of the ASFV 9GL gene (∆9GL) has resulted in the attenuation of different ASFV strains, although the degree of attenuation varies across isolates. Here, we investigated the possibility of the increased safety of the experimental vaccine strain ASFV-G-Δ9GL by deleting two additional virus genes involved in pathogenesis, CD2v, a CD2 like viral encoded gene from the EP402R open reading frame (ORF), and C-type lectin-like viral gene, encoded from the EP153R ORF. Two new recombinant viruses were developed, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, harboring two and three gene deletions, respectively. ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, but not ASFV-G-Δ9GL/ΔCD2v, had a decreased ability to replicate in vitro in swine macrophage cultures when compared with parental ASFV-G-Δ9GL. Importantly, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R induced almost undetectable viremia levels when inoculated into domestic pigs and failed to protect them against challenge with parental virulent ASFV-Georgia, while ASFV-G-Δ9GL offered robust protection during challenge. Therefore, the deletion of CD2-like and C-type lectin-like genes significantly decreased the protective potential of ASFV-G-Δ9GL as a vaccine candidate. This study constitutes an example of the unpredictability of genetic manipulation involving the simultaneous deletion of multiple genes from the ASFV genome. Full article

(This article belongs to the Special Issue Endemic and Emerging Swine Viruses)

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12 pages, 2189 KiB

Open AccessArticle

Frequency and Duration of SARS-CoV-2 Shedding in Oral Fluid Samples Assessed by a Modified Commercial Rapid Molecular Assay

by Licia Bordi, Giuseppe Sberna, Eleonora Lalle, Pierluca Piselli, Francesca Colavita, Emanuele Nicastri, Andrea Antinori, Evangelo Boumis, Nicola Petrosillo, Luisa Marchioni, Giulia Minnucci, Elena D’Agostini, Concetta Castilletti, Franco Locatelli, Alimuddin Zumla, Giuseppe Ippolito, Maria Rosaria Capobianchi and on behalf of INMI ReCOVeRI Study Group

Viruses 2020, 12(10), 1184; https://doi.org/10.3390/v12101184 - 20 Oct 2020

Cited by 18 | Viewed by 7038

Abstract

Background: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and [...] Read more.

Background: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and shedding. Methods: Optimal assay conditions and analytical sensitivity were established for the commercial Simplexa™ COVID-19 Direct assay adapted to OF matrix. The assay was used to test 337 OF and NPS specimens collected in parallel from 164 hospitalized patients; 50 bronchoalveolar lavage (BAL) specimens from a subgroup of severe COVID-19 cases were also analysed. Results: Using Simplexa™ COVID-19 Direct on OF matrix, 100% analytical detection down to 1 TCID50/mL (corresponding to 4 × 10³ copies (cp)/mL) was observed. No crossreaction with other viruses transmitted through the respiratory toute was observed. Parallel testing of 337 OF and NPS samples showed highly concordant results (κ = 0.831; 95 % CI = 0.771–0.891), and high correlation of Ct values (r = 0.921; p < 0.0001). High concordance and elevated correlation was observed also between OF and BAL. Prolonged viral RNA shedding was observed up to 100 days from symptoms onset (DSO), with 32% and 29% positivity observed in OF and NPS samples, respectively, collected between 60 and 100 DSO. Conclusions: Simplexa™ COVID-19 Direct assays on OF have high sensitivity and specificity to detect SARS-CoV-2 RNA and provide an alternative to NPS for diagnosis and monitoring SARS-CoV-2 shedding. Full article

(This article belongs to the Collection Coronaviruses)

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16 pages, 1712 KiB

Open AccessArticle

A Nationwide Study about the Dispersal Patterns of the Predominant HIV-1 Subtypes A1 and B in Greece: Inference of the Molecular Transmission Clusters

by Evangelia Georgia Kostaki, Maria Gova, Georgios Adamis, Georgios Xylomenos, Maria Chini, Nikos Mangafas, Marios Lazanas, Simeon Metallidis, Olga Tsachouridou, Vasileios Papastamopoulos, Dimitrios Chatzidimitriou, Eleni Kakalou, Anastasia Antoniadou, Antonios Papadopoulos, Mina Psichogiou, Dimitrios Basoulis, Dimitrios Pilalas, Ifigeneia Papageorgiou, Dimitra Paraskeva, Georgios Chrysos, Vasileios Paparizos, Sofia Kourkounti, Helen Sambatakou, Vasileios Bolanos, Nikolaos V. Sipsas, Malvina Lada, Emmanouil Barbounakis, Evrikleia Kantzilaki, Periklis Panagopoulos, Vasilis Petrakis, Stelios Drimis, Charalambos Gogos, Angelos Hatzakis, Apostolos Beloukas, Lemonia Skoura and Dimitrios Paraskevis Show full author list Hide full author list

Viruses 2020, 12(10), 1183; https://doi.org/10.3390/v12101183 - 19 Oct 2020

Cited by 6 | Viewed by 3075

Abstract

Our aim was to investigate the dispersal patterns and parameters associated with local molecular transmission clusters (MTCs) of subtypes A1 and B in Greece (predominant HIV-1 subtypes). The analysis focused on 1751 (28.4%) and 2575 (41.8%) sequences of subtype A1 and B, respectively. [...] Read more.

Our aim was to investigate the dispersal patterns and parameters associated with local molecular transmission clusters (MTCs) of subtypes A1 and B in Greece (predominant HIV-1 subtypes). The analysis focused on 1751 (28.4%) and 2575 (41.8%) sequences of subtype A1 and B, respectively. Identification of MTCs was based on phylogenetic analysis. The analyses identified 38 MTCs including 2–1518 subtype A1 sequences and 168 MTCs in the range of 2–218 subtype B sequences. The proportion of sequences within MTCs was 93.8% (1642/1751) and 77.0% (1982/2575) for subtype A1 and B, respectively. Transmissions within MTCs for subtype A1 were associated with risk group (Men having Sex with Men vs. heterosexuals, OR = 5.34, p < 0.001) and Greek origin (Greek vs. non-Greek origin, OR = 6.05, p < 0.001) and for subtype B, they were associated with Greek origin (Greek vs. non-Greek origin, OR = 1.57, p = 0.019), younger age (OR = 0.96, p < 0.001), and more recent sampling (time period: 2011–2015 vs. 1999–2005, OR = 3.83, p < 0.001). Our findings about the patterns of across and within country dispersal as well as the parameters associated with transmission within MTCs provide a framework for the application of the study of molecular clusters for HIV prevention. Full article

(This article belongs to the Special Issue HIV Molecular Epidemiology for Prevention 2020)

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18 pages, 2819 KiB

Open AccessReview

Synthesis, Structure, and Function of Human Adenovirus Small Non-Coding RNAs

by Tanel Punga, Mahmoud Darweesh and Göran Akusjärvi

Viruses 2020, 12(10), 1182; https://doi.org/10.3390/v12101182 - 19 Oct 2020

Cited by 17 | Viewed by 4967

Abstract

Human adenoviruses (HAdVs) are common pathogens causing a variety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an advantage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV [...] Read more.

Human adenoviruses (HAdVs) are common pathogens causing a variety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an advantage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV sncRNAs; VA RNA, mivaRNA and MLP-TSS-sRNA will be discussed in the present review. Even though VA RNA has been extensively characterized during the last 60 years, this multifunctional molecule continues to surprise us as more of its structural secrets unfold. Likely, the recent developments on mivaRNA and MLP-TSS-sRNA synthesis and function highlight the importance of these sncRNA in virus replication. Collectively, we will summarize the old and new knowledge about these three viral sncRNAs with focus on their synthesis, structure and functions. Full article

(This article belongs to the Special Issue Adenovirus Pathogenesis)

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3 pages, 184 KiB

Open AccessEditorial

Special Issue: Bovine Viral Diarrhea Virus and Related Pestiviruses

by Helle Bielefeldt-Ohmann

Viruses 2020, 12(10), 1181; https://doi.org/10.3390/v12101181 - 19 Oct 2020

Cited by 3 | Viewed by 2614

Abstract

The genus Pestivirus, encompassing small positive-strand RNA viruses in the family Flaviviridae, comprises four viruses of very significant economic impact to the cattle, swine and sheep industries worldwide: bovine viral diarrhoea virus (BVDV) type 1 and type 2, classical swine fever [...] Read more.

The genus Pestivirus, encompassing small positive-strand RNA viruses in the family Flaviviridae, comprises four viruses of very significant economic impact to the cattle, swine and sheep industries worldwide: bovine viral diarrhoea virus (BVDV) type 1 and type 2, classical swine fever virus (CSFV) and border disease virus (BDV) [...] Full article

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

27 pages, 7500 KiB

Open AccessArticle

Metatranscriptomic Identification of Diverse and Divergent RNA Viruses in Green and Chlorarachniophyte Algae Cultures

by Justine Charon, Vanessa Rossetto Marcelino, Richard Wetherbee, Heroen Verbruggen and Edward C. Holmes

Viruses 2020, 12(10), 1180; https://doi.org/10.3390/v12101180 - 19 Oct 2020

Cited by 22 | Viewed by 4989

Abstract

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we [...] Read more.

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence. Full article

(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)

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30 pages, 2205 KiB

Open AccessArticle

Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates

by Anne Monette and Andrew J. Mouland

Viruses 2020, 12(10), 1179; https://doi.org/10.3390/v12101179 - 18 Oct 2020

Cited by 34 | Viewed by 5871

Abstract

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming [...] Read more.

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases. Full article

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

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15 pages, 3468 KiB

Open AccessArticle

Identification and Tracking of Antiviral Drug Combinations

by Aleksandr Ianevski, Rouan Yao, Svetlana Biza, Eva Zusinaite, Andres Mannik, Gaily Kivi, Anu Planken, Kristiina Kurg, Eva-Maria Tombak, Mart Ustav, Jr., Nastassia Shtaida, Evgeny Kulesskiy, Eunji Jo, Jaewon Yang, Hilde Lysvand, Kirsti Løseth, Valentyn Oksenych, Per Arne Aas, Tanel Tenson, Astra Vitkauskienė, Marc P. Windisch, Mona Høysæter Fenstad, Svein Arne Nordbø, Mart Ustav, Magnar Bjørås and Denis E. Kainov Show full author list Hide full author list

Viruses 2020, 12(10), 1178; https://doi.org/10.3390/v12101178 - 18 Oct 2020

Cited by 44 | Viewed by 7561

Abstract

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to [...] Read more.

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status. Full article

(This article belongs to the Special Issue Antiviral Drug Combinations)

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15 pages, 2029 KiB

Open AccessArticle

Disruption of Zika Virus xrRNA1-Dependent sfRNA1 Production Results in Tissue-Specific Attenuated Viral Replication

by Hadrian Sparks, Brendan Monogue, Benjamin Akiyama, Jeffrey Kieft and J. David Beckham

Viruses 2020, 12(10), 1177; https://doi.org/10.3390/v12101177 - 18 Oct 2020

Cited by 6 | Viewed by 3718

Abstract

The Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths that result from the exonuclease degradation of the viral 3′ untranslated region (UTR). Over the course of infection, these [...] Read more.

The Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths that result from the exonuclease degradation of the viral 3′ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of an incomplete viral genome degradation of the 3′ UTR by the host 5′ to 3′ exoribonuclease, Xrn1. The halting of Xrn1 in the 3′ UTR is due to two RNA pseudoknot structures in the 3′ UTR, termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we generate a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3′ UTR structure. We find that the X1 virus lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult Ifnar1^−/− mice, we find that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to wild-type ZIKV infection. Despite the attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that the targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches. Full article

(This article belongs to the Special Issue MicroRNAs and Other Small RNAs in Viral Infections)

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20 pages, 3322 KiB

Open AccessArticle

Selective Blockade of TNFR1 Improves Clinical Disease and Bronchoconstriction in Experimental RSV Infection

by Dorothea R. Morris, Maria Ansar, Teodora Ivanciuc, Yue Qu, Antonella Casola and Roberto P. Garofalo

Viruses 2020, 12(10), 1176; https://doi.org/10.3390/v12101176 - 17 Oct 2020

Cited by 12 | Viewed by 3522

Abstract

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infants and young children. Although some clinical studies have speculated that tumor necrosis factor (TNF)-α is a major contributor of RSV-mediated airway disease, experimental evidence remains unclear or conflicting. TNF-α initiates inflammation [...] Read more.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infants and young children. Although some clinical studies have speculated that tumor necrosis factor (TNF)-α is a major contributor of RSV-mediated airway disease, experimental evidence remains unclear or conflicting. TNF-α initiates inflammation and cell death through two distinct receptors: TNF-receptor (TNFR)1 and TNFR2. Here we delineate the function of TNF-α by short-lasting blockade of either receptor in an experimental BALB/c mouse model of RSV infection. We demonstrate that antibody-mediated blockade of TNFR1, but not TNFR2, results in significantly improved clinical disease and bronchoconstriction as well as significant reductions of several inflammatory cytokines and chemokines, including IL-1α, IL-1β, IL-6, Ccl3, Ccl4, and Ccl5. Additionally, TNFR1 blockade was found to significantly reduce neutrophil number and activation status, consistent with the concomitant reduction of pro-neutrophilic chemokines Cxcl1 and Cxcl2. Similar protective activity was also observed when a single-dose of TNFR1 blockade was administered to mice following RSV inoculation, although this treatment resulted in improved alveolar macrophage survival rather than reduced neutrophil activation. Importantly, short-lasting blockade of TNFR1 did not affect RSV peak replication in the lung. This study suggests a potential therapeutic approach for RSV bronchiolitis based on selective blockade of TNFR1. Full article

(This article belongs to the Special Issue From RSV to hMPV: Role of Innate Immunity in Pathogenesis)

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12 pages, 1038 KiB

Open AccessArticle

Can Measurements of Inflammatory Biomarkers Be Used to Spot Respiratory Viral Infections?

by Anirban Sinha, René Lutter, Tamara Dekker, Barbara Dierdorp, Peter J. Sterk, Urs Frey and Edgar Delgado-Eckert

Viruses 2020, 12(10), 1175; https://doi.org/10.3390/v12101175 - 17 Oct 2020

Cited by 5 | Viewed by 2323

Abstract

Accurate detection of human respiratory viral infections is highly topical. We investigated how strongly inflammatory biomarkers (FeNO, eosinophils, neutrophils, and cytokines in nasal lavage fluid) and lung function parameters change upon rhinovirus 16 infection, in order to explore their potential use for infection [...] Read more.

Accurate detection of human respiratory viral infections is highly topical. We investigated how strongly inflammatory biomarkers (FeNO, eosinophils, neutrophils, and cytokines in nasal lavage fluid) and lung function parameters change upon rhinovirus 16 infection, in order to explore their potential use for infection detection. To this end, within a longitudinal cohort study, healthy and mildly asthmatic volunteers were experimentally inoculated with rhinovirus 16, and time series of these parameters/biomarkers were systematically recorded and compared between the pre- and post-infection phases of the study, which lasted two months and one month, respectively. We found that the parameters’/biomarkers’ ability to discriminate between the infected and the uninfected state varied over the observation time period. Consistently over time, the concentration of cytokines, in nasal lavage fluid, showed moderate to very good discrimination performance, thereby qualifying for disease progression monitoring, whereas lung function and FeNO, while quickly and non-invasively measurable using cheap portable devices (e.g., at airports), performed poorly. Full article

(This article belongs to the Special Issue Rhinovirus Infections)

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5 pages, 591 KiB

Open AccessCommunication

Expression Pattern of the SARS-CoV-2 Entry Genes ACE2 and TMPRSS2 in the Respiratory Tract

by Yichuan Liu, Hui-Qi Qu, Jingchun Qu, Lifeng Tian and Hakon Hakonarson

Viruses 2020, 12(10), 1174; https://doi.org/10.3390/v12101174 - 16 Oct 2020

Cited by 25 | Viewed by 3834

Abstract

To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease TMPRSS2 in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in [...] Read more.

To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease TMPRSS2 in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in both small airway epithelium and masticatory mucosa, and high levels of expression in nasal epithelium. The expression of ACE2 is low in mucosal-associated invariant T (MAIT) cells and cannot be detected in alveolar macrophages. TMPRSS2 is highly expressed in small airway epithelium and nasal epithelium and has lower expression in masticatory mucosa. Our results provide the molecular basis that the nasal mucosa is the most susceptible locus in the respiratory tract for SARS-CoV-2 infection and consequently for subsequent droplet transmission and should be the focus for protection against SARS-CoV-2 infection. Full article

(This article belongs to the Collection Coronaviruses)

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16 pages, 3829 KiB

Open AccessArticle

Diversity of tRNA Clusters in the Chloroviruses

by Garry A. Duncan, David D. Dunigan and James L. Van Etten

Viruses 2020, 12(10), 1173; https://doi.org/10.3390/v12101173 - 16 Oct 2020

Cited by 2 | Viewed by 2370

Abstract

Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the [...] Read more.

Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNA^Arg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host’s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis that a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNA^Met that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12–13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae. Full article

(This article belongs to the Collection Unconventional Viruses)

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