Viruses — Open Access Journal

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection. Full article

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation. Full article

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling. Full article

The Arctic marine environment experiences dramatic seasonal changes in light and nutrient availability. To investigate the influence of seasonality on Arctic marine virus communities, five research cruises to the west and north of Svalbard were conducted across one calendar year, collecting water from the surface to 1000 m in depth. We employed metabarcoding analysis of major capsid protein g23 and mcp genes in order to investigate T4-like myoviruses and large dsDNA viruses infecting prokaryotic and eukaryotic picophytoplankton, respectively. Microbial abundances were assessed using flow cytometry. Metabarcoding results demonstrated that seasonality was the key mediator shaping virus communities, whereas depth exerted a diversifying effect within seasonal virus assemblages. Viral diversity and virus-to-prokaryote ratios (VPRs) dropped sharply at the commencement of the spring bloom but increased across the season, ultimately achieving the highest levels during the winter season. These findings suggest that viral lysis may be an important process during the polar winter, when productivity is low. Furthermore, winter viral communities consisted of Operational Taxonomic Units (OTUs) distinct from those present during the spring-summer season. Our data provided a first insight into the diversity of viruses in a hitherto undescribed marine habitat characterized by extremes in light and productivity. Full article

We have developed a generalizable “smart molecular diagnostic” capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use. Full article

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotiniasclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S.sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production. Full article

Type I interferon (IFN-I) is the first line of mammalian host defense against viral infection. To counteract this, the flaviviruses, like other viruses, have encoded a variety of antagonists, and use a multi-layered molecular defense strategy to establish their infections. Among the most potent antagonists is non-structural protein 5 (NS5), which has been shown for all disease-causing flaviviruses to target different steps and players of the type I IFN signaling pathway. Here, we summarize the type I IFN antagonist mechanisms used by flaviviruses with a focus on the role of NS5 in regulating one key regulator of type I IFN, signal transducer and activator of transcription 2 (STAT2). Full article

Parechovirus A (Human parechovirus, HPeV) causes symptoms ranging from severe neonatal infection to mild gastrointestinal and respiratory disease. Use of molecular approaches with RT-PCR and genotyping has improved the detection rate of HPeV. Conventional methods, such as viral culture and immunofluorescence assay, together with molecular methods facilitate comprehensive viral diagnosis. To establish the HPeV immunofluorescence assay, an antibody against HPeV capsid protein VP0 was generated by using antigenic epitope prediction data. The specificity of the anti-HPeV VP0 antibody was demonstrated on immunofluorescence assay, showing that this antibody was specific for HPeV but not enteroviruses. A total of 74 HPeV isolates, 7 non–polio-enteroviruses and 12 HPeV negative cell culture supernatant were used for evaluating the efficiency of the anti-HPeV VP0 antibody. The sensitivity of HPeV detection by the anti-HPeV VP0 antibody was consistent with 5′untranslated region (UTR) RT-PCR analysis. This study established comprehensive methods for HPeV detection that include viral culture and observation of cytopathic effect, immunofluorescence assay, RT-PCR and genotyping. The methods were incorporated into our routine clinical practice for viral diagnosis. In conclusion, this study established a protocol for enterovirus and HPeV virus identification that combines conventional and molecular methods and would be beneficial for HPeV diagnosis. Full article

As part of the Pr55^Gag polyprotein, p6 fulfills an essential role in the late steps of the replication cycle. However, almost nothing is known about the functions of the mature HIV-1 p6 protein. Recently, we showed that p6 is a bona fide substrate of the insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease. This phenomenon appears to be specific for HIV-1, since p6 homologs of HIV-2, SIV and EIAV were IDE-insensitive. Furthermore, abrogation of the IDE-mediated degradation of p6 reduces the replication capacity of HIV-1 in an Env-dependent manner. However, it remained unclear to which extent the IDE mediated degradation is phylogenetically conserved among HIV-1. Here, we describe two HIV-1 isolates with IDE resistant p6 proteins. Sequence comparison allowed deducing one single amino acid regulating IDE sensitivity of p6. Exchanging the N-terminal leucine residue of p6 derived from the IDE sensitive isolate HIV-1_NL4-3 with proline enhances its stability, while replacing Pro-1 of p6 from the IDE insensitive isolate SG3 with leucine restores susceptibility towards IDE. Phylogenetic analyses of this natural polymorphism revealed that the N-terminal leucine is characteristic for p6 derived from HIV-1 group M except for subtype A, which predominantly expresses p6 with an N-terminal proline. Consequently, p6 peptides derived from subtype A are not degraded by IDE. Thus, IDE mediated degradation of p6 is specific for HIV-1 group M isolates and not occasionally distributed among HIV-1. Full article

Rift Valley fever virus (RVFV) is a zoonotic arbovirus of the Phenuiviridae family. Infection causes abortions in pregnant animals, high mortality in neonate animals, and mild to severe symptoms in both people and animals. There is currently an ongoing effort to produce safe and efficacious veterinary vaccines against RVFV in livestock to protect against both primary infection in animals and zoonotic infections in people. To test the efficacy of these vaccines, it is essential to have a reliable challenge model in relevant target species, including ruminants. We evaluated two goat breeds (Nubian and LaMancha), three routes of inoculation (intranasal, mosquito-primed subcutaneous, and subcutaneous) using an infectious dose of 10⁷ pfu/mL, a virus strain from the 2006–2007 Kenyan/Sudan outbreak and compared the effect of using virus stocks produced in either mammalian or mosquito cells. Our results demonstrated that the highest and longest viremia titers were achieved in Nubian goats. The Nubian breed was also efficient at producing clinical signs, consistent viremia (peak viremia: 1.2 × 10³–1.0 × 10⁵ pfu/mL serum), nasal and oral shedding of viral RNA (1.5 × 10¹–8 × 10⁶ genome copies/swab), a systemic infection of tissues, and robust antibody responses regardless of the inoculation route. The Nubian goat breed and a needle-free intranasal inoculation technique could both be utilized in future vaccine and challenge studies. These studies are important for preventing the spread and outbreak of zoonotic viruses like RVFV and are supported by the Canadian-led BSL4ZNet network. Full article

Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection. Full article

Arbuscular mycorrhizal fungi (AMF), including Rhizophagus spp., can play important roles in nutrient cycling of the rhizosphere. However, the effect of virus infection on AMF’s role in nutrient cycling cannot be determined without first knowing the diversity of the mycoviruses in AMF. Therefore, in this study, we sequenced the R. irregularis isolate-09 due to its previously demonstrated high efficiency in increasing the N/P uptake of the plant. We identified one novel mitovirus contig of 3685 bp, further confirmed by reverse transcription-PCR. Also, publicly available Rhizophagus spp. RNA-Seq data were analyzed to recover five partial virus sequences from family Narnaviridae, among which four were from R. diaphanum MUCL-43196 and one was from R. irregularis strain-C2 that was similar to members of the Mitovirus genus. These contigs coded genomes larger than the regular mitoviruses infecting pathogenic fungi and can be translated by either a mitochondrial translation code or a cytoplasmic translation code, which was also reported in previously found mitoviruses infecting mycorrhizae. The five newly identified virus sequences are comprised of functionally conserved RdRp motifs and formed two separate subclades with mitoviruses infecting Gigasporamargarita and Rhizophagusclarus, further supporting virus-host co-evolution theory. This study expands our understanding of virus diversity. Even though AMF is notably hard to investigate due to its biotrophic nature, this study demonstrates the utility of whole root metatranscriptome. Full article

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health. Full article

Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago [1]. Its complete DNA sequence was reported in 1997 [2]. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non-^{essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1} as the reference species for a new SPP1-like viruses genus of the Siphoviridae family. Full article

The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that it infects. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRα) were identified as receptors, respectively, for the trimeric and pentameric glycoprotein H/glycoprotein L (gH/gL) complexes that in large part govern HCMV cell tropism, while CD90 and CD147 were also found to play roles during entry. X-ray crystal structures for the proximal viral fusogen, glycoprotein B (gB), and for the pentameric gH/gL complex (pentamer) have been solved. A novel virion gH complex consisting of gH bound to UL116 instead of gL was described, and findings supporting the existence of a stable complex between gH/gL and gB were reported. Additional work indicates that the pentamer promotes a mode of cell-associated spread that resists antibody neutralization, as opposed to the trimeric gH/gL complex (trimer), which appears to be broadly required for the infectivity of cell-free virions. Finally, viral factors such as UL148 and US16 were identified that can influence the incorporation of the alternative gH/gL complexes into virions. We will review these advances and their implications for understanding HCMV entry and cell tropism. Full article

The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae. Full article

Feline foamy virus (FFV) and feline leukemia virus (FeLV) belong to the Retroviridae family. While disease has not been reported for FFV infection, FeLV infection can cause anemia and immunosuppression (progressive infection). Co-infection with FFV/FeLV allows evaluation of the pathogenic potential and epidemiology of FFV infection in cats with FeLV pathology. Blood and buccal swab samples from 81 cats were collected in Rio de Janeiro. Plasma was serologically tested for FeLV. DNA extracted from peripheral blood mononuclear cells and buccal swabs was used to PCR detect FFV and FeLV. A qPCR was developed to detect and measure FFV proviral loads (pVLs) in cats. FeLV qPCR was performed using previous methods. The median log10 pVL of FFV mono-infected individuals was lower than found in FFV/FeLV co-infected cats in buccal swabs (p = 0.003). We found 78% of cats had detectable buccal FFV DNA in FFV mono-infected and FFV co-infected FeLV-progressive cats, while in FeLV-regressive cats (those without signs of disease) 22% of cats had detectable buccal FFV DNA (p = 0.004). Our results suggest that regressive FeLV infection may reduce FFV saliva transmission, the main mode of FV transmission. We did not find evidence of differences in pathogenicity in FFV mono- and -dually infected cats. In summary, we show that FVs may interact with FeLV within the same host. Our study supports the utility of cats naturally co-infected with retroviruses as a model to investigate the impact of FV on immunocompromised mammalian hosts. Full article

Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 10⁷ and 1 × 10⁸ vRNA copies/mL at 3–10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 10⁶ and 1 × 10⁷ vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies. Full article

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive P_tight promoter at the 5′ end and an antigenomic ribozyme of hepatitis delta virus at the 3′ end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection. Full article