Journal of General Virology最新文献

Orthohantaviruses are emerging zoonotic pathogens that can cause life-threatening diseases in humans. Their tripartite, negative-sense RNA genome is encapsidated by the viral nucleoprotein, but the subcellular localization and dynamics of these viral RNAs and proteins remain poorly characterized. Here, we present a comprehensive microscopy-based analysis of Puumala virus, the most prevalent orthohantavirus in northern and western Europe. Using fluorescence in situ hybridization (FISH) and Multiple Sequential FISH, we mapped the distribution of viral mRNAs, viral genomic RNAs (vRNAs), nucleoproteins and associated host cell factors, quantifying their intracellular abundance, co-localization and subcellular positioning. We observed distinct clustering of vRNAs with varying degrees of nucleoprotein association, a progressive increase in nucleoprotein expression levels during infection and a concomitant rise in the abundance of P-bodies. Moreover, we report a marked spatial reorganization of actin, microtubules and P-bodies, indicating substantial structural remodelling of host cells during orthohantavirus infections. Using a novel end-specific FISH assay, we observed a preferential 5'-end degradation of vRNAs in P-bodies, shedding new light on orthohantavirus RNA turnover within host RNA-processing compartments. Finally, co-localization analyses revealed the formation of potential 'viral factories' composed of nucleoprotein, vRNAs and viral mRNAs, indicating an intricate assembly hierarchy. Collectively, these findings improve our understanding of orthohantavirus replication and highlight the dynamic interplay between virus and host cell components.

Deltaviruses are circular, negative-sense RNA agents that replicate autonomously but depend on heterologous envelope glycoproteins for spread. Only partial sequences of deltaviruses had been reported from marsupials. By reanalysing public metatranscriptomes from the Australian fat-tailed dunnart (Sminthopsis crassicaudata), we assemble the first complete marsupial deltavirus genome and test its replication in human and animal cells. The fat-tailed dunnart deltavirus (FtDDeV) is a 1,680-nt circular RNA that folds into a canonical unbranched rod-like structure and encodes a 195-aa delta antigen (FtDDAg). Genomic and antigenomic HDV-like ribozymes are present and conserve catalytic core motifs. Phylogenetic analyses cluster FtDDAg with the Tasmanian devil sequence, and both are quite close to RDAg from the neotropical rodent species Proechimys semispinosus. A dimeric FtDDeV cDNA replicon supports time-dependent DAg accumulation in human, simian, rodent and Tasmanian devil cells, with faster kinetics in rodents and marsupial cells. FtDDAg accumulation patterns in host nuclei show characteristic viral hubs, observed with other deltaviruses. No obvious coinfecting helper viruses were detected in FtDDeV-positive libraries. Our study extends the confirmed host range of deltaviruses to marsupials and provides a replication-competent clone to investigate helper usage, host restriction and deltavirus evolution.

Coronaviruses evolve rapidly, with recombination and mutation fostering the emergence of variant strains. The avian coronavirus infectious bronchitis virus (IBV) is an important poultry pathogen and a valuable natural model for studying coronaviruses. Australian strains have evolved independently of those infecting chickens elsewhere in the world, so understanding the biology and evolution of these strains can further our understanding of factors driving the emergence of novel coronaviruses. We infected groups of specific pathogen-free Leghorn chickens with six Australian IBVs (from five distinct genotypes) isolated between 1962 and 2013. All six affected the respiratory tract, but only one was nephropathogenic (N1/62). All six induced significant lesions and actively replicated in the upper respiratory tract, but they had lower levels of replication and induced less severe lesions in the middle and lower trachea. There were significant differences between the six strains in the severity of the lesions they induced and in their tissue tropism and effect on tracheal ciliary motility. Strains N1/62 (strain T) and N1/03 caused the most severe tracheal ciliostasis and replicated to the highest levels in tissues. Strain N1/03 caused the most severe lesions at 9 days post-infection. Only strain N1/03 caused lesions in the lower trachea. Overall, strains N1/03 and N1/62 were the most virulent. This study is the first to characterize the histological changes induced by the recently isolated Australian IBVs and compare them directly with older strains. Recombination between field and vaccine strains of IBV has yielded emergent IBVs in Australia that appear to have enhanced virulence for the respiratory tract.

Previous research has demonstrated that influenza A virus (IAV) infection activates activating protein-1 (AP-1) transcription factors as part of the antiviral response. In this study, we identified cFos as the most upregulated AP-1 transcription factor during IAV infection in A549 human lung cells. Surprisingly, the knockdown of cFos resulted in impaired IAV replication. Fluorescence microscopy and functional analyses indicated that cFos is implicated in IAV infection through its nuclear function, rather than its cytoplasmic role as an activator of lipid synthesis. The investigation into the role of cFos in IAV infection revealed increased apoptosis and elevated interferon-β mRNA levels in cFos-knockdown A549 cells during IAV infection. This suggests that cFos may enhance cell survival and reduce interferon-β expression during infection, thereby facilitating IAV proliferation. Furthermore, the levels of viral NA mRNA and the expression of late viral proteins NA and M2 decreased upon cFos-knockdown. Overall, this study identifies cFos as a proviral factor for IAV, through the modulation of innate immunity and apoptosis during infection, and potentially by supporting the viral transcription.

γδ T cells are a highly abundant lymphocyte subset in chickens and play key roles in early immune responses to infection. It has been recently shown that γδ T cells restrict Marek's disease virus (MDV) pathogenesis; however, it remained elusive if they play a role in vaccine protection. In this study, we vaccinated γδ T-cell-knockout chickens with the commercial turkey herpesvirus (HVT) vaccine and challenged them with very virulent MDV. The disease incidence was significantly increased in vaccinated chickens in the absence of γδ T cells. This increase was comparable to a previous study in unvaccinated γδ T-cell-knockout chickens, suggesting that γδ T cells only play a minor role in vaccine protection. Furthermore, the viral load in the spleen was significantly increased in the absence of γδ T cells. Interestingly, viral load in the skin and in dust shed by the animals was drastically increased, suggesting that the absence of γδ T cells affects MDV shedding. In addition, we quantified various immune cell subsets to determine if these could be responsible for the observed phenotypes. Together, our data indicate that γδ T cells only play a minor role in HVT-mediated protection, but their absence drastically affects shedding of this deadly pathogen in vaccinated animals.

Atkinsonella hypoxylon virus (AhV) is a fungi-infecting betapartitivirus and the typical member of the Partitiviridae, a family of persistent viruses that infect a broad range of organisms. Partitiviruses have been largely overlooked following their designation as cryptic viruses. However, evidence is accumulating that they play an important role in the ecology of their hosts. Since the capsid proteins of partitiviruses have been implicated in virus-host interactions, exploring their structural biology may give clues into the evolution, horizontal transmission and host adaptation of partitiviruses. The capsid of AhV shares the same organization of other partitiviruses with 60 dimeric capsid protein protomers arranged with T=1 icosahedral symmetry. The structure, determined by cryo-electron microscopy to 2.4 Å, shows that AhV has a unique iteration on the protrusion domain with an extensive network of hydrophobic interactions among equivalent interdigitating loops at the dimerization interface. AhV also shares a conserved helical core in the shell domain, which we extend to all genera of the recognized partitiviruses using protein structure prediction. The helical core appears to be a conserved element of the picobirnavirus lineage of capsid protein folds and provides a template onto which various elaborations of the protrusion domain have evolved. The involvement of the protrusion in virus-host interactions has previously been proposed, and our findings provide evidence of a structural device enabling capsid protein diversification during the evolution of the Partitiviridae.

The development of an effective prophylactic hepatitis C virus (HCV) vaccine is a priority to achieve global elimination of the virus. Accurate assessment of the neutralizing breadth of antibodies induced by vaccines and a clear understanding of the antigenic differences between viral variants included in vaccines are both critical for vaccine development. Prior studies have indicated that HCV genotypes (gts) do not dictate the sensitivity of HCV envelope glycoprotein (E1E2) variants to neutralizing antibodies. However, most of these prior studies under-sampled variants from gts 2-6. Here, we selected a genetically diverse and representative panel of gt 2-6 E1E2 variants, used them to generate HCV pseudoparticles (HCVpp), and measured neutralization of these HCVpp by neutralizing antibodies and HCV-immune plasma from persons infected with gt 1-6 viruses. We found that neutralization results obtained with this gt 2-6 panel were remarkably similar to results obtained with a previously described, antigenically diverse, gt 1-predominant reference panel of 15 HCVpp. These data confirm that, even considering genetically diverse HCV variants across gt 1-6, E1E2 antigenicity is not dictated by gt, and that the previously published panel of 15 HCVpp represents neutralization of all HCV gts with reasonable accuracy.

Ovine herpesvirus 1 (OvHV-1) was first identified over 50 years ago in sheep with ovine pulmonary adenocarcinoma (OPA). An aetiological role in OPA was later ruled out and OvHV-1 was found to be a common infection in sheep in several countries. Here, we report the sequence and annotation of the complete OvHV-1 genome. The virus has a similar genomic architecture to members of the Macavirus genus of the subfamily Gammaherpesvirinae and is most closely related to bovine gammaherpesvirus 6 (BoGHV6). The OvHV-1 genome comprises a 144,637 bp unique region predicted to encode at least 74 proteins bounded by multiple copies of a 699 bp GC-rich repetitive terminal repeat. Predicted genes include 61 ORFs conserved among all gammaherpesviruses, and 12 genes present only in macavirus genomes, including a homologue of ovine IL-10, previously reported only in ovine gammaherpesvirus 2, and an ornithine decarboxylase, previously described only in BoGHV6. A further gene appears unique to OvHV-1 among macaviruses, encoding a viral-FLIP (FLICE-like inhibitory protein), similar to those found in some other gammaherpesviruses. Notably, several macavirus genes previously predicted in BoGHV6 are defective in OvHV-1. The availability of the genome sequence of OvHV-1 will facilitate studies on its relationship to other macaviruses and its role, if any, in disease.

Senecavirus A (SVA) is a picornavirus that was first isolated in the USA in 2002; however, there is evidence that the virus was circulating in swine herds since 1988. Despite frequent reports of vesicular disease outbreaks caused by SVA infection in swine in Brazil since 2014, there is limited data on the genetic diversity and evolution of the virus in the country. SVA was isolated from swine exhibiting vesicular lesions, with samples originating from farms or slaughterhouses across 57 municipalities in 8 Brazilian states between 2018 and 2022. We obtained 501 SVA genomes through Sanger and Oxford Nanopore sequencing. Phylogenetic analysis revealed that Brazilian SVA sequences are genetically distinct from sequences from other countries, including China, USA and Canada, and form a monophyletic cluster, indicating a common ancestor for the viruses currently circulating in Brazil. Furthermore, there are two main clusters with sequences from the Midwest and Southern regions, suggesting that SVA is evolving independently in the swine population of the country. Pairwise sequence comparisons allowed us to identify seven unique mutations with high frequency in the Brazilian SVA sequences. Notably, mutations were identified in specific regions of the capsid proteins that interact with the host cell receptor (ANTRX1) and in surface-exposed residues, suggesting potential evolutionary changes due to receptor interaction or immune pressure. Recombination analysis provided evidence of at least five recombination events among the Brazilian strains. These findings offer new insights into the evolution of SVA circulating in Brazil and into the global epidemiology/evolutionary dynamics of the virus.

The nucleolus is a multifunctional hub and a common target of viral proteins, yet its role in infections by cytoplasmically replicating RNA viruses remains poorly defined. In rabies virus (RABV), the phosphoprotein (P-protein) isoform P3 localizes to nucleoli and inhibits rRNA biogenesis, whereas P1 lacks nucleolar targeting, even when forced into the nucleus. Here, we show that nucleolar targeting is an isoform- and phylogroup-specific property of lyssavirus P-proteins. Isoforms P3-P5 accumulate in nucleoli, whereas P1 and P2 are excluded. Comparative analyses revealed that P3 nucleolar targeting is conserved in phylogroup I but absent in phylogroup II lyssaviruses. Co-immunoprecipitation assays identified conserved interactions with nucleolin and nucleophosmin (NPM1) but divergent binding to Treacle and nucleolar and coiled-body phosphoprotein 1 (NOLC1). These findings define nucleolar targeting as a gain-of-function of truncated P isoforms, demonstrate its conservation across phylogroup I lyssaviruses and suggest broader engagement with membraneless compartments, highlighting potential therapeutic vulnerabilities.