Journal of General Virology最新文献

Two cases of atypical scrapie (AS) from Dutch sheep were passaged to WT mice and ovine and bovine prion protein (PrP) transgenic mice. Both AS isolates failed to transmit to WT mice but were successfully transmitted to ovine PrP transgenic Tg338 and Tg59 mice, carrying two different sheep PrP alleles. Western blot analysis of the brains from both mouse lines showed a similar protease-resistant band of ~6-8 kDa, which corresponded to the amino acids ~93-155 of the ovine PrP. Sagittal and coronal immunohistochemical profiles of the abnormal scrapie-associated PrP (PrP^Sc) in the mouse brain were constructed and revealed that PrP^Sc was mainly located in white matter structures in the frontal parts of the brain. Cross-passages between the Tg338 and Tg59 mice showed that the same prion strain was isolated in each mouse line.In the primary transmission of one of the AS isolates to Tg338 mice, two mice showed a very short incubation period, a mixed PrP^Sc profile and a classical scrapie banding pattern in the Western blot. Secondary transmission from these mice into WT VM mice and Tg338 mice revealed a similar PrP^Sc profile and incubation period to the classical murine scrapie strain 22A. Both primary and secondary transmissions of the two AS isolates to bovine PrP transgenic mice were negative, so we cannot confirm the previously reported co-presence of classical bovine spongiform encephalopathy (c-BSE) with AS.

Marek's disease virus (MDV) is an alphaherpesvirus responsible for the development of T-cell lymphoma in chickens. Despite the identification of several pro-oncogenic viral molecules encoded by MDV, the processes leading to tumourigenesis remain poorly understood. Extracellular vesicles (EVs) are important mediators of intercellular communication, carrying bioactive molecules that can elicit profound physiological changes in recipient cells. Tumour cells can release significant amounts of EVs, which influence tumour development and growth, metastatic processes and resistance to cancer therapies. These EVs favour cancer cells to evade the immune response, particularly by establishing an immunosuppressive microenvironment. Here, we investigated whether EVs produced by MDV-transformed T lymphocytes affect the proliferation of avian immune cells, a determining feature in neoplastic processes. EVs were purified from an MDV-transformed cell line cultured in vitro. Using a proteomic approach, we confirmed the presence of specific markers and identified a panel of cellular proteins enriched in these EVs. Notably, no viral proteins were detected in the purified EVs. We also demonstrated that EVs are rapidly internalized by recipient chicken cells. Moreover, these EVs can induce a decrease in primary chicken B-cell proliferation, while promoting primary chicken T-cell proliferation. Our findings suggest that EVs released by MDV-transformed cells may contribute to immunosuppression and potentially facilitate lymphoma progression by enhancing T-cell proliferation.

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the virus's ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR assay and an SYBR-based RT-qPCR assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release but instead followed a trend of M>L>S. Overall, this study validates two RT-qPCR assays to measure SEOV RNA, details the accumulation and release of each viral segment and demonstrates the impact of host cell type on hantavirus replication.

Coronaviruses such as SARS-CoV-2 possess the largest positive-sense RNA virus genomes (30 kb). This poses a fidelity problem as the inherent lack of proof-reading capacity of the viral RNA-dependent RNA polymerase results in a high level of mutation. To overcome this issue, coronaviruses encode a 3'-5' exoribonuclease (ExoN) proof-reading activity, which is a property of a complex of two non-structural proteins nsp14 and nsp10. Inactivating ExoN mutants in SARS-CoV-2 are lethal, indicating the importance of this enzymatic activity for virus replication and raising the possibility that small-molecule inhibitors of ExoN activity could be potential antiviral agents. To evaluate this, we used structure-based drug design approaches to identify potential ExoN inhibitors and tested these for activity against infectious SARS-CoV-2. Two compounds had low micromolar EC₅₀ activity and synergized with mutagenic nucleoside analogues. Next-generation sequencing analysis revealed an increased rate of mutation in the presence of these compounds, which is consistent with their mode of action being inhibition of ExoN enzymatic activity.

Nipah virus (NiV), a highly pathogenic zoonotic paramyxovirus, forms two distinct types of membrane-less organelles called inclusion bodies (IBs): cytosolic IBs, which serve as sites of viral RNA synthesis, and those beneath the plasma membrane (IB-PMs), which function in viral particle assembly and budding. We identified the essential domains of the NiV nucleocapsid (N) and phospho (P) proteins required for the formation of cytosolic IB-like structures with liquid-like properties. Dual-site interactions between the N- and C-terminal regions of the N and P proteins were necessary for generating these liquid organelles. In contrast, the matrix protein, along with the N and P proteins, was indispensable for the formation of IB-PM-like structures with low internal fluidity. These findings demonstrate that NiV employs specific protein-protein interactions to generate spatially and functionally distinct IBs, providing new insight into the molecular mechanisms governing viral RNA synthesis and particle formation.

Since the 1990s, the Pacific oyster (Magallana gigas) has experienced repeated mortality events associated with Ostreid herpesvirus 1 (OsHV-1). Although the virus has been genomically characterised, its replication cycle and its interactions with the oyster immune system are still not well understood. In particular, little is known about the dynamics of OsHV-1 gene expression and the immune responses of haemocytes from oysters with varying susceptibility to the virus. While some studies have focused on the expression of specific viral and host genes in whole oysters, none have provided a comprehensive analysis of genome-wide expression across multiple post-infection time points in haemocytes.The lack of oyster cell lines makes studying virus-host interactions in vitro challenging. However, haemocytes, the key immune cells circulating in haemolymph, can be maintained in vitro in the short term and represent a relevant model for analysing infection dynamics. In this study, haemocytes from two M. gigas batches, one highly susceptible and one less susceptible to OsHV-1, were infected in vitro. We tracked the viral and host transcriptomes over a 24-h period post-infection using high-throughput dual transcriptomics.Our results provide a detailed overview of the OsHV-1 transcriptomic landscape in haemocytes from high- and low-susceptible M. gigas over time. In addition, weighted correlation network analysis of host gene expression provided insights into the haemocytes' response to infection and highlighted batch-specific immune responses. This comprehensive transcriptomic study is the first to describe virus-host interactions across multiple stages of infection in haemocytes from Pacific oysters, showing contrasted survival when exposed to OsHV-1.

Influenza D virus (IDV), a new genus within the Orthomyxoviridae family, was initially detected in pigs and cattle. IDV is structurally similar to the influenza C virus (ICV). Influenza A, C and D viruses all have non-human maintenance hosts and likely circulate in several mammalian species. Camelids, as a reservoir for zoonotic viruses, were not extensively studied until the emergence of the Middle East respiratory syndrome coronavirus in 2012. Antibody responses to both ICV and IDV could be detected in dromedary camels from Kenya but not differentiated, owing to cross-reactivity. It was unclear whether these findings reflected a technical issue or suggested a role for camelids in ICV and IDV ecology. In the present study, therefore, alpacas (Vicugna pacos), a camelid species, were experimentally inoculated with ICV (C/Victoria/1/2011) or IDV (D/bovine/France/5920/2014) to assess susceptibility and assess the antibody response. We have demonstrated that alpacas can be experimentally infected with both ICV and IDV with subclinical infection of the upper respiratory tract, suggesting that virus transmission could potentially occur. These findings accord with previous serology results obtained for camelids and indicate a putative role for these species in ICV and IDV ecology.

Novel antiviral drugs targeting DNA viruses are desirable. Previous studies from our laboratory and others have demonstrated that DNA binding bisbenzimide compounds are capable of inhibiting replication of diverse DNA viruses, such as a herpesvirus and prototype or pandemic potential poxviruses. To further the aforementioned studies, we sought to identify a bisbenzimide compound that had been successfully administered to humans and repurpose that compound as a broadly acting antiviral compound. We found that the previously described bisbenzimide antibiotic Ridinilazole was a potent inhibitor of human cytomegalovirus (HCMV) at sub-micromolar concentrations, with no obvious effect on cell viability. However, inhibition of virus replication by Ridinilazole was selective, as Ridinilazole had no obvious effect on the replication of another DNA virus (herpes simplex virus) or an RNA virus (influenza). Based upon our bioinformatic analysis of viral genome content, we propose that the number of putative Ridinilazole binding sites in a viral DNA genome is important for the antiviral action of Ridinilazole. Western blotting and electron microscopy revealed that Ridinilazole had no obvious effect on HCMV protein production, but did decrease the number of HCMV capsids in the cytoplasm. Overall, we identified an antibiotic compound previously used in humans that could be repurposed as an antiviral compound to efficaciously inhibit replication of HCMV.

Marine viruses are ubiquitous entities that impact the biology of a large fraction of prokaryotic and eukaryotic diversity. Dinoflagellates are heterotrophic, mixotrophic and photosynthetic eukaryotes known for their large and complex nuclear genomes, permanently condensed chromosomes and ability to form toxic algal blooms. We used long-read sequencing to explore the genome of the dinoflagellate Oxyrrhis marina and discovered a novel, endogenized ~25 kbp polinton-like virus (PLV). O. marina PLV (OmPLV) resides primarily in low GC content regions and exhibits a distinctive codon usage pattern, suggesting recent endogenization. OmPLV fragmentation was also observed, suggesting that this element is evolving towards a transposable element-like lifestyle. Notably, OmPLV encodes a unique ~1,500 amino acid fused DNA Helicase SF1-pPolB, representing the largest replication proteins reported so far in Preplasmiviricota. Phylogenetic analysis of the major capsid protein positions OmPLV as a novel PLV lineage related to giant viruses. The discovery of OmPLV provides crucial insight into the evolution and diversification of dsDNA viruses and serves as an important reference point for elucidating the role of endogenized viruses in expansion of dinoflagellate genomes.

The 2013 Ebola virus (EBOV) outbreak was the largest in history. Despite recent advances in both vaccines and monoclonal antibody therapies, 12 years later, EBOV still poses a substantial threat. Previously, we published a ligand discovery pipeline combining in silico screening of compounds with a robust and rapid EBOV minigenome assay for early-stage inhibitor validation at Biological Safety Level 2. Here, we present the further use of this pipeline to identify three compounds that also inhibit EBOV minigenome transcription and replication. They are efficacious in the nM range, exhibited low cytotoxicity and were specific, with no effect on either a T7 RNA polymerase-driven firefly luciferase or a Bunyamwera virus minigenome. Furthermore, these small-molecule inhibitors exhibited the ability to block EBOV minigenome activity when applied after establishment of replication complexes, with implications for potential post-exposure EBOV treatment.