Journal of General Virology最新文献

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.

Enteroviruses (EVs) are globally important human and animal pathogens that cause a diverse spectrum of diseases, ranging from febrile illness to paralysis. Despite decades of research, parts of the EV lifecycle remain poorly understood. Replicons, in which reporter genes replace the structural protein-coding region, have proved useful for the study of EV biology. However, it is not possible to study the molecular mechanism(s) of entry, capsid uncoating and genome release without the production of virus particles. To utilize the benefits provided by replicons for the study of viral cell entry, it is necessary to supply the structural proteins separately. Here, we present an EV transencapsidation (TE) system in which reporter replicons are introduced into cells that are modified to express viral structural proteins. The nascent replicons are packaged to form virus particles containing fluorescent or luminescent replicon genomes. This enables the real-time assessment of EV entry and replication through quantification of fluorescence using live-cell imaging. We demonstrate that these TE particles are biologically accurate proxies for EVA71 virions and show their utility for the study of EV entry, uncoating and replication. Additionally, we demonstrate the use of TE particles as platforms for drug discovery and immunological screening, applicable to the development of antiviral therapeutics and the assessment of immunization outcomes.

BK polyomavirus (BKV) causes polyomavirus-associated nephropathy (BKV-nephropathy) and polyomavirus-associated haemorrhagic cystitis following kidney transplantation and allogeneic haematopoietic stem cell transplantation. BKV strains consist of four distinct genotypes (BKV-I, -II, -III and -IV), with more than 80% of individuals seropositive for the BKV-I genotype and lower prevalences of infection or co-infection with the other genotypes. BKV-nephropathy occurs widely in immunosuppressed transplant recipients, with the recommended treatment including reduction of immunosuppressive drugs. High serum titres of BKV-neutralizing antibodies and treatment with BKV-neutralizing intravenous immunoglobulin are associated with reduced levels of BKV-DNAemia in kidney transplant recipients, suggesting anti-BKV antibodies can limit viral load. Thus, we set out to generate broadly neutralizing human mAbs against the viral protein 1 (VP1) major capsid protein of BKV genotypes I-IV using VelocImmune^® transgenic mice that encode for human immunoglobulins. Hybridoma clones from VelocImmune^® mice immunized with combinations of BKV I-IV VP1 and respective genotypes of BKV pseudoviruses (BK-PsVs) were screened using a high-throughput binding assay against BKV-I VP1 expressed in HEK-293 cells. The VP1-binding hybridoma clones were then assessed for neutralization with BK-PsV consisting of respective VP1 proteins from BKV-I, -II, -III or -IV on the virion surface. Overall, 36 broadly cross-neutralizing mAbs against BKV-I, -II, -III and -IV were identified. Importantly, 20 of the cross-reactive immunoglobulins were subjected to nucleotide sequencing, resulting in 6 clonotype families with 14 genetically distinct immunoglobulins. Several of the most effective mAbs were fully humanized with the IgG1 Fc domain with broad neutralization of BK-PsV-I, -II, -III and -IV genotypes with IC50s ranging from ~7 to 200 pM. Thus, these cross-neutralizing mAbs represent potential biologics to be developed into BKV therapeutics.

Cutavirus (CuV), the newest human protoparvovirus (PPV), has gained attention due to its significant association with cutaneous T-cell lymphoma (CTCL) and its precursor, parapsoriasis, whereas the other human PPVs, bufavirus and tusavirus, show no such link. Given this association, it is important to investigate the prevalence of CuV DNA in other tissues, particularly those affected by malignancy or inflammation. This study assessed, by multiplex quantitative PCR, the genoprevalences of all three PPVs in 427 fresh-frozen intestinal biopsies from inflammatory bowel disease (IBD), colorectal cancer, adenomas or healthy mucosa of 185 individuals, as well as in 94 formalin-fixed paraffin-embedded (FFPE) biopsies from malignant and non-malignant breast conditions of 85 patients. The study also compared the DNA prevalences of human herpesvirus (HHV)-6A, -6B and -7 in the breast tissues. CuV mRNA was assayed with reverse-transcription PCR, and corresponding FFPE sections underwent in situ hybridization. CuV DNA was detected in intestinal IBD or healthy mucosa from 6/185 (3.2%) subjects, but no CuV mRNA or in situ signals were detected. In breast biopsies, HHV-6B and HHV-7 DNAs were present in 20.3 and 5.1%, respectively, while all PPVs and HHV-6A were absent. Overall, CuV was absent in all 70 cancer tissues, underscoring its association with CTCL. The low CuV DNA loads and prevalences in intestinal and breast morbidities, and lack of activity, suggest that CuV is unlikely to play a role in these malignancies or inflammatory conditions. In contrast, HHV-6B may be more relevant to breast pathology, even though it is also widely detected in healthy tissues. Nevertheless, our study provides insight into persistent DNA viruses implicated in cancer and highlights their occurrence across various disease manifestations, laying a foundation for future studies.

Rhizomania in sugar beet causes significant yield and sucrose loss worldwide. The disease is caused by Beet necrotic yellow vein virus (BNYVV) and vectored by the plasmodiophorid, Polymyxa betae. Resistance to rhizomania in commercial cultivars is currently dependent upon the use of Rz1 and Rz2 resistant genes in sugar beet. We have developed an ethyl methanesulphonate mutant breeding line (KEMS12; PI672570) that is highly resistant to rhizomania. Using rhizomania-resistant (R) and susceptible (S) sugar beet breeding lines, natural infection and comprehensive RNA sequencing, we have identified the accumulation of a unique set of small non-coding RNAs (sncRNAs) derived from both the sugar beet plant and the BNYVV virus during active infection that may have possible regulatory roles in the resistance and/or susceptibility to rhizomania. Examples of target genes that are differentially expressed in the roots and leaves at early and late infection stages in sugar beet by plant-derived microRNAs (miRNAs) include Bevul.9G209500 (cytoplasm-related catalytic activity), Bevul.2G095700 (potassium transporter) and Bevul.9G160600 (zinc finger), which were up-regulated in the R line (vs. S). Viral-derived sncRNAs predominantly originated from RNA1 and RNA2 and targeted a subset of 69 sugar beet genes with overall expression that showed a strong negative correlation with higher sncRNA abundance. The results presented here for the first time demonstrate putative roles of sugar beet miRNAs in rhizomania resistance and BNYVV-derived sncRNAs and small peptides as potential pathogenicity factors.

After severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a minority of patients experience persistent or emerging symptoms, termed 'long coronavirus disease (COVID)' or post-acute sequelae of COVID-19. The molecular causes of long COVID remain unclear, but disrupted immune functions, such as inflammation and immune deficit, have been posited as factors. In this retrospective cohort study, we measured markers of immune function in a group of patients with long COVID up to 40 months post infection. As proxies for immune function, we measured serum antibody levels, antibody neutralizing capability and production of IFN gamma (IFN-γ) and IL-2 against SARS-CoV-2 and other viral peptides. As expected, serum antibody levels increased over time with vaccinations and reinfections with later variants of SARS-CoV-2. Patients also showed corresponding increasing SARS-CoV-2-specific IL-2 responses and stable IFN-γ responses. We observed no significant differences in immune responses among patients with ongoing long COVID, those who had recovered from it or individuals who recovered from acute COVID-19. Overall, we found no indication of a reduction in these aspects of immune function after SARS-CoV-2 infection. This study provides a valuable foundation for further research aimed at understanding the causes of long COVID.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a notifiable respiratory and reproductive disease of equids that causes significant losses to the equine industry. This study presents a comprehensive analysis of two EAV outbreaks in the UK in 2019, combining virus isolation, sequencing and phylogenetic analysis to provide a holistic understanding of EAV dynamics in these outbreaks. Genetic characterization revealed that all outbreak strains were similar to viruses detected in the UK and Europe from 2004 to 2011, belonging to phylogroup D and clustering in two groups as expected based on epidemiological profiling. Bayesian phylogenetic analysis indicated the direction of transmission. The 2019 EAV strains showed maximum variability in glycoprotein (GP) 3, followed by GP2, non-structural protein 2, GP4 and GP5, with one strain displaying a unique truncation in GP4 at position 149, a feature not previously identified in arteriviruses. Polymorphisms in the CXCL16 gene have been implicated in differential susceptibility to the establishment of long-term carrier states of EAV in stallions. Genotypic analysis of the CXCL16 gene revealed that one horse possessed the homozygous genotype associated with resistance to persistent infection. In contrast, the remaining four horses exhibited the heterozygous genotype, which has been linked to an increased risk of developing a long-term carrier state and contributing to ongoing viral transmission. All infected horses exhibited the presence of neutralizing antibodies in their serum. This study underscores the importance of early detection of silent infections to reduce the spread and prevent clinical outbreaks.

The influenza A H1N1pdm09 virus continues to pose a significant zoonotic threat, with implications for both animal and human health. Italy, which hosts one of the largest swine populations in Europe, is strategically positioned to monitor the evolution of influenza viruses in livestock. This study addresses the genetic diversity and transmission dynamics of H1N1pdm09 in Italian swine, using whole-genome sequencing and dynamic modelling of samples collected from farms across the country. Our findings indicate multiple independent introductions of H1N1pdm09 into Italy. While most were self-limiting, six distinct transmission clusters suggest localized and sustained spread across various regions. Although many introductions were contained, certain lineages demonstrated the ability to circulate within specific areas. Selective pressure analyses showed strong purifying selection across most viral genes in both swine and human hosts, with non-synonymous to synonymous substitution rate (dN/dS) ratios well below 1. The haemagglutinin gene exhibited a higher dN/dS ratio in swine (~0.28) than in humans (~0.22), indicating slightly relaxed selection in swine. Neuraminidase and non-structural proteins were similarly constrained in both hosts. This study underscores the importance of ongoing genomic surveillance to detect viral circulation and mitigate zoonotic risks. Italy's contribution supports global influenza monitoring and reinforces the need for a One Health approach that integrates human, animal and environmental health. These insights are crucial for informing public health strategies and improving preparedness for future outbreaks.

Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.