Journal of General Virology最新文献

Transcription of segmented negative-sense RNA viruses (sNSVs) like rice stripe virus (RSV) is initiated by cap-snatching, where the viral RNA-dependent RNA polymerase cleaves host mRNAs ~10-20 nucleotides downstream of the 5' cap to generate capped RNA leaders (CRLs) that prime viral mRNA synthesis. We previously demonstrated that purified RSV ribonucleoproteins (RNPs) support transcription in vitro, establishing a host-factor-free system to investigate fundamental aspects of this process. In this study, synthetic capped RNAs (scRNAs) with defined sequences were introduced into this system as CRL donors. Analysis of the resultant RSV transcripts revealed that the minimal system accurately mimics key in vivo features of cap-snatching. Specifically, CRL priming and realignment occurred more frequently on the viral RNA template than on the cRNA template, providing strong evidence that these processes are inherent to the viral RNP complex. Quantitative competition assays further revealed that RSV RNPs exhibit sequence-specific selectivity in cap-snatching, preferentially targeting scRNAs with adenosine or cytidine at positions 11-14, with a discernable bias towards adenosine. Surprisingly, no preference was observed for scRNAs containing AC or CA dinucleotides at the cleavage site, despite their potential to generate CRLs capable of base-pairing over two nucleotides with the viral template. Collectively, these findings offer a deeper understanding of the mechanistic aspects of RSV cap-snatching, which may also inform the study of similar processes in other sNSVs.

Koala retrovirus (KoRV) is endemic throughout northern koala populations that are currently in steep decline. We have previously found a strong association between KoRV plasma RNA loads and the risk of secondary diseases, including chlamydiosis. However, it is unclear whether (1) KoRV loads are elevated in sick koalas due to the expansion of leucocyte populations; and/or (2) KoRV induces immunosuppression, increasing susceptibility to disease; and/or (3) KoRV and secondary diseases are related through a third variable such as the physiological stress response. Here, we assess the temporal dynamics of KoRV load over a year and, in relation to chlamydia, to explore the causal direction of their relationship. We also investigated co-variation in faecal glucocorticoid metabolites (FGMs: cortisol and corticosterone) with KoRV load and chlamydia. We found that KoRV load was stable within individuals over time. KoRV load did not increase in wild koalas when they began shedding Chlamydia pecorum or decrease when they then tested negative, through self-clearance or treatment. Koalas that were treated for chlamydiosis maintained higher KoRV loads than their healthy counterparts. We reveal that higher average KoRV loads are correlated with higher average FGM levels (R ²=0.27), which could indicate that higher KoRV loads lead to higher stress levels or that higher cortisol levels increase KoRV replication through a glucocorticoid response element that we have identified in the KoRV genome. However, this association cannot explain the relationship between average KoRV load and chlamydia because average FGM levels were not significantly higher in koalas that contracted chlamydia or initially higher in those with chlamydial disease. Together, these results provide compelling evidence that KoRV load does not respond to a change in disease status and instead that koalas with consistently high KoRV loads are more likely to develop chlamydiosis, potentially through immunosuppression.

Eukaryotic reverse transcriptase genes are mostly incorporated into viruses or transposons. Among the six reported families of reverse-transcribing viruses, three families (Metaviridae/Gypsy, Belpaoviridae/BEL and Pseudoviridae/Copia) have proliferated mostly as transposons, collectively known as LTR retrotransposons. Troyka was reported as a unique lineage of the Metaviridae/Gypsy family. While most LTR retrotransposons generate 4 to 6 bp target site duplications (TSDs) upon integration and contain 5'-TG. CA-3' termini, Troyka generates 3 bp TSDs and contains 5'-CG.CG-3' termini. Here, the distribution and diversity of Troyka were extensively investigated from the available genome sequences. In addition to the six animal phyla reported previously, Troyka was characterized for the first time in Hemichordata, Priapulida, Annelida, Phoronida and Brachiopoda. The unique terminal nucleotides of Troyka are very well conserved. The phylogenetic analysis with various combinations of conserved domains supported the independent position of Troyka from any established retroelement groups, most likely as the sister lineage of the group including Metaviridae/Gypsy, Retroviridae, Caulimoviridae, Lokiretroviruses and Odin LTR retrotransposons. Troyka is here proposed as a new superfamily of LTR retrotransposons.

The Japanese encephalitis virus (JEV), a leading cause of viral encephalitis, exists as similar but non-identical biological clones whose genomic variations/mutations may determine neurovirulence. Two biological clones purified from a brain-derived, clinical isolate were tested in vitro for neurovirulence using human neuronal cells (SK-N-MC) and mouse neuronal cells (NIE-115) and in vivo on a footpad-inoculation mouse model. One clone (JEV-M) demonstrated significantly reduced infectivity in both neuronal cells and the mouse model compared to another clone (JEV-V). Of the 2 E gene point mutations in JEV-M, only the T175C mutation, which translates as an E protein residue 59, amino acid tyrosine to histidine change (Y59H), was found to be the neurovirulence determinant as confirmed by testing with infectious clones with or without these mutations. These novel findings could further our understanding of JEV neuropathogenesis and may be useful for future vaccine development.

Lassa virus (LASV) is circulating in rodents in several countries in West Africa and is the causative agent of the zoonotic disease Lassa fever. Several vaccine candidates have been successfully tested in preclinical and clinical research, while no LASV-specific vaccines or antiviral treatments have been licensed to date. Approximately 500,000 human cases of Lassa fever are estimated to occur every year. However, the high percentage (~80%) of asymptomatic cases and the low frequency of reporting systems in endemic regions demonstrate that Lassa fever cases are highly underreported. Given the frequent spread of the virus by travellers to non-endemic regions, the need for effective vaccines and treatments becomes clear. Here, we describe the generation and preclinical evaluation of two recombinant Lassa virus candidate vaccines, MVA-GP and MVA-NP, which are based on the highly attenuated modified vaccinia virus Ankara (MVA) strain. Constructed in the MVA vector, the MVA-GP vaccine delivers the glycoprotein (GP) of the prototype LASV Josiah strain (lineage IV), whereas the MVA-NP vaccine expresses the nucleoprotein (NP) from the Lassa virus Togo strain (lineage VII). Two immunizations of either MVA-GP or MVA-NP induced substantial polyfunctional Lassa virus-specific CD8⁺ and CD4⁺ T cell responses, respectively, in humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice (HLA-A*0201/DR1 transgenic mice). The identified human Lassa virus-specific T cell epitopes were in agreement with recently discovered T cell epitopes found in Lassa fever survivors. Further studies are warranted to characterize these recombinant MVA-Lassa virus vaccine candidates in other preclinical models and investigate their potential to be characterized in clinical studies in humans.

Circular RNAs (circRNAs) are covalently closed RNA molecules, supporting a wide diversity of functions. While aberrant circRNA expression stands as a recognized hallmark of cancer development, our attention has turned to investigating their role in viral infections, specifically Mardivirus Gallidalpha 2 (GaHV-2, Marek's disease virus) infection. In a previous study focused on the virulent GaHV-2 strain, RB-1B, we extensively catalogued circRNAs produced from virulence genes, notably from the MEQ-vIL-8 locus and the latency-associated transcripts (LATs) gene. Building upon this groundwork, our current investigation uncovers novel loci expressing viral circRNAs in distinct stages of GaHV-2 infection. Furthermore, we extend our focus to viral circRNA signatures in three commonly used Marek's disease vaccines, the avirulent GaHV-2 (CVI988/Rispens strain), non-oncogenic Mardivirus Gallidalpha 3 (GaHV-3) and non-oncogenic Mardivirus Meleagridalpha 1 (MeHV-1) commercially called herpesvirus of turkey. In these vaccine viruses, we identified viral circRNA expression from a locus antisense to the ICP4 immediate early gene, a conserved feature across the three species. This region has been characterized herein for the first time in terms of candidate LATs' exons and introns for GaHV-3 and MeHV-1. LATs' circRNAs were then deeply analysed, and we observed both similarities and distinctions when compared with those of the virulent GaHV-2. Another conserved gene, encoding the DNA packaging protein, was identified as a source of circRNAs in all three species. Eventually, different levels of circRNAs were found to be expressed from the meq locus between virulent and avirulent GaHV-2 strains. Our findings highlight a conserved pattern of virus-derived circRNAs in these related avian alphaherpesviruses. This conservation underscores the potential significance of these transcripts in completing the viral cycle and facilitating viral spread.

The ongoing panzootic of H5N1 high pathogenicity avian influenza virus (HPAIV) has caused the deaths of over half a billion wild birds and poultry and has led to spillover events in both wild and domestic mammals, alongside sporadic human infections. A key driver of this panzootic is the apparent high viral fitness across diverse avian species, which facilitates an increased interface between wild and domestic species. Columbiformes (pigeons and doves) are commonly found on poultry premises, yet little is known about their potential role in contemporary HPAIV disease ecology. Here, we investigated the epidemiological role of pigeons (Columba livia) by determining their susceptibility using decreasing doses of clade 2.3.4.4b H5N1 HPAIV (genotype AB). We investigated infection outcomes and transmission potential between pigeons and chickens. Following direct inoculation, pigeons did not develop clinical signs, and only those inoculated with the highest dose shed viral RNA (vRNA) or seroconverted to H5N1-AB, revealing a 50% minimum infectious dose (MID) of 10⁵ 50% egg infectious dose. Even in the high-dose group, only low-level shedding and environmental contamination were observed, and low-level viral RNAs were present in the tissues of directly inoculated pigeons, with no distinct pathological lesions. Pigeons did not transmit the virus to pigeons or chickens placed in direct contact. We observed distinct differences in sialic acid receptor distribution in the pigeon respiratory tract compared to chickens and ducks. Together, these findings suggest that pigeons have low susceptibility to clade 2.3.4.4b H5N1 HPAIV and are unlikely to contribute significantly to virus maintenance, transmission to poultry or zoonotic infection.

Japanese encephalitis is a neuroinflammatory condition caused by the Japanese encephalitis virus (JEV). Pyruvate kinase muscle isozyme M2 (PKM2) is a key modulator of glucose metabolism. The role of PKM2 in the autoimmune response and inflammation is now increasingly being acknowledged. However, its role in modulating virus replication has not been explored. In the current study, we have explored the role of PKM2 in JEV replication. Our results show that endogenous PKM2 expression is significantly upregulated in JEV-infected mouse neuroblastoma cells. Moreover, overexpression and knockdown studies substantiate the negative effect of PKM2 on JEV replication. Additionally, JEV infection induced signal transducers and activators of transcription 3 (STAT3) activation in the infected neuronal cells. Overexpression of PKM2 enhanced STAT3 activation, while its downregulation reduced STAT3 activation in the JEV-infected neuronal cells. The results suggested that the overexpression of PKM2 exhibited elevated levels of TNF-α and IL-1β, whereas the downregulation of PKM2 decreased their expression. The in silico studies revealed the potential interaction between PKM2 and non-structural protein 1 (NS1), which was subsequently validated in vitro by co-immunoprecipitation assay. The microscopic studies also unveiled the cellular co-localization of PKM2 and NS1 in the endoplasmic reticulum of infected cells. Altogether, these findings indicate that PKM2 negatively regulates JEV replication by inducing the expression of proinflammatory cytokines such as TNF-α and IL-1β. The study also establishes PKM2 as a binding partner of the NS1 protein. Thus, the study paves the path towards understanding the multifaceted role of PKM2 in JEV pathology.

Chikungunya virus (CHIKV) is an enveloped RNA virus that causes Chikungunya fever in humans. It is classified as an arbovirus (arthropod-borne virus) and is transmitted by mosquitoes. Therefore, CHIKV can replicate in many types of cells derived from mammals or insects. In this study, we tried to establish the widely useable CHIKV pseudotype system, adapting various viral species, and we demonstrated the production of Chikungunya pseudotype virus bearing the envelope protein from two different viral families, Coronaviridae or Rhabdoviridae, i.e. severe acute respiratory syndrome coronavirus 2 spike protein or vesicular stomatitis virus glycoprotein, respectively. We found that the capsid protein of CHIKV is not always necessary in the formation of CHIKV-based pseudotypes, but that the capsid protein increases the efficiency of expression of the sub-genomic RNA which codes the labelled genes. Our established pseudotype virus-producing system supplied a sufficient titre of virions for application to most virological experiments that showed more than 10⁴ focus-forming units per millilitre. The pseudotype infections were strictly dependent on compatibility between the viral envelope protein and its receptor, and there was no false-positive background infection. Our established pseudotype virus system can be used as a robust platform to study various virus infections and for screening and in-depth evaluation of neutralizing antibodies and antiviral agents.