Journal of General Virology最新文献

Pumilio proteins (PUM1 and PUM2) are essential post-transcriptional regulators of gene expression found across plants, animals and yeast. They bind Pumilio response elements (PREs) on messenger RNAs (mRNAs) to modulate mRNA stability and translation. PUMs have been implicated in diverse cellular processes, including stem cell maintenance, neurogenesis and cell cycle regulation. They have also been reported to negatively regulate innate immunity genes and to participate in viral RNA sensing. Previous high-throughput interactome studies revealed that PUMs bind severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We found that SARS-CoV-2 transcripts contain multiple conserved PREs, some of which are preferentially bound by PUM2. Surprisingly, altering PUM levels does not affect the production of progeny virions. However, depletion of PUM1 slightly increases intracellular viral RNA levels, suggesting that PUM1 either plays a mild antiviral role against SARS-CoV-2 or regulates host factors that promote viral replication. Notably, PUM1 also negatively regulates innate immunity gene expression both at steady state and during SARS-CoV-2 infection. Our findings support a complex immunomodulatory role for PUM1, acting both as a negative regulator of innate immunity genes and a mild inhibitor of SARS-CoV-2 RNA accumulation. However, in cell culture, these roles appear negligible based on viral progeny output. Whether the multiple PREs found in the SARS-CoV-2 genome contribute to evasion of PUM1 activity remains an open question.

Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) are widely used in research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their susceptibility to viral infection. However, the extent to which genetic differences among mouse strains affect inflammatory responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein remains unclear. In this study, we compared the susceptibility of five commonly used mouse strains - DBA2, ICR, C3H/HeOuJ, BALB/c and C57BL/6J - to RBD-induced pulmonary inflammation. Histopathological analysis revealed that DBA2 and C57BL/6J mice exhibited significantly heightened inflammatory responses, characterized by increased angiotensin-converting enzyme 2 (ACE2) expression, macrophage infiltration, upregulation of proinflammatory cytokines (e.g. IL-1β, TNF-α) and activation of the IL-6/STAT3 and nicotinamide adenine dinucleotide phosphate oxidase pathways. These responses were markedly attenuated by pretreatment with an anti-ACE2 antibody, supporting a potential role of RBD-ACE2 interactions in driving inflammation, although ACE2-independent mechanisms cannot be excluded. Our findings suggest that DBA2 and C57BL/6J mice are particularly sensitive to RBD exposure and represent cost-effective and physiologically relevant models for studying ACE2-mediated lung inflammation and for evaluating therapeutic interventions targeting coronavirus disease 2019-related inflammatory mechanisms.

The realm Duplodnaviria includes viruses of archaea, bacteria and eukaryotes, with linear dsDNA genomes. Duplodnavirians share a distinct morphogenetic module of four hallmark genes encoding the HK97-fold major capsid protein, a genome packaging ATPase-nuclease (large terminase subunit), a portal protein and a capsid maturation protease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the realm Duplodnaviria, which is available at ictv.global/report/duplodnaviria.

H5Nx clade 2.3.4.4b high pathogenicity avian influenza viruses (HPAIVs) have decimated wild bird and poultry populations globally since the autumn of 2020. In the UK and in continental Europe, the H5N8 subtype predominated during the first epizootic wave of 2020/21, with few detections of H5N1. However, during the second (2021/22) and third (2022/23) epizootic waves, H5N1 was the dominant subtype. The rapid shift in dominance from H5N8 to H5N1 was likely driven by a combination of virological, immunological and/or host-related factors. In this study, we compared viral fitness and immunological responses in ducks, a key reservoir species, using dominant genotypes of H5N1 (genotype AB) and H5N8 (genotype A) from the second wave. While viral shedding dynamics were similar for both viruses, H5N8 was more pathogenic. Antigenic analysis of post-infection duck sera revealed that the haemagglutinin protein was antigenically similar across clade 2.3.4.4b H5 HPAIVs, but neuraminidase proteins displayed different patterns of cross-reactivity. We also modelled a scenario where ducks were pre-exposed to H5N1 (genotype C) or H5N8 (genotype A) from the first wave and subsequently challenged with either homologous or heterologous subtypes from the second wave (genotype AB or A). Despite the absence of seroconversion, pre-exposure to different subtypes resulted in varying clinical outcomes following challenge. These findings indicate that both viral and immunological factors likely played significant roles in the emergence and spread of H5Nx HPAIVs in wild bird populations.

Four Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-encoded microRNAs (miRNAs) have been characterized previously. Here, we report the fifth AcMNPV-encoded miRNA, AcMNPV-miR-5 (Ac-miR-5), which downregulates the viral gene ac66. Target genes were predicted through sequence analysis and validated using luciferase reporter assays. The regulatory effects of Ac-miR-5 on ac66 expression were assessed by reverse transcription quantitative PCR and Western blot. The impact of Ac-miR-5 overexpression on virus infection was analysed by TCID₅₀ assay and quantitative real-time PCR in Sf9 cells. The results showed that Ac-miR-5 downregulates ac66 at both the mRNA and protein levels. Meanwhile, the budded virion production and DNA replication were decreased. Furthermore, microscopy revealed a decrease in the number of polyhedra formed. These findings suggest that Ac-miR-5 overexpression restricts viral load, potentially contributing to the establishment of a stable viral infection within the host cell.

Tomato spotted wilt virus (TSWV) is a major yield-limiting pathogen of peanut in the southeastern USA. To assess viral variability, field isolates were collected from symptomatic peanut plants at three locations in Alabama. Sequence analysis identified mutations in the non-structural movement protein (NSm) and the nucleocapsid protein (N). Subcellular localization studies showed that NSm fused to GFP (NSm:GFP) localized to plasmodesmata and co-localized with callose deposits by 2 days post-infiltration (dpi). By 4 dpi, NSm:GFP formed cytoplasmic aggregates, and callose deposition appeared more consistent with basal plasmodesmatal patterns. The N protein localized to the nuclear periphery and cell margins at 2 dpi and later aggregated in the cytoplasm by 4 dpi. The early callose accumulation associated with NSm expression suggests a potential plant defence response, warranting further investigation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been linked to several neurological symptoms in coronavirus disease 2019 (COVID-19) patients; however, the molecular mechanisms underlying virus-induced neuroinflammation are not well identified. For example, the effect of SARS-CoV-2 infection of the substantia nigra pars compacta (SNpc) of the midbrain has not been addressed, in spite of its importance in dopaminergic signalling and neurodegenerative abnormalities. The purpose of this study was to understand the SARS-CoV-2-induced inflammatory response in the SNpc region of the brain. We inoculated (intranasally) transgenic mice expressing human ACE2 under control of the human keratin 18 promoter (K18-hACE-2 mice) with a 4×10³ TCID₅₀ (mild) dose of SARS-CoV-2. Ten days post-inoculation, SARS-CoV-2 was detected in the SNpc of mice, along with increased levels of IL-1β, B1R and ADAM17, and reduced microglial/macrophage occurrence. miR-330-5p expression was significantly reduced in virus-positive SNpc tissue. Luciferase reporter assays supported ADAM17 as a direct target of miR-330-5p. There was no significant difference in miR-330-5p expression levels in the experimental autoimmune encephalomyelitis mice compared to control mice, demonstrating a crucial role for SARS-CoV-2-induced miR-330-5p in brain pathology. Our study uncovers for the first time that SARS-CoV-2 can invade the SNpc and downregulate miR-330-5p expression levels, causing an enhanced ADAM17 expression and possible neuroinflammatory signalling. The results implicate miR-330-5p as a prospective therapeutic target for alleviating midbrain inflammation associated with SARS-CoV-2 infection.

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.