Journal of General Virology最新文献

Epstein-Barr virus (EBV) ubiquitously infects humans, establishing lifelong persistence in B cells. In vitro, EBV-infected B cells can establish a lymphoblastoid cell line (LCL). EBV's transcripts in LCLs (latency III) produce six nuclear proteins [EBV nuclear antigens (EBNAs)], two latency membrane proteins (LMPs) and various microRNAs and putative long non-coding RNAs [BamHI A rightward transcripts (BARTs)]. The BART and EBNA transcription units are characterized by extensive alternative splicing. We generated LCLs with B95-8 EBV-BACs, including one engineered with 'barcodes' in the first and last repeat of internal repeat 1 (IR1), and analysed their EBV transcriptomes using long-read nanopore direct RNA-seq. Our pipeline ensures appropriate mapping of the W promoter (Wp) 5' exon and corrects W1-W2 exon counts that misalign to IR1. This suggests that splicing across IR1 largely includes all W exons and that Wp-derived transcripts more frequently encode the EBNA-LP start codon than Cp transcripts. Analysis identified a short variant of exon W2 and a novel polyadenylation site before EBNA2, provided insights into BHRF1 miRNA processing and suggested co-ordination between polyadenylation and splice site usage, although improved read depth and integrity are required to confirm this. The BAC region disrupts the integrity of BART transcripts through premature polyadenylation and cryptic splice sites in the hygromycin expression cassette. Finally, a few transcripts extended across established gene boundaries, running from EBNA to BART to LMP2 gene regions, sometimes including novel exons between EBNA1 and the BART promoter. We have produced an EBV annotation based on these findings to help others better characterize EBV transcriptomes in the future.

The highly pathogenic Nipah virus (NiV), a World Health Organization priority pathogen with pandemic potential, remains a critical public health threat due to its capacity to cause fatal encephalitis and respiratory disease. Despite its 1998 emergence, no approved therapeutics exist against NiV infection, underscoring the urgent need to identify genomic regulatory elements as antiviral targets. Our study focuses on the extended 5' UTRs characteristic of NiV transcripts, a distinctive genomic feature whose functional significance remained enigmatic. Comparative reporter assays showed these UTRs strongly inhibit downstream ORF translation through mechanisms distinct from internal ribosome entry site activity. Mutagenesis studies identified upstream ATG elements in multiple transcripts as critical regulators of translational efficiency, with the C 5' UTR exhibiting maximal suppression. A functional hotspot spanning within the C 5' UTR was mapped as the primary ribosomal initiation blockade, and ribosome leaky scanning was confirmed as the mechanism enabling dual-protein expression. Notably, therapeutic targeting of this regulatory element with antisense oligonucleotides significantly impaired viral replication. These findings provide fundamental insights into henipaviral translation regulation while identifying concrete antiviral targets, particularly the druggable C 5' UTR element, advancing efforts to combat this biosafety level 4 pathogen.

Infection with SARS-CoV-2 continues to be a threat to human health. Despite successful immunization campaigns, effective treatment of COVID-19 remains an essential need to help patients and prevent the spread of new virus strains. Viroporins are intracellular ion channels that are essential for virus replication and release, thus presenting promising pharmaceutical targets. Mutations found in variants of concern (VOC) are expected to increase the virulence of the new virus strains. Recognizing the effects of these mutations at the molecular level is essential for the development of improved therapies. Here, we characterized the putative viroporin ORF3a found in VOCs of SARS-CoV-2, using expression constructs containing a myc-tag for identification, and an optional membrane-directing signal peptide. Additionally, constructs containing N-terminal fluorescence protein tags were prepared. Expression and cell surface transport in HEK-293 cells were studied using Western blot and dot blot assays, and the cellular distribution of fluorescent-marked ORF3a was studied using subcellular organelle markers and high-resolution fluorescence microscopy. Viroporin activity of all ORF3a constructs was assessed using cell viability and metabolic assays, as well as patch-clamp recordings of recombinant ORF3a. All ORF3a mutants were expressed well in the recombinant system, and the presence of a signal peptide increased expression on the cellular surface. Intracellular distribution was similar for all variants. The VOC mutants ORF3a-S171L and ORF3a-Q57H showed reduced cytotoxic activity and sensitivity to the viroporin inhibitor rimantadine, respectively, suggesting these positions to be relevant for ORF3a function and a starting point for the search of novel antiviral drugs.

Chikungunya is a febrile infection caused by the Chikungunya virus (CHIKV), an alphavirus which has emerged as a serious public health problem globally. Despite extensive research, our understanding of different host factors facilitating effective CHIKV infection is not clear yet. NEDD4, a member of the E3 ubiquitin ligase, is one such protein. Here, the importance of NEDD4 has been explored during CHIKV infection in vitro. It was observed that the level of NEDD4 is downregulated after CHIKV infection. Interestingly, the CHIKV-nsP3 level and the viral load were decreased significantly when NEDD4 was silenced, while a 93% decrease in the viral load was observed in the case of NEDD4 overexpression, indicating the importance of an optimum level of NEDD4 for effective CHIKV infection. Further study revealed that there was interaction between the NEDD4 and CHIKV-nsP3 proteins through co-immunoprecipitation (CO-IP) during CHIKV infection. Additionally, in silico data illustrated that the WW domain of NEDD4 can bind to the nsP3, as well as the macrodomain of nsP3 (nsp3-MD) of CHIKV. These data were further confirmed by the pull-down assay with purified nsP3-MD. The finding suggested that the host protein NEDD4 might interact directly with nsP3-MD during the CHIKV infection. However, the presence of a faint band of NEDD4 along with nsP3-MD in the pull-down assay may indicate the involvement of some other residues for this interaction. These in silico data were further confirmed by the CO-IP experiments, where all domains of nsP3, MD (macrodomain), AUD (alphavirus unique domain) and HVD (hypervariable domain) were found to interact with NEDD4. Additional experiments with a truncated form of MD, MD1 (1-100 residues of amino acid), revealed that this region is not able to maintain the interaction with NEDD4, indicating the crucial role of the C-terminal region of MD for this binding. In conclusion, these findings offer valuable insights about the importance of NEDD4 during CHIKV infection and the residues of nsP3 for its interaction, which might be useful to design future therapeutics against CHIKV.

Flaviviridae is a family of viruses that are mainly transmitted by mosquito vectors of the genus Aedes, which cause febrile illnesses and, in severe cases, haemorrhagic or neurodegenerative conditions. Over time, these viruses have been reported as emerging pathogens, leading to epidemic outbreaks in various regions worldwide. Additionally, climate change has facilitated the migration of these vectors to regions where they were not previously found. Unfortunately, there are currently no effective treatments or vaccines to prevent or combat Orthoflavivirus infections. Consequently, a deeper understanding of the viral biology and the human host immune response is crucial for advancing the development of therapeutic targets. Amongst the molecules involved in the innate immune response to viral infections are antimicrobial peptides (AMPs), which have been studied for decades. However, their role in Orthoflavivirus infections remains poorly understood. Several researchers have proposed the stimulation or exogenous administration of AMPs during various viral infections, highlighting these molecules as potential innovative therapeutic targets. This study compiles current knowledge on AMPs with a specific focus on Orthoflavivirus infections, emphasizing the importance of these promising therapeutic approaches.

A hallmark of the dengue virus (DENV) infection is the manipulation of host cell membranes, lipid trafficking and lipid droplets (LD), all cellular functions that depend on the cytoskeleton and the cytoplasmic streaming system. We previously reported the interaction between the DENV non-structural (NS1) protein and members of the kinesin motor complex in the Aedes albopictus cell line C6/36. In this work, we present evidence indicating that the protein kinesin light chain 1 (KLC1) is indeed a susceptibility factor for the DENV replicative cycle in mosquito cells. The interaction between NS1 and KLC1 was confirmed by proximity ligation and co-immunoprecipitation assays in cells harvested 24 hpi. In addition, transmission immunoelectron microscopy showed KLC1 decorating the surface of vacuoles in association with NS1. Increased levels of KLC1 were observed starting at 6 hpi, suggesting that virus infection stimulates KLC1 synthesis. Silencing KLC1 expression results in a reduction in viral genome synthesis, decreased secretion of NS1 and a reduction of virus progeny by nearly 1 log. In agreement, similar affectations were observed in infected cells transfected with a peptide that competes and interferes with the interaction between KLC1 and its cargo molecules. Of note, both silencing the expression and interfering with the function of KLC1 resulted in a disorganization of LD, which decreased in number and increased in area, in mock or infected cells. These results, taken together, suggest that KLC1 is a host susceptibility factor for DENV in mosquito cells and appears to play an important role in the proper transport and homeostasis of LD required for flavivirus replication. However, modest colocalization was observed between NS1 and LD, and the significance of the KLC1 and NS1 interactions needs to be further investigated.

This article summarises the activities of the International Committee on Taxonomy of Viruses Bacterial Viruses Subcommittee, detailing developments in the classification of bacterial viruses. We provide here an overview of all new, abolished, moved and renamed taxa proposed in 2024, approved by the Executive Committee, and ratified by membership vote in 2025. Through the collective efforts of 74 international contributors of taxonomy proposals in this round, 43 ratified proposals have led to the creation of one new phylum, one class, four orders, 33 families, 14 subfamilies, 194 genera and 995 species. These proposals mark significant progress in refining the taxonomy of bacterial viruses. Key updates include the creation of new orders and families that include existing taxa to better reflect genomic and evolutionary relationships. As sequencing and bioinformatics approaches continue to advance, further expansion and refinements in viral taxonomy can be anticipated in the coming years.

Mpox (formerly known as monkeypox) virus (MPXV) is the zoonotic pathogen of mpox disease in humans. Its increasing emergence outside of its endemic area has heightened the importance of investigating the virus' prevalence and maintenance in sylvatic reservoirs. The common brown rat (Rattus norvegicus) can inhabit almost anywhere in the UK, posing a threat to zoonotic transmission to humans. Two independent studies were carried out; the first investigated the susceptibility of brown rats to MPXV infection with a clade IIb mpox strain via two challenge routes: intranasal and intradermal. The second study considered the transmission of MPXV between challenged and naïve brown rats. All animals were asymptomatic to mpox disease, although enzyme-linked immunosorbent assay (ELISA) confirmed subclinical infection in challenge groups. In the susceptibility study, reverse transcription PCR (RT-PCR) detected mpox DNA in the lung tissue and throat swabs within the intranasally inoculated group, in addition to viable virus observed from the intranasal throat swabs. In contrast, no virus was detected in either tissues or swabs in the intradermally inoculated group or control group. RT-PCR results from the transmission study detected mpox DNA in tissues and throat swabs taken from challenged animals. Viable virus was observed from tissues and swabs of intranasally challenged animals with infectious titres of ~10²-10⁴ TCID₅₀ per millilitre. ELISA assays in the transmission study showed replicable results compared to the first susceptibility study in directly challenged animals alongside evidence of seroconversion in co-housed naïve animals. In conclusion, brown rats are susceptible to MPXV infection, as they have been demonstrated to maintain viable virus in the absence of clinical signs. Viral transmission of MPXV from infected rats to naïve rats was not observed by RT-PCR, although naïve rats did show antibody responses when exposed to infected rats indicating exposure to virus.