The murine hepatitis virus (MHV) is an important model system for studying coronavirus (CoV) molecular and cell biology. Despite this, few reagents for MHV are available through repositories such as ATCC or Addgene, potentially limiting the widespread adoption of MHV as a tractable model system. To overcome some challenges inherent in the existing MHV reverse genetics systems, we developed a plasmid-launched transformation-associated recombination (TAR) cloning-based system to assemble the MHV (strain A59; MHV-A59) genome. Following assembly in yeast, virus replication was launched by transfecting the fully assembled genome into HEK-293T cells. MHV-A59 recovered using this TAR cloning-based approach (WT MHV-A59) replicated with kinetics identical to the virus recovered using a ligation- and T7-based approach (WT MHV-A59). Additionally, WT MHV-A59 can be detected at least 10 h post-transfection without requiring additional nucleocapsid (N) provided in trans. Lastly, we demonstrated the tractability of this TAR cloning-based system by recovering MHV-A59 expressing an 11 amino acid-containing HiBiT tag fused to the C-terminus of spike (S). While this virus, S MHV-A59, replicated with reduced kinetics compared to WT MHV-A59, the kinetics of virion production could be measured over time directly from the supernatant. This report represents the first plasmid-launched, TAR cloning-based system for MHV-A59. Furthermore, it describes a new reporter virus that could be used to study early steps during MHV-A59 entry and be used in the screening of antiviral compounds. To support future research with MHV-A59, we have made the necessary plasmids for this system available through ATCC.

RNA structures that are functionally important are defined as -acting RNA elements because their functions cannot be compensated for in trans. The -acting RNA elements in the 3' UTR of coronaviruses are important for replication; however, the mechanism linking the -acting RNA elements to their replication function remains to be established. In the present study, a comparison of the biological processes of the interactome and the replication efficiency between the 3' UTR -acting RNA elements in coronaviruses, including severe acute respiratory syndrome coronavirus 2, suggests that (i) the biological processes, including translation, protein folding and protein stabilization, derived from the analysis of the -acting RNA element interactome and (ii) the architecture of the -acting RNA elements and their interactomes are highly correlated with coronavirus replication. In addition, alteration of the interactome using the compound 5-benzyloxygramine can cause reduced coronavirus replication, reinforcing the connection between -acting RNA elements and replication by interactome. Together, these results link -acting RNA elements to the coronavirus replication and establish a model to analyse the -acting RNA elements in the replication of RNA viruses by interactome.

Translation errors, impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal, including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways, the ubiquitin-proteasome system and selective autophagy. In humans, the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain-Barré syndrome). Non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses, catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm, ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here, we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell, although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells, while transient overexpression of UBA1 favours faster replication kinetics, with higher virus titres and protein levels detected. Moreover, UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.

Species A rotaviruses (RVs), which belong to the family and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.

Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here, we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors, we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody, with Chinese hamster ovary cells expressing 20-50 mg l after 5 days when cells were grown to a density of ~1×10 cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both and .

The complexity and speed of evolution in viruses with RNA genomes makes predictive identification of variants with epidemic or pandemic potential challenging. In recent years, machine learning has become an increasingly capable technology for addressing this challenge, as advances in methods and computational power have dramatically improved the performance of models and led to their widespread adoption across industries and disciplines. Nascent applications of machine learning technology to virus research have now expanded, providing new tools for handling large-scale datasets and leading to a reshaping of existing workflows for phenotype prediction, phylogenetic analysis, drug discovery and more. This review explores how machine learning has been applied to and has impacted the study of viruses, before addressing the strengths and limitations of its techniques and finally highlighting the next steps that are needed for the technology to reach its full potential in this challenging and ever-relevant research area.

Human immunodeficiency virus (HIV) is an exemplar virus, still the most studied and best understood and a model for mechanisms of viral replication, immune evasion and pathogenesis. In this review, we consider the earliest stages of HIV infection from transport of the virion contents through the cytoplasm to integration of the viral genome into host chromatin. We present a holistic model for the virus-host interaction during this pivotal stage of infection. Central to this process is the HIV capsid. The last 10 years have seen a transformation in the way we understand HIV capsid structure and function. We review key discoveries and present our latest thoughts on the capsid as a dynamic regulator of innate immune evasion and chromatin targeting. We also consider the accessory proteins Vpr and Vpx because they are incorporated into particles where they collaborate with capsids to manipulate defensive cellular responses to infection. We argue that effective regulation of capsid uncoating and evasion of innate immunity define pandemic potential and viral pathogenesis, and we review how comparison of different HIV lineages can reveal what makes pandemic lentiviruses special.

Nudiviruses (family ) are double-stranded DNA viruses that infect various insects and crustaceans. Among them, Heliothis zea nudivirus 1 (HzNV-1) represents the rare case of a lepidopteran nudivirus inducing a sexual pathology. Studies about molecular pathological dynamics of HzNV-1 or other nudiviruses are scarce. Hence, this study aims to provide a transcriptomic profile of HzNV-1 in an ovary-derived cell line of (HZ-AM1), during early (3, 6 and 9 h post-infection) and advanced (12 and 24 h post-infection) stages of infection. Total RNA was extracted from both virus- and mock-infected cells, and RNA-seq analysis was performed to examine both virus and host transcriptional dynamics. Hierarchical clustering was used to categorize viral genes, while differential gene expression analysis was utilized to pinpoint host genes that are significantly affected by the infection. Hierarchical clustering classified the 154 HzNV-1 genes into four temporal phases, with early phases mainly involving transcription and replication genes and later phases including genes for virion assembly. In addition, a novel viral promoter motif was identified in the upstream region of early-expressed genes. Host gene analysis revealed significant upregulation of heat shock protein genes and downregulation of histone genes. The identification of temporal patterns in viral gene expression enhances the molecular understanding of nudivirus pathology, while the identified differentially expressed host genes highlight the key pathways most hijacked by HzNV-1 infection.

is a family of plant viruses with tripartite, positive-sense RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain sub-genomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and may be transmitted between hosts mechanically, via pollen, or non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomatoes, cucurbits, bananas, fruit trees, common beans and alfalfa. Since the adoption of metagenomic high-throughput sequencing methodologies, there has been a notable increase in the number of species in the genus . This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family which is available at ictv.global/report/bromoviridae.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection, hospitalization and death in infants worldwide. No fully effective RSV therapy using direct antivirals is marketed. Since clinical efficacy data from naturally infected patients for such antivirals are not available yet, animal studies are indispensable to predict therapeutic intervention. Here, we report the impact of an RSV fusion inhibitor, JNJ-49214698, on severe RSV-associated acute lower respiratory tract infection (ALRTI) in neonatal lambs. Randomized animals were treated once daily with 25 mg/kg JNJ-49214698, starting either before RSV infection, 1 day post-infection or as late as peak lung viral load on Day 3 post-infection. Treatment efficacy was assessed by scoring clinical signs of illness, development of RSV-induced gross and microscopic lung lesions and measuring virus titres in the lungs. Treatment with JNJ-49214698 was very effective in all treatment groups. Even in animals for which treatment was delayed until peak viral load was reached, a reduced amount and severity of gross and microscopic lesions, as well as RSV titres and RNA levels, were found. These results strongly suggest that treatment with small-molecule fusion inhibitors is an effective strategy to treat patients who are diagnosed with an RSV-induced ALRTI.

Boid inclusion body disease (BIBD) caused by reptarenaviruses affects captive constrictor snake collections worldwide. The disease manifests by the formation of cytoplasmic inclusion bodies in various tissues. Curiously, a snake with BIBD nearly always carries a swarm of reptarenavirus small and large segments rather than a single pair, and the composition of the swarm can vary between tissues. The role of reptarenavirus coinfections in BIBD pathogenesis remains unknown, and it is unclear whether reptarenavirus infection affects the susceptibility to superinfection or to secondary infections. For mammarenaviruses, co- and/or superinfection can occur if the infecting viruses are genetically divergent enough, and we hypothesized reptarenaviruses to behave similarly. To study this hypothesis, we employed boa constrictor kidney- and brain-derived cell cultures to perform a set of co- and superinfection experiments with one hartmanivirus and five reptarenavirus isolates. While all tested viruses replicated well in the boid kidney cells, experiments on the brain-derived cells showed differences in the replication efficacy between the viruses, suggesting that reptarenaviruses could differ in their target cell spectra. The quantification of viral RNA released from infected cells as a proxy for virus replication did not reveal overt differences between mono- and coinfections. Passaging of coinfected cell cultures revealed that one of the reptarenavirus isolates requires a coinfecting reptarena- or hartmanivirus to establish a persistent infection. Superinfection experiments on persistently reptarenavirus-infected cell lines suggested some interference between genetically similar viruses. We hypothesized that such interference would be mediated by the viral Z protein (ZP) specifically locking the genetically similar viral polymerase in a catalytically inactive state. Curiously, experiments on ZP-expressing cell lines indicated ZP overexpression not to significantly affect the amount of released viral RNA. Our experiments showed very little co- or superinfection interference between genetically dissimilar reptarenaviruses, reflecting the naturally occurring reptarenavirus coinfections in snakes with BIBD.

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that affects the silkworm, , causing substantial economic losses in sericulture. This study investigates the mechanisms underlying budded virus egress, focusing on the roles of the ubiquitin-proteasome pathway (UPP) machinery. BmNPV produces two virion types: budded virions (BVs) and occlusion-derived virions (ODVs), which differ in their envelope origins and functions. Recent findings suggest similarities in the budding pathways of BmNPV and Autographa californica multiple nucleopolyhedrovirus (AcMNPV), involving plasma membrane budding and multivesicular body (MVB) pathways. The study reveals that specific UPP-related proteins, including 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), polyubiquitin, proteasome alpha subunit 6 (PSMA6) and proteasome zeta subunit (PSMZ), are involved in BV egress. Using recombinant viruses and UPP inhibitors, we demonstrate the necessity of these proteins for GP64 secretion and effective BV release. RNA interference and cell surface display of GP64 analyses further validate the critical role of UPP in BmNPV BV egress and protein secretion. This research enhances our understanding of the mechanisms behind BmNPV MVB budding and GP64 secretion while also identifying potential targets for controlling the virus in sericulture.

Lassa virus (LASV) is an Old World (OW) mammarenavirus that causes Lassa fever, a life-threatening acute febrile disease endemic in West Africa. Lymphocytic choriomeningitis virus (LCMV) is a worldwide-distributed, prototypic OW mammarenavirus of clinical significance that has been largely neglected as a human pathogen. No licensed OW mammarenavirus vaccines are available, and the current therapeutic option is limited to the off-label use of ribavirin, which offers only partial efficacy. This situation underscores the urgent need to develop novel antivirals against human pathogenic mammarenaviruses. Previously, we showed that afatinib, a pan-ErbB tyrosine kinase inhibitor, inhibited multiple steps of the life cycles of OW LASV and LCMV, as well as the New World Junín virus vaccine strain Candid#1. In the present study, we investigated the inhibitory effect of U-73122, a phospholipase C inhibitor that acts downstream of ErbB signalling, on LCMV multiplication. U-73122 inhibited WT recombinant (r) LCMV multiplication in cultured cells. Preincubation of cell-free LCMV virions with U-73122 resulted in impaired virion infectivity. U-73122 also inhibited the infection of rLCMVs expressing heterologous viral glycoproteins, including the vesicular stomatitis Indiana virus (VSIV) glycoprotein, whereas WT VSIV infection was not affected by U-73122 treatment. Our results show the novel bioactivity of U-73122 as an LCMV inhibitor and indicate the presence of a virion-associated molecule that is necessary for virion infectivity and can be exploited as a potential antiviral drug target against human pathogenic mammarenavirus infections.

In , phosphoproteins (P) are essential polymerase cofactors, forming oligomers and interacting with viral components to facilitate replication. Previous studies have demonstrated that a P-derived peptide (PFr) from the respiratory syncytial virus (RSV), containing the oligomerization domain (OD) and C-terminal domain (CTD), effectively inhibits RSV replication. Here, we extend this approach to paramyxoviruses, including HPIV3, MeV and MuV. Customized PFrs exhibited potent inhibitory effects against their respective viruses, with IC values below 100 nM, while showing minimal cytotoxicity. These findings highlight the potential of targeting P oligomerization as a broad-spectrum antiviral strategy for paramyxoviruses and other mononegaviruses.

It is important to be able to retrospectively determine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections with high accuracy, both for post-coronavirus disease 2019 (COVID-19) epidemiological studies, and to distinguish between Long COVID and other multi-syndromic diseases that have overlapping symptoms. Although serum antibody levels can be measured to retrospectively diagnose SARS-CoV-2 infections, peptide stimulation of memory T cell responses is a more sensitive approach. This is because robust memory T cells are generated after SARS-CoV-2 infection and persist even after antibodies wane below detectability thresholds. In this study, we compare T cell responses using FluoroSpot-based methods and overnight stimulation of whole blood with SARS-CoV-2 peptides followed by an ELISA. Both approaches have comparable sensitivity and specificity but require different equipment and samples to be used. Furthermore, the elimination of peptides that cross-react with other coronaviruses increases the assay specificity but trades off some sensitivity. Finally, this approach can be used on archival, cryopreserved PBMCs. This work shows comparative advantages for several methods to measure SARS-CoV-2 T cell responses that could be utilized by any laboratory studying the effects of the coronavirus disease 2019 pandemic.

Baculovirus is one of the most complex viruses found in nature. Proteomic analysis of budded viruses (BVs) indicates that they are formed by at least 50 different structural proteins. The function of most of these structural proteins and their specific localization in individual virions remain unknown. In the present study, we have conducted single-molecule localization microscopy analysis of the spatial distribution of the nucleocapsid protein P24 and the envelope proteins GP64 and E25. Our results show that P24 and GP64 are polarized to one end of the baculovirus, while E25 distributes more homogenously along the viral envelope. This is the first study using optical microscopy to demonstrate the polarized distribution of structural proteins in individual baculoviruses.

Enteric hepatitis, represented by the hepatitis A virus (HAV) and hepatitis E virus (HEV), remains a significant global public health concern. While much progress has been made, many aspects of the biology and pathophysiology of HAV and HEV are still not fully understood. One of the major challenges is the absence of a reliable system for virus replication. Additionally, the lack of standardized and widely accessible diagnostic tests contributes to the underestimation of the true prevalence of these viruses. Factors such as climate change, environmental shifts, globalization and increased population mobility further complicate the spread of these infections by affecting pathogen transmission, water quality and the distribution of vectors. This review approaches the emergent research challenges and trends of enteric hepatitis and focuses on developing more efficient diagnostic tools, exploring the role of zoonotic transmission and addressing the impact of environmental and climate changes on disease dynamics, underscoring the need for collaborative, interdisciplinary efforts to effectively combat enteric hepatitis in a rapidly changing world.

A contaminated viral stock results in considerable loss of time, resources and financial means and is generally discovered only by chance after years of research. Thus, it is necessary to implement a technique that can detect contamination without prior knowledge or assumptions, such as next-generation sequencing (NGS). Here, we describe the discovery of a cross-contaminated viral stock from a biological repository of an African Zika virus isolate with Toscana virus after performing NGS on infected cells. In addition, we also describe the consequences that we faced using this contaminated stock. These included the economic and time loss to the lab that needed to repeat all previously performed experiments, the generation of biologically flawed results with a subsequent potential retraction and the severe risk of infection of lab members who manipulated the contaminated stock.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes encephalitis in humans and infects crocodiles, resulting in rashes and neurological signs. In Zambia, two distinct lineages of WNV have been detected in neighbouring areas: lineage 2 in mosquitoes and lineage 1a in farmed crocodiles. Considering the risk of direct or vector-mediated WNV transmission from crocodiles to mammals, it is necessary to elucidate the pathogenicity of WNV strains derived from crocodiles. In this study, WNV was successfully isolated from naturally infected farmed crocodiles (Croc110/2019/1/ZM, Croc110). We then investigated its proliferation and pathogenicity in mice in comparison with a WNV isolate from mosquitoes in Zambia (Zmq16) and two reference strains, including one highly pathogenic (NY99) and one low pathogenic (Eg101) strain. Although viral proliferation in Vero and mammalian neuronal cells was comparable among the strains, Croc110 exhibited low cell-to-cell transmission efficiency. , more than 70% of mice (C57BL/6) intracerebrally inoculated with Croc110 displayed neurological signs, and Croc110-infected mice exhibited similarly high mortality rates as NY99- and Zmq16-infected mice. Meanwhile, comparable virus growth was observed among the strains in the brain. However, the virulence of Croc110 was significantly lower than that of Zmq16 and NY99 following intradermal (ID) and intraperitoneal inoculation. Consistently, Croc110 displayed lower growth than Zmq16 and NY99 in the brain and peripheral tissues after ID inoculation. Our study revealed that the crocodile-derived WNV strain is less neuroinvasive in mice, and it exhibits distinct pathogenicity from the highly pathogenic mosquito-derived WNV strain circulating in Zambia.

White spot syndrome virus (WSSV) poses a significant threat to shrimp aquaculture, leading to substantial economic losses. This study aims to evaluate the virulence and evolution of recent WSSV outbreaks in Japan. Shrimp infected with WSSV were collected from Okinawa, Miyakojima and Miyazaki prefectures, yielding a total of seven isolates. Through injection and immersion tests, the lethal dose 50% endpoints were determined. Genomic analysis revealed isolates with sizes ranging from 288 to 299 kbp, sharing ~99% nucleotide identity with the reference genome (CN01: NC_003225.3). Variant analysis identified 1197 forms, primarily single-nucleotide polymorphisms, with Miyakojima isolates displaying the highest diversity. Frameshift mutations, notably in ORFs such as wsv006, wsv011, wsv091 and wsv403, were observed across all isolates. Phylogenetic analysis indicated clustering of Miyakojima isolates, suggesting similar outbreak intensities. Furthermore, isolates exhibited smaller genomic sizes compared with the reference genome, indicating ongoing WSSV evolution. Notably, a high frameshift mutation in wsv403, a viral E3 ubiquitin ligase, implies its potential role in the observed outbreaks, particularly in Miyakojima. This study addresses the research question regarding the virulence and evolutionary dynamics of WSSV outbreaks, proposing a hypothesis that genetic variations contribute to the severity and spread of WSSV in shrimp aquaculture.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that causes Kaposi's sarcoma, a cancer of endothelial origin. KSHV uses the activity of host molecular chaperones like Hsp70 and Hsp90 for the folding of host and viral proteins required for productive infection. Hsp70 and Hsp90 chaperones form proteostasis networks with several regulatory proteins known as co-chaperones. Of these, Hsp90-Hsp70-organizing protein (HOP) is an early-stage co-chaperone that regulates the transfer of folding substrate proteins between the Hsp70 and Hsp90 chaperone systems. While the roles for Hsp90 and Hsp70 in KSHV biology have been described, HOP has not previously been studied in this context despite its prominent interaction with both chaperones. Here, we demonstrate a novel function for HOP as a new host factor required for effective lytic replication of KSHV in primary effusion cell lines.