Journal of General Virology

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide, yet no effective antiviral therapeutics are currently available. Although environmentally induced capsid conformational changes associated with infectivity have been reported in murine noroviruses (MNVs), comparable conformational switching has not been demonstrated in HuNoVs. In this study, we generated HuNoV GII.3 virus-like particles (VLPs) using a baculovirus expression system and identified two distinct T = 3 particle populations coexisting within VLP preparations derived from a single strain through cryo-electron microscopy single-particle analysis. Comparative structural analysis revealed that these two T = 3 capsid conformations correspond to the resting and rising states of the protruding (P) domain. Rearrangement of the P domain alters intermolecular interactions between adjacent capsid subunits, resulting in distinct capsid surface architectures. In the resting state, intermolecular contacts were mediated predominantly by the P2 subdomain, with limited contribution from the P1 subdomain. In contrast, the rising state exhibited a shift toward an alternative interaction interface primarily involving the P1 subdomain. These findings demonstrate previously unrecognized structural polymorphism in the HuNoV capsid and provide evidence that conformational switching may occur in HuNoVs. Our results offer new insights into norovirus capsid dynamics and may inform future structure-based vaccine and antiviral drug development.

Capsid stability is vital for virion survival as the capsid must withstand varying environmental challenges such as pH and temperature to allow the virus to reach a target cell. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of importance to human health globally, with no approved vaccine or antiviral available. Despite this, the molecular mechanisms behind norovirus capsid stability and capsid rearrangement prior to RNA translocation are understudied. Using murine norovirus as a model, we utilised thermal stress to create a thermally stable virus population. By introducing three identified substitutions in the major capsid protein VP1 from this virus population into an infectious clone, we were able to create a heat and pH stable virus that had delayed viral uncoating during the infectious lifecycle. Cryo-EM reconstructions of the triple substitution virus demonstrated that enhanced inter-chain hydrogen bonding was vital for increased capsid stability. Finally, mutagenesis to remove the enhanced inter-chain hydrogen bonding reverted capsid stability back to wild-type levels. This work contributes to fundamental calicivirus biology by demonstrating areas of importance in capsid stability down to amino acid resolution. Furthermore, this work could inform vaccine design for a thermostable norovirus vaccine in the future.

RNA molecules form specific 3D structures that facilitate a variety of functions through interactions with other macromolecules. Many RNA viral genomes maintain these structures to interact with and evade host immunity machinery. One such element, the competitive inhibitor RNA (ciRNA), discovered in the protein coding region of the poliovirus serotype 1 (PV1) genome, inhibits a host antiviral protein, ribonuclease L (RNase L). Although some functionally essential structural motifs of the PV1 ciRNA have been studied, the extent of its evolutionary conservation and other structural requirements remained unexplored. Here we combined bioinformatic and biochemical techniques to further define the requirements of a functional ciRNA and assess its phylogenetic distribution. We systematically mutated ciRNA structural features, verifying that ciRNA inhibitory activity requires a conserved loop E motif and a long-range base-pairing interaction, but its peripheral stems are dispensable and in fact a circularly permuted version maintains function. A structure-based homology search identified potential ciRNAs across the Picornaviridae family, but only a subset of those tested were functional - all are in Enterovirus coxsackiepol. When structural features needed for function were transposed from PV1 ciRNA to an RNA unable to inhibit RNase L, the chimeric RNAs did not gain wild-type function, and chemical probing data revealed that these nonfunctional RNAs are unable to form the correct secondary structure. Overall, the dual constraints of encoding a protein and forming a specific functional structure appear to not only limit the sequence diversity, but also the phylogenetic distribution, of ciRNAs.

Rodents are an important reservoir of zoonotic viruses and are ubiquitously present in densely populated urban areas. Betacoronaviruses in the Embecovirus lineage are well known to infect both humans and animals and have established rodent reservoirs. Here three Betacoronavirus gravedinis genomes were sequenced and characterized in white footed mice (Peromyscus leucopus, commonly white footed mice) collected in New York City, the second most populous city in North America. The genomes were distinct from mouse hepatitis virus (MHV), the prototype mouse betacoronavirus, and highly similar and identical in one case to previously characterized B. gravedinis sequences from white footed mice in Connecticut. Codon aware evolutionary models were used to identify specific sites under positive selection within the spike protein of B. gravedinis. A novel method was developed to predict the probable geographic distribution of the virus using publicly available data from the Global Biodiversity Information Facility to generate a weighted distribution map highlighting overlapping potential host ranges based on the evolutionary distance using a high resolution cytocrome B (CYTB) phylogeny of rodent species with potentially overlapping ranges. Our models predict three current hotspots of circulation in North America under different possible transmission regimes, and an additional fourth hotspot was predicted to arise in a warming future. This study highlights the continued need for biodiversity-informed surveillance of potential zoonotic pathogens in rodents.

Hepatitis C virus (HCV) depends on host lipid metabolism and lipid droplets (LDs) for genome replication, assembly, and particle production, yet how LD structure and lipid utilization change over the course of infection remains incompletely understood. Here, we investigated the temporal remodeling of LD-associated metabolic pathways during HCV JFH-1 infection of human hepatoma Huh7 cells. HCV infection transiently increased LD fluorescence intensity at 24 hours post-infection (hpi), followed by normalization or relative loss of LD signal at later time points. Concomitantly, LDs became progressively clustered and enlargement during late infection, despite reduced protein levels of the canonical LD fusion proteins CIDEA, CIDEB, and CIDEC, suggesting that HCV-induced LD enlargement occurs through CIDE-independent mechanisms. Transcriptomic, RT-qPCR, and immunoblot analyses revealed time-dependent regulation of genes and proteins involved in LD structure, triglyceride synthesis, lipolysis, lipid uptake, and mitochondrial fatty acid utilization. Subcellular fractionation demonstrated preferential accumulation of fatty acids in mitochondrial fractions at 24-72 hpi. This redistribution was accompanied by increased oxygen consumption rate, elevated extracellular acidification, and progressive reactive oxygen species accumulation, indicating infection-associated metabolic activation and oxidative stress. Pharmacological inhibition of DGAT1-dependent LD biogenesis, LIPA-dependent lysosomal lipid hydrolysis, LIPE/HSL-dependent lipolysis, or CPT1-dependent mitochondrial fatty acid transport markedly reduced mitochondrial fatty acid accumulation and suppressed HCV-induced respiratory activity. Inhibition of LIPA or LIPE/HSL reduced both HCV RNA and core protein levels, whereas inhibition of CPT1 or DGAT1 had more pronounced effects on core protein than on viral RNA. Together, these findings support a model in which HCV dynamically remodels LDs, mobilizes LD-associated fatty acids, and redirects them toward mitochondria to support infection-associated metabolism and downstream stages of the viral life cycle. Lipid hydrolysis and mitochondrial fatty acid trafficking therefore represent potential host-directed targets for limiting HCV infection. SIGNIFIGANCEHepatitis C virus depends on host lipid metabolism for replication, assembly, and production of infectious particles, but how it uses lipid droplets over time remains incompletely understood. This study shows that hepatitis C virus dynamically remodels lipid droplets, causing an early increase in lipid storage followed by droplet enlargement and mobilization of fatty acids during later infection. The released fatty acids preferentially accumulate in mitochondria, where they are associated with increased cellular respiration and oxidative stress. Blocking lipid droplet formation, lipid breakdown, or fatty acid transport to mitochondria reduced this metabolic response and decreased viral RNA or core protein accumulation. Inhibition of lysosomal acid lipase and hormone-sensitive lipase suppressed both viral RNA and protein levels. These findings identify lipid droplet breakdown and mitochondrial fatty acid trafficking as important host processes used by hepatitis C virus and as potential targets for host-directed antiviral intervention.

BackgroundHerpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. MethodsThe samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. ResultsTwo samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787bp) and UL30 (2,846bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. ConclusionThis study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.

The conserved herpesvirus protein kinase (CHPK) is encoded by all members of the Orthoherpesviridae and contributes to replication in cell culture but is not strictly required. Mareks disease virus (MDV) CHPK is dispensable for replication in cultured cells yet essential for horizontal transmission in chickens. To elucidate its role during natural infection, we performed RNA sequencing (RNA-seq) and mass spectrometry (MS)-based phosphoproteomics on spleen and feather follicle epithelial skin cells from chickens infected with wild-type or CHPK-null MDV. RNA-seq detected only a limited number of viral transcripts in the spleen--including latency-associated transcripts (LATs) and the major oncogene Meq--with minimal differences between wild-type and CHPK-null infections. In feather follicle epithelial skin cells, the full repertoire of viral genes was expressed, but only seven genes showed differential expression between wild-type and CHPK-null viruses. In striking contrast, MS-based phosphoproteomics identified many differentially phosphorylated proteins, including 21 viral proteins. These findings indicate that CHPKs critical functions in skin replication and subsequent horizontal transmission are primarily mediated through post-translational modifications (PTMs) rather than transcriptional regulation. Among the CHPK-targeted viral proteins were three MDV-unique proteins, eight conserved within the Alphaherpesvirinae, and ten conserved across the Orthoherpesviridae. In silico analysis revealed that many differentially phosphorylated serine and threonine residues lie near or within predicted nuclear localization signals (NLS) and nuclear export signals (NES). Functional validation confirmed that several of these motifs actively control nucleocytoplasmic shuttling of the respective viral proteins. Collectively, these data suggest that MDV CHPK orchestrates the subcellular localization of multiple viral proteins in epithelial skin cells via phosphorylation, thereby enabling efficient replication and horizontal transmission in the natural host. AUTHOR SUMMARYUnderstanding the mechanisms by which herpesviruses replicate and spread within their natural hosts and identifying the viral genes essential for these processes are fundamental to developing effective antiviral strategies. Mareks disease virus (MDV), a highly contagious alphaherpesvirus, remains a major economic threat to the global poultry industry while serving as a powerful natural animal model for studying herpesvirus pathogenesis and transmission in vivo. Using an established in vivo enrichment method for infected cells, we conducted a comprehensive analysis of viral gene expression, protein abundance, and post-translational modifications (PTMs) during natural infection. Remarkably, RNA sequencing revealed virtually no differences in viral transcription between wild-type and CHPK-null viruses in either spleen or feather follicle epithelial skin cells. In contrast, phosphoproteomics showed that CHPK extensively regulates the phosphorylation of multiple viral proteins specifically in skin epithelial cells. In silico and functional analyses further indicate that these CHPK-mediated phosphorylations occur near or within nuclear localization (NLS) and nuclear export (NES) signals, directly controlling the nucleocytoplasmic shuttling of key viral proteins. This work suggests CHPK as a master regulator of viral protein subcellular localization during replication in the natural host and highlights CHPK orthologs as promising broad-spectrum therapeutic targets against herpesviruses.

Virus-like particles (VLPs) are an abundant component of microbiomes with critical ecological roles such as population control through viral predation and horizontal gene transfer. Studying the collection of viruses in microbiomes (the virome) through metagenomics has provided important insights into the composition and functions of VLPs in different environments. However, the current gold-standard method for VLP purification, CsCl density gradient ultracentrifugation (CsCl), is low throughput, time consuming and suffers from biases which limits the ability to study viromes in larger sample sets and can interfere with data interpretation. Here we present an anion exchange (AEX) chromatography-based approach for the purification of VLPs from microbiome samples that allows for significant increases in throughput and reproducibility while achieving VLP purity levels similar to or higher than CsCl. We used microbiome samples of known composition to first establish and evaluate the AEX approaches and compare them to CsCl. We implemented the AEX approach both for fast performance liquid chromatography (FPLC) and in multi-well plates. We compared the VLPs purified with CsCl and AEX using shotgun metagenomic sequencing and found that AEX performs similarly to or better than CsCl for purification of VLPs. AEX purified VLP-fractions captured significantly more viral DNA compared to CsCl. We also found that both AEX and CsCl were capable of capturing viruses present at extremely low relative abundances (<0.001%). Additionally, we found that DNase digestion and CsCl may bias against filamentous phage morphologies. Finally, we purified VLPs from conventional murine feces using AEX and CsCl. AEX purified murine fecal VLPs had a much higher viral DNA content (85%) than CsCl (41%). While there were some differences in viral contigs assembled from AEX and CsCl VLP metagenomes, these method unique viral contigs made up only small proportions (<8%) of the relative abundance in the VLP metagenomes. AEX, particularly in the multi-well format, enables the ultrapurification of VLPs from tens to hundreds of samples in a single day thus facilitating virome studies with the large sample numbers needed for translational and clinical research.

Background: Antimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. Methods: We evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. Results: DNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. Conclusions: DNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations.

High Pathogenicity Avian Influenza (HPAI) H5N1 clade 2.3.4.4b has spread beyond birds to affect seals across the Southern Ocean and sub-Antarctic region, with southern elephant seals (Mirounga leonina) particularly devastated. The virus, likely introduced via spillover from infected migratory birds, has killed tens of thousands of adult seals and pups throughout most of their range, though Macquarie Island remains unaffected so far. We used twenty years of elephant seal movement data from the southern Indian and Pacific oceans to assess whether seal-to-seal transmission could spread HPAI H5N1 between breeding colonies, despite the vast distances separating them (Marion Island, Iles Crozet, Iles Kerguelen, and Macquarie Island). There was substantial overlap in seals' at-sea distributions during their winter post-moult trips, when seals travel for weeks at average speeds of 3.5 km/h. Two transmission pathways were examined: (1) terrestrial "stepping stone" routes, where infected seals could pass the virus between colonies during short intervals to remain infectious were feasible from Marion Island to Kerguelen but not from Kerguelen to Macquarie Island; and (2) at-sea encounters between seals, which occurred frequently enough to enable transmission. The findings suggest that once established at Macquarie Island, the virus could potentially spread further to New Zealand's sub-Antarctic islands and mainland New Zealand. While seal-to-seal transmission appears possible, we conclude this is unlikely. Nonetheless, understanding at-sea contact rates enhances knowledge of H5N1 epidemiology and demonstrates the value of combining long-term population monitoring with movement data to understand wildlife disease dynamics.

Orthoflaviviruses undergo significant structural changes through maturation and infection, yet the molecular mechanisms remain incompletely understood. Using St. Louis encephalitis virus (SLEV) as a model, a reemerging mosquito-borne orthoflavivirus endemic in the Americas, we elucidated the structures of immature and mature SLEV particles at resolutions of 4.4 [A] and 3.3 [A], respectively, using cryo-EM. SLEV is characterized by glycosylated E and prM proteins, the presence of lipid pockets, and is stabilized by an intricate network of inter- and intra-protein interactions between E and (pr)M across maturation stages. Several interactions were mediated by histidines that play different roles depending on SLEV maturation and pH. Non-lethal single mutations of H285R and H443R in E protein affect SLEV replication in mammalian and mosquito cell lines, and delay death in mouse models of infection. These histidine residues are conserved across orthoflaviviruses, illustrating the complexity of orthoflavivirus particles and indicating a possible strategy for attenuation.

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

Bovine ephemeral fever virus (BEFV), a member of the Rhabdoviridae family, is an arthropod-borne pathogen that causes acute febrile disease in cattle. The structural basis of its genome encapsidation and virion assembly remains unexplored, with the current knowledge largely limited to predictions derived from bioinformatic comparisons with other rhabdoviruses. Furthermore, the structural principles that permit the formation of variable-diameter nucleocapsids resulting in the distinctive bullet-shaped morphology of rhabdoviruses remain poorly understood. Here, we report the cryo-electron microscopy structure of the BEFV nucleoprotein (N) in complex with RNA, in the absence of other viral components. The complex predominantly forms circular decameric oligomers that we propose to act as nucleation intermediates during assembly of the bullet-shaped nucleocapsids. Direct subunit interactions are limited to a small polar surface area, with additional intersubunit links mediated by flexible N- and C-terminal loops. These interfaces generate a structurally plastic oligomeric lattice in which neighbouring N subunits can undergo substantial rigid-body rotations and positional rearrangements while preserving conserved local contacts and continuous RNA encapsidation. Such quasi-equivalent interactions provide a plausible mechanism for accommodating the progressive changes in helical diameter required for the transition from the highly curved bullet tip to the wider cylindrical trunk of rhabdovirus nucleocapsids. The assembly is stabilised by the bound RNA molecule, where nine RNA bases are accommodated by each N subunit. The RNA-binding mechanism is consistent with that of VSV, the closest BEFV homologue characterised structurally, but differs at about half of the RNA-binding residues, demonstrating the versatility of the nucleoprotein scaffold in interacting with ssRNA. Comparative analysis with other rhabdoviruses, as well as negative-sense RNA viruses with constant-diameter nucleocapsids, such as Ebola, further confirms the structural features that enable bullet-shaped versus cylindrical nucleocapsid assembly.

Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737532v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14a54a6org.highwire.dtl.DTLVardef@fb6621org.highwire.dtl.DTLVardef@ee2e2aorg.highwire.dtl.DTLVardef@118f91d_HPS_FORMAT_FIGEXP M_FIG Created with biorender.com and PyMOL Molecular Graphics System, version 2.5.0. Schrodinger, LLC. C_FIG

Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus with an unusually broad host and vector range. Despite increasing mosquito-to-human spillover, the viral determinants governing host adaptation remain poorly defined. We examined changes in JCV replication during serial passage in mosquito cells and sought to link adaptive changes in viral fitness to specific genetic mutations. In mosquito-derived C6/36 cells, JCV exhibited a distinct lag-burst phenotype in which viral replication remained nearly undetectable for 10 days before abruptly increasing. Strikingly, following reinfection of fresh C6/36 cells, JCV that had been passaged once in mosquito cells exhibited immediate, robust replication with no detectable lag phase. Sequencing before and after passage identified multiple M segment mutations associated with enhanced replication. Using a plasmid-based reverse genetics system, individual mutations were introduced into recombinant JCV and evaluated for their effects on replication in mosquito cells. All tested mutations independently enhanced replication efficiency, demonstrating that adaptation can arise through multiple independent genetic pathways. However, no individual mutation fully reproduced the phenotype acquired naturally through mosquito cell passage. Together, these findings demonstrate that JCV rapidly adapts to mosquito cells under minimal selective pressure and highlight the potential for emergence of increasingly well-adapted viral variants.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

Background: The gut environment is hostile to life, yet the human virome, dominated by bacteriophages, persists. Adaptations to the major capsid protein (MCP) may explain this. Phage MCPs conserve the HK97 fold, ideal for detecting convergent features across phage populations. Prior capsid stability research focused on individual phages, limiting broader pattern identification. Methods: MCPs from the Gut Phage Database (GPD) (n=8,478) and INPHARED (n=4,905) were predicted using ProtPhage + Phold and clustered using MMseqs2 (GPD=902 vs INPHARED=606). Structural predictions, conservation analysis, and capsomere modeling were used to characterize cysteine environments. Results: Biochemical analysis identified cysteine enrichment in GPD MCPs. Phylogenetic mapping was consistent with convergent evolution of high-cysteine MCPs. Over 50% of cysteines were [&ge;]90% conserved within and between clusters. Simulated capsomeres showed 83% of cysteines are buried (RSA <10%). Conclusions: These findings suggest gut phages may have convergently evolved cysteine-based capsid stabilization, with implications for engineering therapeutic phages.

Lone star virus (LSV) is a bandavirus first isolated from Amblyomma americanum ticks in the United States (U.S.) and is phylogenetically related to severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV), and Bhanja virus, each of which has been associated with severe human disease. In contrast to these better-characterized bandaviruses, LSV remains poorly studied, and its pathogenic potential is not well defined. Recent detection of LSV RNA in cerebrospinal fluid from an immunocompromised patient in Idaho, U.S., with fatal meningoencephalitis further highlights the need for experimental systems to investigate LSV biology. Here, we rescued recombinant (r) LSV from cloned cDNA and used it to characterize LSV. rLSV replicated similarly to the parental isolate in mammalian cells and caused rapid, systemic, and lethal disease in IFNAR-/- mice, with widespread detection of viral (v) RNA across multiple tissues, hepatic and splenic pathology, and induction of inflammatory cytokines. In contrast, C57BL/6J mice controlled infection and exhibited no clinical disease. To place LSV within a broader comparative framework, we generated rSFTSV from cloned cDNA and compared rLSV, rSFTSV, and HRTV in cell culture and IFNAR-/- mice. Our studies revealed distinct disease kinetics among these related tick-borne bandaviruses and showed that HRTV-induced immunity protected against homologous HRTV rechallenge and heterologous rSFTSV challenge, but not rLSV challenge. Together, these findings establish reverse-genetics platforms and small-animal models for comparative bandavirus studies, define key features of LSV pathogenesis, and place this neglected virus within a framework of related bandaviruses that differ in virulence and immunological overlap.

Climate-driven increases in sea surface temperature have been associated with the expansion of Vibrio species and a corresponding rise in vibriosis cases in both human populations and aquaculture systems. Coastal waters across the south of England are increasingly becoming suitable for the growth and establishment of both human- and aquaculture-associated Vibrio species, potentially increasing vulnerability to the types of infections and disease outbreaks already reported elsewhere in the world. In this study, we report the presence of a diverse and well-established Vibrio community within the Teign Estuary, (Southwest, UK), including the human-pathogenic species V. parahaemolyticus, V. cholerae (non-O1/non-O139), V. alginolyticus, and V. diabolicus, as well as the important aquaculture pathogens V. jasicida, V. aestuarianus, and V. anguillarum. We identified V. diabolicus, a species that was indistinguishable from V. alginolyticus using conventional biochemical identification methods and could only be accurately resolved by whole-genome sequencing and developed novel PCR targets to differentiate these species in the lab. Using the insect infection model Galleria mellonella, we demonstrate that environmental isolates of V. cholerae (non-O1/non-O139), V. parahaemolyticus, and V. alginolyticus possess virulence potential. We also investigated the effects of sewage effluent on the growth of Vibrio isolates from the Teign Estuary and found that sewage can preferentially promote the growth of Vibrio species. Furthermore, several Vibrio isolates were multidrug resistant and carried antimicrobial resistance genes, highlighting the potential role of environmental Vibrio populations in the Teign Estuary as reservoirs of antimicrobial resistance. Together, these findings demonstrate how rising sea surface temperatures and sewage pollution may influence the emergence, persistence, and public health and aquaculture significance of Vibrio species in UK coastal waters.