Journal of General Virology

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5-3 exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3-untranslated region (3-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3-stem loop (3SL). Despite the importance of the 3-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses (MbFV) including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus (ZIKV). We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin (sHP) directly upstream of the 3-SL, and 3-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient. ImportanceFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3-UTR RSEs that promote its stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

Avian influenza virus (AIV) epidemiology is well-documented in temperate regions but remains poorly understood in isolated ecosystems like tropical oceanic islands. On these islands, seabirds nest in dense interspecific colonies where the role of different species as reservoirs and dispersers of AIV may vary greatly. Here, we examine the role of noddies (Anous spp.) as potential reservoirs for low pathogenic AIV and evaluate their potential as sentinel species for highly pathogenic AIV introduction on tropical oceanic islands. We analyzed blood samples from 11 seabird species across eight islands in the southwestern Indian Ocean (2015-2020). Noddies exhibited high, stable seroprevalence (30-45%), comparable to reservoir host species in temperate regions. The detection of two N7-positive noddies, sampled the same year on two distinct islands, provided direct molecular evidence that AIV actively circulates on these island colonies. While most other species showed low exposure, Bridled Terns (Onychoprion anaethetus) had exceptionally high seroprevalence (80%), though their reservoir status requires further investigation due to limited sampling. Given noddies consistent exposure and regional distribution, we recommend prioritizing islands with large noddy populations for AIV surveillance. Continued investigation of viral dynamics within and among islands is now called for to elucidate the ecological drivers of AIV maintenance and transmission.

Okra (bhendi) is a widely cultivated food crop in warm regions of the world, with India contributing approximately 60% of global production. However, okra cultivation in India is severely constrained by viral diseases, among which infections caused by the geminiviruses bhendi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV), in association with their satellites, represent major limitations to crop productivity. In recent years, the geminivirus-encoded C4 protein has emerged as a key pathogenicity determinant in this viral family, with functions that include suppression of multiple layers of plant antiviral defence and induction of disease symptoms. Here, we comparatively characterize the C4 proteins of BYVMV and OELCuV by determining their targeting signals and subcellular localization, and by assessing their ability to induce developmental abnormalities and suppress the cell-to-cell spread of RNA silencing. Our results reveal that the two C4 proteins display distinct subcellular localization patterns, yet both are capable of inducing developmental alterations, likely through different mechanisms, and of suppressing the intercellular spread of RNA silencing, possibly via interaction with a common host factor. Together, these findings suggest that C4 might be a critical virulence factor in okra-infecting geminiviruses and act as a symptom determinant. The C4 proteins encoded by BYVMV and OELCuV therefore emerge as promising targets for the development of antiviral management strategies in okra.

Background and AimsHepatitis C virus (HCV) infections were previously treated with interferon (IFN) but today direct acting antivirals (DAAs) with cure rates >95% are available. DAA treatment failure is primarily attributed to resistance associated mutations (RAMs), often imposing a fitness cost. Interferon treatment outcome was shown to be associated with the interferon sensitivity determining region (ISDR), which is part of the replication enhancing domain (ReED) in non-structural protein (NS) 5A. We found that accumulation of mutations in the ReED was indicative of elevated viral genome replication fitness. This study investigates the impact of HCV replication fitness on antiviral treatment outcomes. MethodsWe utilized chimeric HCV subgenomic replicons containing RAMs and ReED sequences from patients after interferon treatment or DAA failure to assess replication fitness of patient isolates in presence and absence of inhibitors. ResultsReplication fitness did not impact on IFN sensitivity in cell culture but resulted in higher remaining antigen levels for highly replicating variants at a given IFN concentration. Furthermore, we identified ReED variants substantially increasing HCV replication in several patients who failed DAA therapy across different genotypes. High replicator ReEDs rescued the fitness loss caused by RAMs like Y93C/H (NS5A) and S282T (NS5B). While high replication fitness did not intrinsically increase drug sensitivity (IC50), it allowed the virus to sustain robust replication despite antiviral pressure. ConclusionsElevated replication fitness might support interferon treatment due to increased antigen presentation, facilitating adaptive immune responses. Furthermore, ReED mediated increase in replication fitness could contribute to DAA treatment failure by preserving higher replication upon treatment and compensating for RAM associated fitness costs. Thus, patients failing DAA treatment should be monitored for RAMs and ReED mutations.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants (Elephas maximus) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 x 106 viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at -80 {degrees}C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398) and the previously reported EEHV1B sequence (KC462164), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

In the present study, we assessed the pathogenicity of H5N8 avian influenza viruses belongs to the clade 2.3.4.4b in chicken. Birds of three different dose groups, 102, 104, and 106 EID50 were used in the study. No mortality was observed in 102 EID0 group. Percent cumulative mortality of 104 and 106 EID50 group was 66.67 and 100 %, respectively. Varying duration of MDT of 3.2 and 2 days was observed in 104 and 106 EID50 group, respectively. The CID50 of virus was found to be 104.5 EID50. High no. of viral RNA copies were found both in oropharyngeal and cloacal swabs and in various organs of birds infected in 104 and 106 EID50 group. Significant gross and histological changes and presence of viral antigen in various organs were observed in 104 and 106 EID50 group. So, the study concludes that Indian HPAI, H5N8 isolates are highly pathogenic in nature to chicken by affecting most organs systemically. CID50 of this H5N8 virus indicates poor adaption in chicken and it implies poor transmission possibility of this virus for host species in field condition. Though this virus is highly pathogenic in nature as that of HPAI, H5N1 viruses, absence of endothelial staining in most organ attributes variation in replication process and pathogenesis from HPAI, H5N1 viruses. Hence, further studies need to be done to elucidate the pathobiology of this virus in various bird species. HighlightsO_LIH5N8 virus belong to the clade 2.3.4.4b, Indian isolate is highly pathogenic in nature as that of HPAIV, H5N1. C_LIO_LIThe dose inocula, 102 EID50 is noninfectious to chicken. C_LIO_LIThe dose inocula, 104 and 106 EID50 had caused significant mortality in the inoculated chicken with MDT of 2 and 3.2 days, respectively. C_LIO_LIH5N8 virus was detected with high viral titres in clocal and oral shedding and in multiple organ with the dose inocula, 104 and 106 EID50. C_LIO_LI104 and 106 EID50 of H5N8 inocula virus caused significant gross and histological changes in multiple organs and viral antigens were detected in respective organs. C_LI

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle in the United States. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication facilitating high-throughput antiviral and serological screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural (NS) gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro, presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titers in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Collectively, these results establish the TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis, and serological screens and potentially antiviral screens.

RNA viruses are common in tephritid fruit flies including the Queensland fruit fly, Australias most significant horticultural pest. For many their transmission, tissue tropism and load across host development remain unexplored. Yet these factors are important for host biology, ecology and pest management. We investigated Bactrocera tryoni orbivirus (OV), Bactrocera tryoni xinmovirus (XV), Bactrocera tryoni toti-like virus (TLV) and Bactrocera tryoni iflavirus species 2 (IVsp.2) that commonly coinfect B. tryoni laboratory populations. OV and XV transmission was vertical within and on eggs, while TLV transmission was vertical within eggs. IVsp.2 was not detected in eggs but was present in adults; however, IVsp.2 was horizontally transmitted, with viral load increasing with cohabitation time with infected flies. Horizontal transmission was not observed for the other viruses. OV had a similar load across all tissues, while XV was consistently more abundant in ovaries. TLV had a high viral load in the brain whereas IVsp.2 was abundant in the thorax, foregut and midgut. Besides differences in eggs, the viruses were detected in all other developmental stages, but viral load patterns differed: viral load remained constant for TLV, fluctuated for OV and XV, and was low in pre-adult stages and high in adults for IVsp.2. Our findings demonstrate distinct transmission strategies and tissue tropism among the viruses, providing new insights into their epidemiology and role in host biology. Furthermore, contrary to prevailing views that viruses are generally horizontally transmitted, most known RNA viruses of B. tryoni are vertically transmitted affecting the evolution of host-virus interactions.

Background & Aims: People with HIV (PWH) who achieve hepatitis C virus (HCV) cure may retain persistent metabolic alterations, particularly those with advanced fibrosis or cirrhosis. This study aimed to characterize plasma metabolomic and lipidomic profiles associated with cirrhosis in PWH at one and five years post-HCV therapy. Methods: Two cross-sectional studies evaluated PWH one (n=48) and five (n=30) years post-HCV therapy. Cirrhosis was defined as a liver stiffness measurement (LSM)[≥]12.5 kPa. Metabolomics and lipidomics were performed using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS), respectively. Data were analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA) and generalized linear models (GLM), adjusting for relevant covariates. Results: At one and five years, 32 (66.7%) and 10 (33.3%) participants, respectively, had cirrhosis. OPLS-DA identified 235 and 229 metabolites with variable importance in projection (VIP)scores >1. At one year, cirrhosis was associated with elevated levels of glycerophospholipids, sphingomyelins, and amino acids, and lower levels of triglycerides. At five years, cirrhotic PWH exhibited higher levels of glycerophospholipids and acyl-carnitines, together with lower levels of triglycerides and amino acids. Conclusions: PWH with cirrhosis post-HCV cure exhibits a persistently altered metabolic profile stable for five years, suggesting ongoing liver disease progression. These findings underscore the need for continued long-term monitoring of this population.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

Pneumonia virus of mice (PVM), the mouse homolog to respiratory syncytial virus (RSV), is increasingly used as surrogate model to study pneumovirus pathogenesis in a more natural pathogen-host relation. Two major strains of PVM, strain 15 and J3666 are currently used in laboratories, with preferences for either one or the other based on the well-documented isolation history of strain 15 or the suggested higher virulence of strain J3666. Using conventional and long read sequencing, we found that the PVM strain J3666 represents two distinct virus populations, which are defined by sequence and structure of the G and SH genes encoding the putative attachment and small hydrophobic proteins, in addition to further nucleotide polymorphisms. Specifically, a nucleotide polymorphism at position 65 in the G gene results in either an upstream open reading frame (uORF) preceding the main ORF in frame, or an extension of the major G ORF by 18 codons. The impact of the different forms of the J3666-G genes on PVM was examined by generating recombinant PVMs differing exclusively in the distinctive 5 portion of the respective G gene. This revealed that the population expressing a G protein with an extended main G ORF was more virulent, whereas the presence of a uORF attenuated virulence. The virulence of PVM correlated with increased expression levels of G, whereas attenuation was rather associated with downregulated expression of G due to the presence of a uORF. Thus, modulation of G protein levels may be an important mechanism by which pneumoviruses modulate virulence. ImportanceThe pneumonia virus of mice strain J3666 is considered a more virulent and more suitable model for severe lower respiratory tract infections. The organization of the gene for the attachment protein G is reported to contain a small upstream open reading frame (uORF) preceding the main G ORF in frame. The translated G protein is predicted to comprise 396 amino acids. We report that this virus strain may be a mixture of two different populations, each with differing virulence. The more virulent population encodes a G protein of potentially 414 amino acids instead of a small uORF. This G gene organization is associated with an increased G protein expression. Importantly, this organization of the G gene is in line with that of several newly identified pneumoviruses, i.e., canine and swine pneumoviruses. These viruses may comprise a distinct group within the Pneumoviridae family.

Efficient recovery of negative-stranded RNA viruses from plasmid cDNA requires robust bacteriophage T7 RNA polymerase expression, commonly supplied by Modified Vaccinia Ankara expressing T7 polymerase (MVA T7). Here, we describe the generation and characterization of two fluorescent MVA T7 viruses expressing either enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1). Both fluorescent-expressing MVA T7 viruses retain plaque morphology, growth kinetics, and transgene expression comparable to parental MVA T7. Importantly, both fluorescent-expressing MVA T7 viruses support rescue of replication-competent recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. Fluorescent protein expression from MVA T7 viruses facilitates stock generation and titration and allows detection of remaining helper virus during rVSV recovery and amplification. Altogehter, EGFP- and mRFP1-expressing MVA T7 viruses provide a practical tool for monitoring T7-driven rescue of recombinant RNA viruses and identifying helper virus carryover. IMPORTANCEReverse genetics systems for negative-stranded RNA viruses rely on robust bacteriophage T7 RNA polymerase expression, commonly provided by a Modified Vaccinia Ankara virus expressing T7 polymerase (MVA T7). However, generation of virus stocks, monitoring MVA T7 helper virus infection and detecting residual virus during rescue of negative-stranded RNA viral can be challenging. We generated fluorescent-expressing MVA T7 variants that enable direct visualization of helper virus infection without compromising viral growth kinetics, transgene expression, or efficient rescue of recombinant Vesicular Stomatitis Virus (rVSV) while enabling direct visualization of MVA T7 infection. MVA T7 viruses expressing fluorescent proteins simplify stock generation and titration, facilitate optimization of negative-stranded RNA rescue conditions, and allow rapid identification of helper virus carryover during generation and amplification of recombinant virus, improving the efficiency, reproducibility, and quality control in reverse genetics systems relying on T7 expression widely used across virology.

BackgroundOropouche virus is an emerging arbovirus increasingly associated with neurological complications, but its human cellular tropism and potential routes to the central nervous system remain poorly defined. This study aimed to characterize infection across clinically relevant human cell types and to investigate interactions with a human blood-brain barrier model and human neuronal/glial cells. MethodsA panel of human cell lines and primary human cells relevant to systemic and neurological disease was infected with Oropouche virus. Viral replication and production of infectious particles were quantified using molecular assays and infectivity titrations, and viral protein expression was assessed by immunoblotting and immunofluorescence. Barrier crossing was evaluated using a Transwell brain endothelial model with permeability monitoring, and infection dynamics in neuronal/glial cultures derived from human neural progenitors were quantified by imaging-based analyses. Group comparisons used non-parametric tests with Dunn-Bonferroni correction and Mann-Whitney tests; neuronal/glial cell counts were analysed using linear models with Fisher tests for interaction terms and multiplicity-adjusted post hoc comparisons. ResultsOropouche virus productively infected hepatocyte-like and intestinal epithelial cells, with high viral RNA output and release of infectious progeny. Primary synoviocytes, chondrocytes and skeletal muscle cells were permissive but produced lower infectious titers. Brain endothelial cells were inoculated and virus was progressively detected in the basolateral compartment, while endothelial permeability remained unchanged, indicating barrier crossing without disruption. In neuronal/glial cultures, both neurons and astrocytes were susceptible; infection was associated with marked cytopathic changes and a preferential, accelerated decline in neuron abundance over time. ConclusionsThese findings demonstrate broad human cell tropism and support blood-brain barrier crossing without major loss of barrier integrity, alongside pronounced neuronal vulnerability. The described models provide a platform to dissect mechanisms of neuroinvasion and to evaluate targeted antiviral strategies.

Dengue infections are considered an increasing threat to mankind due to their rapid global spread rate. The development of a widely accepted drug/vaccine is hindered due to an incomplete understanding of the virus lifecycle. Present data suggest that a cytoskeleton protein, called MYH9 binds to the 3UTR, at A4 region, a highly conserved part of the UTR across the serotypes. The levels of this protein were found to be elevated in the cells infected with the virus and the above increase is commensurate with the virus load. This protein is found to accumulate at the endoplasmic reticulum (site of virus replication) and interacts with dsRNA (a replicative intermediate), suggesting its involvement in replication. Inhibition of this proteins expression by its siRNA reduced viral load, supporting its role in viral replication. Immunofluorescence studies indicate that this protein accumulates at the cell periphery and pulldown studies suggest that this protein interacts with the viral envelope protein, suggesting a role in the dengue viruss cellular entry, possibly by acting as a receptor. Use of an anti-MYH9 drug, ML-7 indicated the reduction of the virus load, prevented the accumulation at the periphery and aided in regaining the cell morphology of virus infected cells, confirming its role in replication and entry. Collectively, these studies demonstrate a dual function of MHY9 in the virus life cycle, which may serve as a general paradigm for the other viruses and hence to develop specific drugs.

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

In this study the phenotypic consequences of naturally occurring single nucleotide polymorphisms (SNPs) in the MERS-CoV Spike protein were investigated. The impact of Spike mutations on virus entry and neutralisation of contemporary MERS-CoV strains is not currently well understood. Naturally occurring mutations were identified by aligning 584 MERS-CoV Spike sequences from either human clinical isolates collected between 2012 - 2024 or from viruses passaged in human cells. Fifteen SNPs of interest occurring in the NTD, RBD and adjacent to the S1/S2 cleavage site were selected for further characterisation based on their location in the Spike protein, frequency and identification in previous studies. A representative clade B, lineage 5 wildtype Spike sequence, which reflected those carried by MERS-CoV viruses circulating in the Middle East, was used in this study. The mutations of interest were introduced to the wildtype backbone to generate Spike variants. A lentiviral-based pseudotyping system was then used to investigate the impact of these Spike mutations on entry and neutralisation. I529T, E536K and L745F were shown to improve MERS-CoV entry. L411F, T424I, L506F, L745F and T746K were found to increase resistance to neutralisation by pooled patient sera. This study has identified novel naturally occurring Spike mutations that resulted in phenotypic differences in virus entry and neutralisation of contemporary MERS-CoV strains. Continued investigation of the phenotypic consequences of naturally occurring MERS-CoV Spike mutations is essential for assessing the risk to public health, especially given the pandemic potential of this virus. ImportanceThe main aim of this study was to investigate the impact of naturally occurring MERS-CoV Spike mutations on virus entry and neutralisation. The phenotypic consequences of mutations occurring in the Spike protein of contemporary MERS-CoV strains are not currently well understood. Improving our understanding is of particular importance due to MERS-CoV continuing to pose a public health risk, with frequent spillover events and mounting evidence of human-to-human transmission since the virus emerged in 2012. A major concern is that as MERS-CoV continues to evolve, it may become more infectious, resulting in increased transmission between humans. To add to this, surveillance is limited and there are currently no specific medical countermeasures available to treat MERS-CoV disease. The MERS-CoV Spike pseudotyping system developed in this study is a useful tool that could be used alongside surveillance systems to rapidly assess novel Spike mutations in functional assays. This MERS-CoV pseudotyping system could also be used to aid the development of medical countermeasures such as vaccines, antivirals and antibody therapies.

Human intestinal enteroids (HIE) and zebrafish larvae (ZF) models support the replication of certain strains of human norovirus (HuNoV), the leading cause of viral gastroenteritis worldwide. The replication of 17 HuNoV (9 genotypes) positive stool specimens from patients ranging from 5 months to 83 years old was evaluated in both HIE and ZF models. The yolk of 3-day post-fertilization ZF larvae were microinjected with 3 nL 10% clarified stool suspension. Each day post-infection (dpi), 10 larvae were pooled as one sample, and two specimens were collected daily until 5 dpi. Viral RNA was extracted from harvested larvae and quantified using reverse transcription (RT) droplet digital PCR. For the HIE model, J4FUT2 K1 enteroid monolayers were inoculated with 100-fold dilution of each stool specimen. Viral RNA levels were quantified at 1 and 72 h post-infection using RT real-time PCR. All genogroup (G) I specimens (n=5) replicated in both models showing a 1 to 2.5 log increase in HuNoV RNA. Of the 12 GII specimens tested, 8 replicated in the ZF model and 7 replicated in the HIE model. Specimens that failed to replicate in each model were not the same, although most of them were GII.4 Sydney [P16]. One additional strain, GII.17[P17], replicated only in the ZF model. Among the GII strains, viral RNA increase ranged from 1.6 to 3.3 log in the ZF and 0.4 to 3.68 log in the HIE model. These data highlight variability in HuNoV replication efficiency across models, potentially indicating the influence of model-specific factors on replication. ImportanceHuman intestinal enteroid and ZF models have been independently shown to support replication of different strains of HuNoV, the leading cause of viral gastroenteritis worldwide. However, whether the same HuNoV positive stool specimens replicate similarly in both systems has not been examined. In this study, stool specimens positive for a range of HuNoV genotypes were tested in both models. While specimens from the GI genogroup consistently replicated in both systems, specimens from the GII genogroup showed variable replication, with some replicating in only one model and a couple of specimens not replicating in either model. These findings demonstrate that model specific biological factors influence HuNoV replication and highlight the significance of using complementary model systems to better understand HuNoV pathogenesis.

Usutu virus (USUV) is a mosquito-borne flavivirus that has recently expanded northwards in Europe and become endemic in the UK [1-3]. USUV emergence often precedes the closely related West Nile virus (WNV), potentially reflecting differences in epidemiological parameters [4, 5]. One key parameter is the extrinsic incubation period (EIP), the time required for a mosquito to become infectious following an infected blood meal. Here we present the first ever estimate of the temperature-dependent EIP for USUV in the vector Culex pipiens molestus. We were able to quantify the shortening of the EIP with temperature by re-analysing published laboratory data with bespoke Bayesian model that accounted for key features of the experimental design. Under typical UK summer temperatures, the median EIP (EIP50) of USUV is shorter than that of WNV, and the potential transmission season of USUV is both longer and geographically more extensive. Under RCP8.5 climate projections, WNV transmission suitability is expected to match or exceed current USUV levels between 2055 and 2065, highlighting the future threat to the UK from emerging mosquito-borne pathogens. Our findings support USUV as a precursor for WNV in northern Europe and provide a robust characterisation of a key epidemiological parameter of USUV, enabling accurate modelling of its transmission dynamics.

There are no approved medical countermeasures for combatting Nipah virus (NiV) which causes regular outbreaks in humans and animals in South and Southeast Asia with mortality rates in humans ranging from 40% to more than 90%. Recently, it was shown that 4-fluorouridine (4-FlU; EIDD-2749), an orally available ribonucleoside analog, protected guinea pigs and nonhuman primates from lethal challenge with Lassa virus and that 4-FlU has in vitro antiviral activity against NiV. Here, we assessed the postexposure protective efficacy of 4-FlU in a lethal hamster model of NiV infection. Daily treatment with 4-FlU beginning 3 days after exposure to NiV resulted in complete protection from lethal infection. Our findings support the further development of 4-FlU as a therapy for NiV disease.

Small ruminants (sheep and goats) are one of the few mammals in which an exogenous retrovirus (XRV) and closely related endogenous retroviral elements (ERV) coexist within the same host genome. The betaretroviruses Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause pulmonary and nasal adenocarcinomas, respectively, and share extensive sequence similarity with their endogenous counterparts. Consequently, molecular surveillance must rely on assays that can unequivocally distinguish true exogenous infection from ERV-derived templates; failure to do so compromises diagnosis, phylogenetic inference, and epidemiological conclusions. We retrieved all complete JSRV, ENTV-1/2, and related ERV genomes deposited in public repositories and performed a comprehensive alignment. Only a limited number of genomic segments were capable of distinguishing exogenous from endogenous sequences. We refer to these as discriminating regions (DRs). Phylogenies built using DRs revealed that several entries annotated as XRV are, in fact, ERV-derived or chimeric artefacts generated by short-amplicon reconstruction. A systematic literature review of over 100 articles identified 286 distinct primers and probes used for the XRV amplification. In-silico mapping of each oligonucleotide onto the full alignment showed that only 28 % reliably differentiate XRV from ERV. We experimentally validated the predictive power of this approach for 17 primer/probe sets, confirming that non-discriminating assays produce false-positive signals from endogenous templates. The misannotation of ERV sequences as exogenous viruses has resulting in the population of databases with dubious entries, fostering erroneous hypotheses such as vector-borne transmission of JSRV and ENTV. To address this issue, we propose a concise set of criteria for assay design, validation, and database annotation emphasizing DR targeting, specificity testing against endogenous templates, and transparent reporting. Although this framework was developed for small ruminants, it is readily applicable to any host-virus system in which exogenous viruses coexist with endogenous viral elements. This will strengthen viral surveillance, phylogenetics, and the One Health initiatives.