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.

BackgroundHepatitis B virus infection remains a major public health challenge, particularly among people living with human immunodeficiency virus, due to shared transmission routes and the potential for accelerated liver disease progression. Molecular characterization of circulating HBV strains is essential for understanding viral epidemiology, mutation patterns, and implications for diagnostics and vaccination. MethodsThis study investigated the prevalence of hepatitis B infection and molecular characteristics of the hepatitis B virus surface gene among HIV-infected individuals receiving antiretroviral therapy in Nairobi County, Kenya. Plasma samples were screened for hepatitis B surface antigen using enzyme-linked immunosorbent assay. Hepatitis B viral DNA was extracted from HBsAg-positive samples and the surface gene region amplified by polymerase chain reaction. Amplified products were subjected to Sanger sequencing. Sequence assembly, genotype determination, and mutation analysis. ResultsThe prevalence of HIV/HBV co-infection among HIV-positive individuals was determined to be 8.97%. Genotype analysis revealed the circulation of HBV genotype A (sub-genotypes A1 and A4) and genotype D (sub-genotypes D4 and D10) among the studied population. Amino acid sequence analysis of the major hydrophilic region of the surface gene identified several mutations, with R122K and Y134F being the most frequently observed substitutions. ConclusionHepatitis B infection remains prevalent among HIV-infected individuals receiving antiretroviral therapy in Nairobi County. The circulation of multiple hepatitis B virus genotypes and the presence of mutations within the surface gene highlight the importance of continuous molecular surveillance to monitor viral evolution and its potential implications for hepatitis B virus diagnosis, vaccination strategies, and clinical management in HIV-infected populations

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.

African swine fever virus (ASFV), the etiologic agent of African Swine Fever (ASF), is a high-consequence pathogen requiring experiments to be conducted in containment in non-endemic countries, thereby restricting diagnostic development, the creation of reference standards, and proficiency testing (PT). Safe and reliable inactivation methods are essential to expand diagnostic capacity while preserving nucleic acid integrity for molecular assays in unaffected countries. This study employed gamma irradiation to achieve complete inactivation of ASFV without compromising downstream molecular detection, as gamma irradiation offers deep penetration and uniform dose delivery. ASFV-cell culture supernatants were subjected to gamma irradiation doses ranging from 2 to 50 kGy. Viral replication was evaluated using TCID{square}{square} and serial passages, revealing a consistent dose{square}dependent reduction in infectivity across increasing irradiation dose levels and a complete loss of ASFV infectivity at 30 and 50 kGy. Molecular detection remained unaffected at all of the tested doses as confirmed by qPCR Ct values and sequence identity of the p72 gene. Whole genome sequencing demonstrated >99% genome coverage and consistent read depth profiles across irradiated and non-irradiated samples, indicating preservation of genomic integrity at all tested doses. These findings demonstrate that gamma irradiation at 50 kGy fully inactivates ASFV-cell supernatants while maintaining nucleic acid quality suitable for molecular diagnostics. The resulting inactivated material meets quality assurance requirements for molecular reference standards and PT panels and can be safely distributed to laboratories outside high containment facilities, supporting broader diagnostic readiness and harmonization of ASFV testing.

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.

We prepared siRNA libraries against the H5N2 virus NP gene, and the PA, PB1 and PB2 genes that express the proteins that form the virus polymerase complex. The antiviral activity of the siRNA libraries in H5N2 virus infected cells was initially assessed by using qPCR to measure the corresponding mRNA levels in the siRNA-treated cells. In this way siRNA molecules within each library were identified that exhibited to a greater than 70% reduction in levels of each target mRNA. A selection of these siRNA molecules was further evaluated for their antiviral activity in a multi-cycle H5N2 MDCK cell model. The siRNA molecules identified were successful in blocking virus transmission and lead to a reduction in influenza virus progeny virus production. This antiviral activity correlated with both the inhibition of nuclear export of the newly formed RNP complexs that arise from the transcriptional activity of the input virus, and the inhibition of the polymerase activity of the newly formed virus polymerase complexes. This study highlights the potential use of siRNA as a strategy to block virus transmission by targeting the avian influenza virus polymerase complex.

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.

Tomato fruit blotch virus (ToFBV) is an emerging multipartite positive-sense RNA virus associated with blotchy symptoms on tomato fruits and classified within the genus Blunervirus (family Kitaviridae). Despite its increasing agricultural relevance, the study of ToFBV has been hindered by the lack of mechanical transmissibility and the difficulty in reproducing infections under controlled conditions. In this work, we report a preliminary step toward the development of the first infectious agroclone system for ToFBV, based on full-length cDNA copies of its four genomic RNAs. We demonstrate that the cloned viral genome is capable of initiating cell autonomous replication in Nicotiana benthamiana, as indicated by the accumulation of negative-sense RNA intermediates in infiltrated tissues. To further validate the system, RNA3 was engineered to express GFP, enabling visualization of infection foci and confirming active viral replication in both N. benthamiana and tomato. Functional assays of RNA4-encoded proteins demonstrated that it encodes a movement protein capable of complementing movement-deficient viral vectors and a putative suppressor of post-transcriptional gene silencing (PTGS). Together, these results establish a versatile reverse genetics platform for ToFBV, providing new insights into the replication and functional organization of blunerviruses and enabling future studies on virus-host interactions, pathogenicity, and control strategies.

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.

BackgroundDuring 2021-2022, high pathogenicity avian influenza (HPAI) caused mass mortality in wild birds across Europe, with Northern Gannets (Morus bassanus) among the most affected. Following the outbreak, unusual alterations in the species characteristic pale iris were observed in some individuals. MethodsOpportunistically captured gannets on Bass Rock (n=52), selected to represent a range of iris pigmentation, were examined. Slit-lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry and photography were performed. Iris pigmentation was classified as normal, mottled or black. Eleven birds underwent avian influenza virus (AIV) serology. Histopathology was performed on two eyes. ResultsAbnormal iris pigmentation was found in 74% of adult and immature gannets, with 61% affected bilaterally. Additional signs consistent with uveitis were present in 77% of affected birds. Iris pigmentation abnormalities were positively associated with AIV H5 seropositivity (Fishers exact test, P=0.018). Histopathology from affected eyes showed increased melanin deposition and disorganisation, including loss of a distinct anterior layer of melanocytic cells and hypertrophy of melanocytes within the iris stroma. LimitationsField conditions limited uniform lighting and concurrent serology. ConclusionsIris pigmentation changes were associated with prior HPAI exposure and frequently accompanied by signs of uveitis, suggesting iris alterations may indicate past infection and potential chronic sequelae.

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

Colibacillosis, caused by Avian Pathogenic Escherichia coli (APEC), result in substantial economic losses in global poultry production. The emergence of multidrug-resistant (MDR) APEC poses zoonotic risks through horizontal transfer of antimicrobial resistance (AMR) genes. Bacteriophage therapy emerges as a safe alternative to antibiotherapy; however, comprehensive characterization of phages targeting MDR-APEC from diverse geographical regions remains limited. We isolated five lytic bacteriophages from poultry fecal samples collected from five Indian states and characterized them through morphological analysis, physiological stability testing, whole-genome sequencing, and in vivo efficacy assessment. Host range was determined against APEC isolates, and therapeutic potential was validated in Galleria mellonella infection model. All five phages showed Myovirus-like morphology and stability across physiologically relevant temperatures (up to 55-70{degrees}C) and pH conditions (3-11). Their genome size ranges from 170 to 356 kb, belonging to three distinct genera; Dhakavirus, Gaprivervirus, and Asteriusvirus. Genomic analysis confirmed absence of antimicrobial resistance, virulence, toxin, or lysogeny genes. 51 APEC strains were isolated, of which 23 (45.1%) were MDR. Individual phages lysed 37-51% of tested APEC and 17-39% of MDR strains. Three Escherichia phages (fBSZT1, fUAMT1, fPKPT2) significantly improved larval survival to 60-80% at MOI 10 in G. mellonella infection models compared to untreated controls. This study establishes a well-characterized phage bank targeting MDR-APEC strains, providing foundation for developing phage-based interventions to reduce antibiotic dependency and mitigate AMR transmission risks under One Health framework.

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.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

Lamprey reddening syndrome (LRS) is an emerging disease affecting pouched lamprey (Geotria australis; kanakana/piharau), a culturally and ecologically significant species in Aotearoa New Zealand. Characterised by skin haemorrhaging and elevated mortality, the aetiology of LRS has remained unresolved despite previous investigations. We used a metatranscriptomic approach to characterise viral communities in 28 lamprey from New Zealand and Tasmania, Australia, comparing diseased and presumably healthy individuals. This analysis revealed eight fish-infecting RNA viruses, seven of which were novel, including two highly divergent coronaviruses. One of these coronaviruses possessed a bi-segmented genome structure, and three lamprey were co-infected with both coronaviruses. While these coronaviruses were detected in both healthy and diseased individuals, lamprey with reddening exhibited markedly higher viral abundance, driven by elevated RNA transcripts of both viruses. This pattern suggests that increased coronavirus replication in diseased individuals may be influenced by host stress to environmental factors or co-infection with other pathogens, rather than acting as a sole causative agent of disease. Beyond identifying candidate viral associations, this study expands the known virosphere of an ancient vertebrate lineage and demonstrates the utility of genomics-informed diagnostics for investigating disease in threatened wildlife.

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.

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.

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.

Influenza D virus (IDV), the most recently identified member of the Orthomyxoviridae, was first isolated from pigs but cattle have been identified as the reservoir host. To date, IDV has not been confirmed to cause human disease. Like the haemagglutinin (HA) of influenza A virus (IAV) and the haemagglutinin-esterase fusion (HEF) protein of influenza C virus (ICV), the IDV HEF is produced as a precursor protein (HEF0) that must be proteolytically cleaved by host cell proteases (into HEF1 and HEF2) to gain its fusion capacity. The proteases that activate IAV HA have been extensively studied, but those responsible for activation of IDV HEF were unknown. Identifying these proteases is key to understanding early virus-host interactions and host restriction. Therefore, we generated ICV and IDV pseudotyped viruses (PVs) in HEK 293T producer cells with or without co-transfection of plasmids expressing different type II serine proteases. Subsequent transduction of swine testicular (ST) cells indicated strong activation of both ICV and IDV PVs by the human airway trypsin-like protease (HAT) and its swine homologue (swAT). Furthermore, like influenza A/Puerto Rico/8/34 (H1N1) virus, addition of exogenous protease is not essential for IDV replication in MDCK II cells, most likely due to endogenous expression of matriptase. In conclusion, our data unveil new information on host cell proteases that activate ICV and IDV HEF proteins. Importantly, the data suggest that protease specificity is not a factor in restriction of IDV replication in the human upper respiratory tract.

Hepatitis D virus (HDV) is a satellite virus that utilizes hepatitis B virus (HBV) as a helper for infectious particle formation. HDV was originally identified as a novel antigen in liver biopsies of HBV patients, and later studies showed the "delta" antigen (DAg) to be the sole protein encoded by HDV. Until the discovery of HDV-like agents in birds and snakes in 2018, HDV was a unique example of animal satellite viruses. We identified Swiss snake colony virus 1 (SwSCV-1) in the brain of a Boa constrictor, and through comparison we found the genome organization of SwSCV-1 to resemble that of HDV. However, in addition to the DAg open reading frame (ORF), the genome of SwSCV-1 includes another >500 nt ORF, "ORF2". To study whether the putative ORF2-encoded protein plays a role in the SwSCV-1 life cycle, we established an infectious clone of the virus with a point mutation in the methionine initiation codon of ORF2. The mutation did not significantly affect initiation of replication, establishment of persistent infection, or infectious particle formation upon superinfection with a helper virus. Using additional methods, we gathered further evidence confirming that ORF2 is not actively translated in boa constrictor cells. We further showed that unlike HDV, SwSCV-1 expresses a single form of the DAg. Although the proteins encoded by SwSCV-1 and HDV only include one and two forms of the DAg, respectively, whether other kolmioviruses express additional forms of DAg or related proteins in some cell types or host species merits further research. IMPORTANCEApproximately 40 years after the discovery of hepatitis D virus (HDV), satellite viruses with similar genome organization were found in various animals, thereby giving rise to family Kolmioviridae. HDV encodes a single protein, the delta antigen (DAg), which comes in small and approximately 20 amino acids longer large form. The genome of some HDV species and many of the newly found kolmioviruses contains additional open reading frames (ORFs), potentially enabling protein expression. Here, we studied the viral proteins expressed during Swiss snake colony virus 1 (SwSCV-1) infection of boa constrictor cells. Our findings show that unlike HDV, SwSCV-1 encodes only a single form of DAg. In addition, our study suggests that, like in HDV, the additional ORF in SwSCV-1 genome does not give rise to a protein. Although we could not demonstrate expression of additional viral proteins during SwSCV-1 infection, it is important to study the proteome of other kolmioviruses.