Journal of General Virology最新文献

{"title":"An <i>in silico</i> study of tymovirus virocells.","authors":"A J Gibbs, M J Gibbs, H M Gibbs, M Hajizadeh","doi":"10.1099/jgv.0.002329","DOIUrl":"https://doi.org/10.1099/jgv.0.002329","url":null,"abstract":"<p><p>We used computational tools to study the tymoviral proteome. Past gene sequence comparisons had shown that tymovirus genomes encode five proteins already identified in other viruses; however, our AlphaFold3 modelling found, in addition to their five <i>α</i>-helical and <i>β</i>-sheet structures, five regions of 'intrinsically disordered protein' (IDP). Notably, one of these IDPs is the movement protein (MP), distinguishing it from the structured MPs of other plant viruses. The MP is the largest IDP having some features of structured proteins including genus-specific conserved motifs. All tymovirid methyltransferases (Mtases) have a novel structure that includes a membrane-targeting domain (MTD) at their C-termini. Modelling showed that polymers of 3 or 11 copies of the Mtase:MTD are favoured; the former form complexes with the viral RNA-dependent RNA polymerases, whereas the latter form short rounded cylinders with an equatorial groove. The cylinders are similar in size to the necks of vesicles previously found in the outer chloroplast membranes of all tymovirus-infected plants. The DisLipPred program found that the outer groove of the polymeric cylinders was probably lipophilic and so able to associate stably with plasma membranes. The vesicles are probably the 'replication organelles' of tymoviruses and the cylinders possibly the 'replication organelle organizers'. Modelling also indicated that the tymovirus helicase may function as a tetramer and that the coat protein may form trimer intermediates for assembling the virions. Our study has revealed some of the complexity and three-dimensionality of the viral components of tymovirus virocells.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13630270/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149110855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"First isolation of a novel tottorivirus from wild boar faeces in Japan.","authors":"Mami Oba, Ai Hirabayashi, Natsuko Teshima, Hitoshi Takemae, Toru Oi, Fujiko Fukuda, Shoichi Sakaguchi, Takanori Shiga, Naoyuki Aihara, Junichi Kamiie, Hiroho Ishida, Hironobu Murakami, Tetsuya Mizutani, Takeshi Noda, Makoto Nagai","doi":"10.1099/jgv.0.002334","DOIUrl":"10.1099/jgv.0.002334","url":null,"abstract":"<p><p>Viruses of the genus <i>Tottorivirus</i> (family <i>Picornaviridae</i>) have been identified in pigs but no isolates have been reported to date. In this study, a novel tottorivirus strain, designated Tottori/tc/Wild Boar/Japan/Ishi-6/2020 (Ishi-6), was isolated for the first time from a faecal sample of a wild boar using swine testis cells, where cytopathic effects (CPEs) were observed. Ultrathin-section transmission electron microscopy revealed virus-like structures arranged around vesicular membranes in infected cells. The Ishi-6 replicated in Porcine Kidney-15 and Cloned Porcine Kidney cells with CPEs but no replication was observed in non-porcine cell lines. Sequence analysis showed that Ishi-6 shared high aa sequence identity with a U.S. pig slurry-derived strain in the polyprotein, especially 2C+3 CD region with the highest identity (95.16%). In contrast, the P1 region was more closely related to a Chinese pig-derived strain (74.18%), while showing low identity to Japanese pig-derived tottorivirus strains. Phylogenetic analysis divided tottoriviruses into three clusters based on the polyprotein sequence, whereas different clustering patterns were observed for the P1 and 2C+3 CD regions. The incongruent clustering of Ishi-6 between these regions suggested recombination, which was further supported by recombination analysis identifying a breakpoint near the VP1-2A junction, involving U.S. pig slurry-derived and Chinese pig-derived strains. Real-time reverse transcription PCR targeting the 5' UTR of Ishi-6 was performed on faecal samples from 280 wild boars and 70 domestic pigs collected between 2018 and 2023. Nine wild boar samples collected between 2018 and 2022 tested positive, while no positive sample was obtained from pigs. These findings indicate that Ishi-6 is a putative recombinant tottorivirus that may have circulated in Japanese wild boar populations. Tottoriviruses exhibit considerable genetic diversity, likely driven by recombination, and continued surveillance is warranted, including their pathogenicity in pigs.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13645378/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149127752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A high-quality telomere-to-telomere lumpy skin disease virus genome assembly.","authors":"Caroline Wright, Noemi Polo, Sarwar Azam, Graham Freimanis, G Taru Sharma, Bryan Charleston, Carrie Batten, Tim Downing, Jonas Albarnaz","doi":"10.1099/jgv.0.002348","DOIUrl":"10.1099/jgv.0.002348","url":null,"abstract":"<p><p>Lumpy skin disease virus (LSDV) is an emerging livestock capripoxvirus (CaPV) that continues to cause substantial economic losses across Africa, Asia and Europe. However, important uncertainties remain regarding LSDV genome structure, particularly at the telomeric inverted terminal repeats (ITRs) that are central to host interaction, replication and adaptive evolution. Structural variation at the ITRs is driven by recombination during virus replication. Previous assemblies have relied primarily on short-read sequencing, which provides limited resolution of repeat-rich telomeric regions and propagates structural ambiguities into downstream annotation and comparative analyses. Here, we present a high-quality telomere-to-telomere assembly of the LSDV Oman 2009 isolate, generated using a hybrid approach that integrates short Illumina reads with higher-accuracy long Nanopore reads. The resulting 151,091 bp genome contains 157 annotated ORFs and fully resolves complex repeat-rich structures at both ITRs. This new reference corrects misassemblies present in earlier LSDV genomes and confirms clade-specific gene truncations. This genome provides a robust foundation for improved genomic surveillance, accurate read mapping, mutation detection and evolutionary inference of LSDV and demonstrates the value of long-read approaches for resolving complex CaPV genome structures.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13668194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149182038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular characterization and infectious clone construction of beet capulavirus 1 infecting tomato in Germany.","authors":"Omid Eini, Paolo Margaria, Dennis Knierim, Claudia Pommerenke, Monika Heupel, Björn Krenz, Mark Varrelmann, Wulf Menzel","doi":"10.1099/jgv.0.002347","DOIUrl":"10.1099/jgv.0.002347","url":null,"abstract":"<p><p><i>Capulavirus betae</i> (beet capulavirus 1, BCV1) is a recently described species of the genus <i>Capulavirus</i> (family <i>Geminiviridae</i>) that was first identified in sugar beet in France. Capulaviruses are new group of geminiviruses because they are transmitted by aphids. Here, we report the identification and molecular characterization of an isolate of BCV1 infecting cultivated tomato in Germany and describe the construction of an infectious clone. In August 2025, tomato plants displaying severe virus-like symptoms, including leaf deformation, leaf curling, stunting and chlorosis were collected from two production sites in North Rhine-Westphalia. High-throughput RNA sequencing revealed a single viral contig representing a complete circular DNA genome with 99.5% nt identity to BCV1. The presence of the virus was additionally confirmed in five out of six individual tomato samples by specific PCR. Rolling-circle amplification (RCA) supported the circular nature of the genome and direct sequencing of RCA products confirmed the HTS-derived sequence. An infectious clone was generated and used to induce systemic infection after agrobacterium-mediated inoculation of sugar beet, tomato and <i>Nicotiana benthamiana</i>, thereby demonstrating that the cloned genome is biologically active in taxonomically distinct hosts. The RNA-seq data revealed a novel splicing event in the complementary-sense transcripts which was confirmed by RT-PCR. Together, these results expand the known host range and geographic context of BCV1 and establish an experimental system for studying a capulavirus pathosystem.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13649057/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149137043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Snake kolmiovirus encodes a single form of delta antigen and shows no evidence of translation from ORF2.","authors":"Leonóra Szirovicza, Udo Hetzel, Tomas Strandin, Anja Kipar, Jussi Hepojoki","doi":"10.1099/jgv.0.002327","DOIUrl":"10.1099/jgv.0.002327","url":null,"abstract":"<p><p>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-<i>like</i> 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 <i>Boa constrictor</i> and through comparison we found the genome organization of SwSCV-1 to resemble that of HDV. However, in addition to the DAg 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.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13644933/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149127776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Into the wild: a long-term study of begomovirus community dynamics in a natural landscape.","authors":"João P Herrera da Silva, César A D Xavier, Phedra G S Oliveira, Márcio T Godinho, Júlia B Lage, José C F da Silva, Alison T M Lima, F Murilo Zerbini","doi":"10.1099/jgv.0.002340","DOIUrl":"10.1099/jgv.0.002340","url":null,"abstract":"<p><p>Begomoviruses have been the subject of many evolutionary studies that explored their temporal dynamics; however, most of the studies relied on sequences retrieved from public repositories rather than hypothesis-driven sampling. These datasets are often temporally or spatially unbalanced, which can bias molecular clock calibration, especially when the population exhibits strong genetic structure. Furthermore, most studies have focused on agricultural environments where anthropogenic activity may influence viral diversification. Longitudinal studies with systematic sampling in the natural environment are essential to understand begomovirus ecology and evolution. We monitored a begomovirus community over 12 years (2011-2014, 2016-2022) in a 300 m² Atlantic Forest regeneration area in southeastern Brazil, sampling 385 <i>Sida acuta</i> plants and obtaining 371 complete genomic segments (262 DNA-A and 109 DNA-B). We quantified community diversity and estimated substitution rates and demographic dynamics. Five begomoviruses were detected at the site, with Oxalis yellow vein virus (OxYVV; <i>Begomovirus oxalisflavi</i>) and Sida yellow leaf curl virus (SiYLCV; <i>Begomovirus sidaflavacontorsionis</i>) dominating the community. Species composition changed over time: OxYVV was the most frequent virus during the early years but was outnumbered by SiYLCV from 2016 onwards, with alternating prevalence in later years. Community diversity peaked in 2021 when all five viruses were detected co-occurring. The diversification of SiYLCV exhibited a higher relative contribution of recombination than mutation (ρ/θ>1), contrary to all other begomoviruses studied to date. Although we hypothesized that the changes in community composition observed in 2016 resulted from a genetic bottleneck in the OxYVV population, no evidence of such an event was detected. Instead, we observed a demographic expansion of SiYLCV, consistent with its increase in frequency. This study reveals the complex community dynamics of begomoviruses in natural landscapes and underscores the role of wild plants as reservoirs of viral diversity, providing a baseline for comparisons with agroecosystems to inform management strategies.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13657429/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149169157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Herpes simplex virus type 2 induces S-phase entry and arrest to regulate the cell cycle for efficient replication.","authors":"Huimin Wang, Ranqing Cheng, Miaomiao Li, Yuncheng Li, Siyu He, Longchao Xu, Yuhao Chen, Zhenhua Huang, Binlian Sun, Yalan Liu, Qinxue Hu, Mudan Zhang","doi":"10.1099/jgv.0.002346","DOIUrl":"10.1099/jgv.0.002346","url":null,"abstract":"<p><p>Viruses often hijack core host cell processes to optimize their replication. In the current study, we conducted comparative proteomics analyses of epithelial cells infected by the prevalent human pathogen herpes simplex virus type 2 (HSV-2), followed by experimental validation. The results reveal that HSV-2 infection induces significant reprogramming of the host cell cycle, characterized by a pronounced accumulation of cells in the synthesis (S)-phase. Mechanistically, we discovered that HSV-2 promotes gap 1 (G1)/S transition by downregulating the cyclin-dependent kinase inhibitor p21 via a proteasome-mediated pathway. Following this, the virus induces S-phase cell cycle arrest, characterized by reduced levels of cyclin-dependent kinase 2 (CDK2) and its active phosphorylated form and decreased activity of the cyclin A2-CDK2 complex. Consequently, this dysfunctional 'pseudo-S-phase' state significantly enhances HSV-2 replication. These findings reveal a previously unrecognized dual mechanism employed by HSV-2 to manipulate the host cell cycle, thereby advancing our understanding of how pathogens disrupt cellular homeostasis to facilitate their replication.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13645362/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149127833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Botrytis virus X ORF2 suppresses RNA silencing in a trigger-restricted manner and is associated with altered Dicer-like gene expression in <i>Botrytis cinerea</i>.","authors":"Fayruza Lalany, Sarah C Drury, Mamadou L Fall, Peter Moffett","doi":"10.1099/jgv.0.002343","DOIUrl":"10.1099/jgv.0.002343","url":null,"abstract":"<p><p>RNA interference (RNAi) is a central antiviral defence mechanism in fungi, yet relatively few mycoviral suppressors of RNA silencing (VSRs) have been functionally characterized, particularly in phytopathogenic hosts. Botrytis virus X (BVX), a positive-sense RNA virus in the family <i>Alphaflexiviridae</i>, infects <i>Botrytis cinerea</i> and encodes five predicted ORFs, most of which have unknown functions. Here, we screened BVX ORFs 2-5 for RNA silencing suppressor activity using complementary GFP-based assays in <i>Nicotiana benthamiana</i> and examined the leading candidate in the fungal host <i>B. cinerea</i>. BVX ORF2 (X2) enhanced GFP transcript and protein accumulation in assays where silencing was triggered by sense RNA but failed to suppress silencing triggered by hairpin-derived small interfering RNAs or microRNA-guided targeting, indicating a trigger-restricted suppressor phenotype. In <i>B. cinerea</i>, transgenic expression of X2 was associated with reduced induction of the RNAi-associated genes <i>BcDCL1</i> and <i>BcDCL2</i> compared with empty-vector controls, with the strongest effect observed on <i>BcDCL1</i>. In a virus-infected fungal background, X2 expression was also associated with increased viral RNA accumulation. Together, these results identify BVX X2 as a BVX-encoded, trigger-restricted suppressor of RNA silencing and link its expression to altered RNAi-related gene induction and increased viral RNA accumulation in <i>B. cinerea</i>.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13668195/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149182115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ORF2 of hepatitis E virus: genetic and antigenic variation across genotypes and implications for immunity, diagnostics and vaccine development.","authors":"Sherif A El-Kafrawy, Sigit S Putra, Ayman T Abbas, Umama A Abdel-Dayem, Thamir A Alandijany, Hisham N Altayb, Esam I Azhar","doi":"10.1099/jgv.0.002342","DOIUrl":"10.1099/jgv.0.002342","url":null,"abstract":"<p><p>Hepatitis E virus (HEV) causes over 19 million infections annually, yet the molecular determinants governing its diverse clinical phenotypes and cross-genotype immunity remain incompletely synthesized. The ORF2 capsid protein is the sole structural component of the virion, the primary neutralizing antibody target and the basis for all commercial diagnostics and the only licensed vaccine (Hecolin®). While current literature often treats HEV as a monolithic antigenic entity, ORF2 sequences diverge by 15-25% across the eight recognized genotypes (GT1-GT8), with critical substitutions concentrated in the protruding (P) domain neutralizing epitopes. This comprehensive review integrates recent structural, virological and clinical data to evaluate the multidimensional impact of ORF2 variability. We highlight how the differential expression of three ORF2 isoforms (infectious ORF2i, secreted decoy ORF2g and cleaved ORF2c) complicates immune recognition. Furthermore, we critically analyse the functional consequences of P domain divergence, which results in 2-4-fold reduced cross-genotype neutralization by GT1-derived vaccines against the globally emerging GT3 and drives false-negative rates of 12-30% in commercial serological assays. Finally, we map ORF2 genetic signatures to distinct clinical phenotypes, including GT1-associated pregnancy mortality and GT3-driven chronicity in transplant recipients. By synthesizing these fragmented domains, this review identifies critical knowledge gaps, particularly concerning the zoonotic emergence of GT7/GT8 and provides a prioritized research roadmap for the development of true pan-genotype vaccines and universal diagnostic algorithms.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13649056/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149136956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sensitive reverse transcription-digital droplet PCR-based discrimination of hepatitis delta virus RNAs reveals differential effects of interferon-α on viral RNA species.","authors":"Caroline Pons, Emilie Charles, Cassandre Authier, Elise Chabot, Roxanne Fouillé, Faustine Bernardin, Claudie Eber, Léa Corbet, Maud Michelet, Marine Oudot, Charlotte Bach, Michel Rivoire, Guillaume Passot, Thomas F Baumert, Karim Majzoub, Eric Huntzinger, Bertrand Seraphin, David Durantel, Eloi R Verrier, Julie Lucifora","doi":"10.1099/jgv.0.002324","DOIUrl":"10.1099/jgv.0.002324","url":null,"abstract":"<p><p>Hepatitis delta virus (HDV) replication relies on the production of distinct viral RNA species, including genomic (HDV-G), antigenomic (HDV-AG) and mRNAs (HDV mRNAs), which differ in abundance and function. However, the lack of sensitive and quantitative approaches allowing their simultaneous discrimination has limited the precise analysis of HDV RNA dynamics, particularly under antiviral conditions. Here, we combined nascent RNA and RNA stability assays with a novel reverse transcription digital droplet PCR (RT-ddPCR) approach enabling strand-specific quantification of HDV RNAs. Using this methodology, we confirmed the previously described kinetics of viral RNA production and assessed the ratio of each RNA species in HDV-infected cells. Specific small interfering RNA (siRNA) treatment demonstrated the specificity of our assay in discriminating HDV RNA species. Next, applying this technique to the study of virus-host interactions, we showed that IFN-α treatment decreases the synthesis of HDV-G and may additionally accelerate HDV-AG and HDV-G decay, while HDV mRNA stability remained unaffected in infected HuH7.5-Na+-taurocholate cotransporting polypeptide (NTCP) cells. Immunoprecipitation assays coupled to strand-specific RT-ddPCR revealed no difference in the association of cellular RNA polymerase II (Pol II) with HDV-G and HDV-AG. In parallel, transcriptomic analyses of differentiated HepaRG and HuH7.5-NTCP cells following IFN-α treatment identified IFN-stimulated gene candidates with potential anti-HDV activity. Among them, we showed that HELZ2 overexpression does not affect HDV RNA stability but interferes with HDV-AG and HDV-G RNA synthesis. Altogether, this RT-ddPCR-based approach provides a versatile tool to investigate the effects of current and emerging antiviral strategies on individual HDV RNA species.</p>","PeriodicalId":15880,"journal":{"name":"Journal of General Virology","volume":"107 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13644934/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149127781","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}