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 Sida acuta 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; Begomovirus oxalisflavi) and Sida yellow leaf curl virus (SiYLCV; Begomovirus sidaflavacontorsionis) 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.

Capulavirus betae (beet capulavirus 1, BCV1) is a recently described species of the genus Capulavirus (family Geminiviridae) 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 Nicotiana benthamiana, 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.

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.

Viruses of the genus Tottorivirus (family Picornaviridae) 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.

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.

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 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.

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.

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 α-helical and β-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.

Feline leukaemia virus subgroup A (FeLV-A) has generally been regarded as having a narrow, predominantly feline host range. Here, we characterize a naturally occurring FeLV-A isolate, strain F8701, that exhibits unexpectedly broad mammalian cell tropism. Using an infectious molecular clone of F8701, we show that F8701 infects canine cells efficiently and can utilize the canine orthologue of thiamine transporter 1, the canonical FeLV-A receptor. Compared with reference FeLV-A strains, F8701 displayed markedly higher infectivity in multiple non-feline cell lines, including canine and human cells. Sequence analysis of the envelope glycoprotein revealed that F8701 carries the previously described 83D/91D di-aspartic acid residue combination in variable region A, together with an additional K105N substitution whose functional contribution remains to be determined. Interference assays confirmed that F8701 belongs to FeLV subgroup A, indicating that broad cellular tropism can occur within a naturally occurring FeLV-A background. Although replication in cultured canine cells was observed, whether F8701 can establish productive infection or transmission in dogs in vivo remains unknown. These findings highlight the natural plasticity of the FeLV-A envelope and suggest that receptor compatibility can contribute to the expansion of mammalian cellular tropism, while additional host-level barriers are likely to determine cross-species infection in vivo.

Bacilladnaviridae is a family of viruses with single-stranded, circular DNA genomes that range from 3.4 to 6 kb and contain a short double-stranded region of 0.5-1 kb. The genome is replicated by the rolling-circle mechanism. Bacilladnavirids encode a jelly-roll capsid protein which forms non-enveloped icosahedral capsids with a T=3 symmetry. All cultured bacilladnavirids infect diatoms, a group of unicellular algae (Stramenopiles), and cause lysis of the host cells. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bacilladnaviridae, which is available at ictv.global/report/bacilladnaviridae.

Vertical transmission has been identified as a viable route of transmission in the spread of chronic wasting disease (CWD), yet demonstration of trafficking mechanism(s) at the maternal-foetal interface has been limited. Conventional assays, including immunohistochemistry (IHC), Western blot and ELISA, often fail to detect low concentrations of the aberrant prion protein (PrP[CWD]). Contemporary in vitro amyloid conversion assays can detect low concentrations of prion seeding activity but lack the ability to localize prion seeds within specific cellular structures of tissues. In this study, we developed an enhanced immunohistochemical approach, as IHC uniquely enables cellular localization, to reveal prion deposition in maternal-foetal tissues from experimentally infected Reeves' muntjac. After confirming improved sensitivity of amplification-IHC (AMP-IHC) in lymphoid tissues, we applied the assay to placentomes and uterine samples, comparing the results to matched tissue sections assessed by conventional IHC. AMP-IHC consistently detected PrP[CWD] deposition that was undetected by conventional IHC, including its presence within the uterine glandular epithelium and glandular lumen. These observations provide the first histological evidence depicting prion association with uterine glands - structures responsible for secreting vital nutrients during early pregnancy. The presence of PrP[CWD] in these structures supports the hypothesis that the developing conceptus can be exposed to prions in utero. Our findings expand upon earlier studies demonstrating vertical CWD transmission and highlight AMP-IHC as a valuable tool for identifying low-level prion accumulation in tissues where conventional IHC proves insufficient.

Rabies virus (RABV) is a neurotropic virus of the genus Lyssavirus that causes a nearly always fatal encephalitis in humans once it reaches the central nervous system. Dog bites are a major transmission route of RABV, and following superficial skin exposure, RABV first encounters epidermal Langerhans cells (LCs), yet their role in early infection remains poorly understood. Here, we show that exposure of immature human epidermal LCs to RABV leads to RABV association with cells in the absence of productive viral replication. RABV-exposed LCs display reduced cytokine and type I IFN responses and lack induction of IFN-stimulated genes, indicating suppression of antiviral signalling pathways. Together, these findings suggest that RABV is efficiently taken up by immature LCs, which dampens the antiviral immune responses of LCs, potentially facilitating immune evasion at the skin entry site.

The tape measure protein (TMP) dictates the tail length of tailed bacteriophages. However, the sequence features that drive the process of length fine-tuning have not yet been described. Tandem repeats (TRs), stretches of aas organized as multiple adjacent copies of the same or very similar sequence motif, are contributors to TMP length variation. Homorepeats (polyX regions) are a specific type of TRs composed of stretches of identical aas, forming low-complexity regions that contribute to protein structural flexibility and dynamics. Here, we performed a large-scale analysis of polyX regions across 12 million phage proteins to test whether these homorepeats help explain the variable length of flexible phage tails. PolyX tracts were detected in 41% of all phage proteins and in 95.5% of TMPs, with polyA, polyG, polyS and polyT dominating the composition. Furthermore, the number of polyX tracts in TMPs scaled directly with protein length. About half of the TMPs have both polyX and TRs (23,469 out of 45,500 TMPs), but nearly half of the polyX-containing TMPs lacked any TRs (19,999 out of 43,468 TMPs), showing that polyX are independent features rather than by-products of larger repeats. Morphological comparisons showed that phages with long and flexible tails (Siphovirus morphotype) combine polyX and TRs, whereas shorter or contractile-tailed phages rely mostly on polyX. Last, the total aa content in polyX regions correlated significantly with experimentally measured tail lengths (R[2]=0.811, P=1.56×10[-][4]). Our results identify polyX tracts as an important and tunable component of TMPs that enable precise control of phage tail length.

The emergence of the panzootic clade 2.3.4.4b highly pathogenic H5N1 avian influenza in 2020 marked a major expansion in the host range of influenza A viruses (IAVs), raising concerns about further cross-species transmission events and zoonotic spillover. The introduction of 2.3.4.4b viruses into US dairy herds has resulted in widespread circulation, accompanied by reduced milk yield, mastitis and high viral loads in milk. Notably, virus circulation in dairy cattle represents a novel route for mammalian adaptation and transmission that has already led to more than 40 human cases in the USA since 2024. As haemagglutinin (HA) is the primary determinant of viral host range and tissue tropism, we investigated whether viruses containing the glycoproteins of avian H5N1 subclade 2.3.4.4b viruses could infect mammary tissue from Aberdeen Angus, Holstein Friesian and Limousin cattle. Using mammary gland explants, we inoculated tissues with attenuated reassortant viruses expressing the HA and neuraminidase (NA) of three 2.3.4.4b viruses that predated the emergence of H5N1 in the US cattle: A/chicken/England/053052/2021 (AIV07), A/chicken/Scotland/054477/2021 (AIV09) and A/chicken/England/085598/2022 (AIV48). Detection of viral NP antigen by immunohistochemistry in explants from both the teat and gland cistern for all three breeds following inoculation with AIV09 and AIV48 indicates that mammary tissue from each of the three tested cattle breeds is susceptible to infection by viruses bearing H5N1 glycoproteins. Lectin staining showed expression of both α2,3- and α2,6-linked sialic acids in the mammary tissue of all donors, indicating that all three breeds have the potential to support infection with both avian- and mammalian-adapted IAVs. Together, these findings demonstrate that the mammary glands from both beef and dairy cattle breeds are susceptible to infection with viruses bearing avian-origin H5N1 glycoproteins and suggest that bovine mammary tissue may provide an environment in which diverse IAVs could co-infect the same tissue. This possibility underscores the need for further investigation and the inclusion of both beef and dairy cattle in ongoing H5N1 surveillance and risk-assessment frameworks.

Most human-acquired prion diseases result from infection with M1 or V2 prions from sporadic Creutzfeldt-Jakob disease. To prevent transmission via surgical instruments, sterilization methods targeting these prions are essential. In this study, stainless steel wires coated with M1 or V2 prions were used to evaluate inactivation protocols. None of the individual treatments - autoclaving, SDS or NaOH - eliminated infectivity except for autoclaving against V2 prions. Autoclaving at 134 °C revealed distinct thermal stability between M1 and V2, with M1 exhibiting greater thermal stability. Importantly, combining methods, which we herein called two-step processing methods, achieved practical inactivation: SDS treatment followed by autoclaving at 121 °C was highly effective. Commercial alkaline detergents also proved effective against M1 prion under a combination of high pH and temperature. The inability to detect infection in our model mice using our assay system demonstrates that the infectivity titre decreased to 10[-8] in M1 and 10[-7] in V2. These findings indicate that synergistic approaches can achieve prion inactivation under practical conditions, supporting the need for revised sterilization protocols to prevent iatrogenic transmission.