Bluetongue virus (BTV) is a complex, transboundary arboviral pathogen that circulates globally in ruminant populations. Bluetongue disease is a World Organisation for Animal Health listed disease affecting economically important farmed ruminants. BTV is a dsRNA virus from the Sedoreoviridae family that has a segmented genome. Classification of BTV traditionally focussed on antigenic characteristics. More recently, however, Sanger sequencing methods have been adopted for typing based on specific genome segment analysis and targeted whole-genome sequencing (WGS) is being more widely applied. Despite these advances, standardised approaches for analysing genomic sequencing data to better define individual BTV strains are lacking. New perspectives are needed to update and unify the current array of approaches surrounding BTV genetic characterisation nomenclature. A transition from single-segment-based serotyping and genotype descriptors to adopting WGS-derived descriptors that better describe a detected virus rather than an individual segment is required. This change will improve the ability of researchers to understand and communicate virus movement pathways with improved granularity. This review paper aims to condense the extensive literature on BTV characterisation, genomic sequencing and virus transmission from an Australian perspective. Recommendations are made to assist with clarity and uniformity around virus characterisation, including genomic definitions and more standardised BTV nomenclature.
High-throughput sequencing (HTS) technologies have transformed life sciences by enabling rapid, large-scale analysis of nucleic acids, providing unprecedented insights into genomics and transcriptomics. Accurate determination of RNA termini remains, however, a major challenge. We have addressed this limitation by fine-tuning a commercial HTS library preparation protocol. The optimized strategy was validated on plant virus isolates representing diverse taxonomic groups and genome architectures and benchmarked against conventional RACE, demonstrating its effectiveness and robustness. Investigations in members of the genus Ilarvirus allowed to prove the occurrence of non-templated nucleotide additions at the 3' end of the minus-strand viral RNAs in vivo, expanding previous results obtained in vitro in related members in the Bromoviridae and supporting that this feature may be common across the family.
The natural history of chronic hepatitis B virus (HBV) infection comprises distinct stages resulting from virus-host interactions. A late and pivotal event in this process is hepatitis B e antigen (HBeAg) seroconversion, marked by the abrogation of HBeAg expression, a significant reduction in viral load and the accumulation of mutations throughout the genome, particularly within the Core region. While HBeAg loss is associated with mutations in the basal core promoter and preCore regions, these alone do not account for the decreased viral load observed during this stage. To elucidate the contribution of Core variability to HBV replicative capacity, we engineered replication-competent chimeric genomes by reciprocally exchanging the core gene between a WT clone and three HBeAg-negative patient-derived isolates. These constructs were functionally characterized in Huh-7 cells to assess replication intermediates, antigen expression and viral transcriptional activity. Our findings demonstrate that mutations within the Core protein can either impair or enhance HBV replication, depending on their specific mutational patterns. Importantly, all viral replication intermediates were restored to WT levels when the WT Core protein was introduced into the HBeAg-negative genomes. Strong positive correlations between covalently closed circular DNA (CCC DNA) and other viral markers indicate that the Core protein exerts its regulatory effect primarily through regulation of CCC DNA levels. Notably, the absence of Core expression increased CCC DNA transcriptional activity, supporting a repressive role of the WT Core protein in gene expression. Collectively, these findings highlight the pivotal regulatory role of Core protein mutations in modulating HBV replication dynamics and gene expression during the HBeAg-negative phase and underscore its potential as a promising target for novel antiviral strategies.
Maize chlorotic mottle virus (MCMV) represents a major quarantine pathogen that poses a serious threat to global maize production, yet its spatiotemporal evolutionary dynamics remain incompletely characterized. In this study, we reconstructed the global molecular epidemiology of MCMV by analysing 117 complete genome and 214 coat protein (CP) gene sequences. We employed whole-genome data for Bayesian phylodynamic inference while utilizing CP sequences for phylogenetic reconstruction and population genetic analyses. Our phylodynamic analyses estimated a mean evolutionary rate of 2.07×10-4 substitutions per site per year, with the global most recent common ancestor traced to the Americas around 1938. Following its emergence, the MCMV population diversified into two major clades: a basal American lineage (Clade I) and a recently emerged, rapidly diversifying lineage (Clade II, originating ~1952). Within Clade II, we identified a monophyletic East African cluster - representing the most extensively sampled geographic population - that is phylogenetically nested within a broader assemblage of Asian isolates. This East African population, dating to the mid-1980s, exhibits signatures of a recent founder effect, characterized by minimal intra-regional genetic differentiation and lower nucleotide diversity (π=0.003) relative to Asian (0.008) and American (0.026) populations. Phylodynamic demographic reconstructions reveal that regional establishment in East Africa coincided with a pronounced global expansion in effective population size (N e) from the mid-1990s through the mid-2000s, subsequently followed by demographic stabilization. These findings provide structural and temporal insights into the global population structure and spatiotemporal dynamics of MCMV, establishing a foundation for international surveillance strategies and phytosanitary control measures.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to circulate globally through the persistent emergence of novel variants. Vaccination has been regarded as one of the selective environments that can influence SARS-CoV-2 evolution by exerting immune pressure. This study investigated the evolutionary dynamics of the SARS-CoV-2 Delta variant in hamsters immunized with varying doses of a full-length spike protein vaccine, potentially reflecting the heterogeneous levels of immunity. In hamster models, higher vaccine doses prevented viral replication in the lungs but only partially suppressed replication in the nasal passages. After excluding intra-host single-nucleotide variants (iSNVs) detected in non-vaccinated controls, a negative binomial model adjusting for read depth revealed a significant vaccine dose-dependent increase in iSNV occurrence in the Spike, ORF1a, ORF1b and ORF3a genes in nasal samples and positive selection signals were predominantly observed in the highest vaccine dose group. The iSNVs were observed in diverse and distinct combinations that were unique to each individual, reflecting host-specific intra-host mutation patterns. Some aa substitutions detected in at least two individuals in the vaccinated group were more frequently observed in the Omicron variant. In silico analyses incorporating individual-specific iSNVs demonstrated that reduced binding affinity to class 1 and 3 neutralizing antibodies was observed exclusively in variants identified from certain vaccinated individuals. In conclusion, these findings indicate that heterogeneous vaccine-induced immune pressure can shape intra-host SARS-CoV-2 evolution in a dose-dependent and host-specific manner and highlight the potential role of partial immunity and increased iSNVs in the upper respiratory tract in driving the emergence of putative immune-evasive viral variants.
The International Committee on Taxonomy of Viruses (ICTV) is considering a proposal submitted in July 2025 to create an optional additional taxonomic rank below the level of species. This was designed to increase the precision of taxonomic assignment where clinical or other regulatory distinctions need to be made between viruses assigned to the same species. To understand current classification practice and to gauge opinion on the proposal to formalize a below-species taxon, current ICTV study group members (n=746) representing over 100 Study Groups were invited to complete a survey, from whom 240 completed forms and comments were collected in November 2025. The survey also recorded opinions on the use of designators, such as 'type', 'isolate' and 'subspecies' for below-species assignments and the typographic format of their names, for example, whether to be italicized and/or Latinized.Overall, a 6 : 1 majority favoured the introduction of an optional below-species taxonomic rank managed by the ICTV, and was particularly supported by those working in animal and veterinary virology fields where various forms of community-supported below-species classifications are used. There was also support to formalize and taxonomically assign existing classifications of viruses and define their nomenclature. Names might be formatted using the Latin alphabet but with less support for italicization or for Latinization of the below-species epithet. The survey data and associated comments will be considered in further discussions at the next ICTV Executive Committee meeting in November 2026. We thank all of their survey respondents for their invaluable and expert contributions to this consultative process.
Morbilliviruses utilize signalling lymphocytic activation molecule (SLAM) and nectin-4 as their major cellular receptors and exhibit distinct host specificities despite sharing common receptor usage. While species-specific differences in SLAM have been extensively investigated, the contribution of nectin-4 to morbillivirus host range remains poorly understood because nectin-4 is highly conserved among mammals. Here, we systematically compared the receptor activities of nectin-4 orthologs from human, dolphin, bat, dog and seal for representative morbilliviruses, including measles virus (MV), cetacean morbillivirus (CeMV), canine distemper virus (CDV), phocine distemper virus (PDV) and Myotis bat morbillivirus (MBaMV). Cell-cell fusion, virus infection and plaque assays demonstrated that most nectin-4 orthologs efficiently supported infection by all tested viruses. In contrast, dolphin nectin-4 exhibited markedly reduced receptor activity for MV, PDV and MBaMV, while remaining fully functional for CeMV and CDV. This phenotype was consistently observed with multiple clinical MV isolates. Mutational analyses identified amino acid residue 63 as a key determinant of this receptor specificity. Substitution of glutamic acid at position 63 in dolphin nectin-4 with glycine restored MV receptor activity to a level comparable to that of human nectin-4. These findings demonstrate that nectin-4 is generally a broadly permissive morbillivirus receptor but that specific amino acid differences can substantially influence receptor usage. Our results indicate that receptor compatibility contributes to morbillivirus host range and cross-species transmission while being insufficient on its own to fully explain viral host specificity.
Feline coronavirus (FCoV) infections, including fatal feline infectious peritonitis (FIP), have been documented in captive cheetahs (Acinonyx jubatus), but longitudinal data on viral circulation and genetic diversity in this species remain limited. In this study, we performed a 3.5-year molecular epidemiological investigation of a closed captive cheetah cohort at a single zoological facility in Japan. Non-invasive monitoring of 17 individually identified cheetahs by reverse-transcription quantitative PCR of faecal samples revealed long-term maintenance of FCoV in the population, with highly variable individual-specific viral RNA detection patterns over time. Phylogenetic analysis of near-complete genomes recovered from an asymptomatic shedder and an FIP-affected individual placed the cheetah-derived viruses within the type I FCoV clade. Targeted sequencing of the partial nucleocapsid gene identified 17 nt haplotypes and revealed viral sequence diversity and temporal changes in the dominant consensus haplotypes detected within individual cheetahs. These temporal changes could not distinguish reinfection from within-host evolution or undetected within-sample viral diversity. Analysis of the spike (S) gene showed marked regional differences in sequence diversity: the S1/S2 cleavage site region contained 16 nt haplotypes, whereas the S2 1058/1060 region contained only 4 nt haplotypes and showed lower diversity. Tissue-derived sequences from the FIP-affected cheetah were assigned to defined haplotypes within the population-level haplotype framework. Together, these data show that FCoV was maintained for multiple years in this closed cheetah cohort and that faecal surveillance combined with targeted sequencing can track temporal changes in the dominant FCoV sequences detected in captive non-domestic felids.
Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that poses a growing global threat, causing severe disease in humans and livestock, including hepatitis, haemorrhage and encephalitis. Documented cases of vertical transmission in humans and livestock highlight risks during pregnancy. Although RVFV can directly infect the placenta, how it enters trophoblast cells remains unclear. Here, we investigated the role of low-density lipoprotein receptor-related protein 1 (LRP1), a known RVFV entry factor, in human trophoblast infection. Using JEG-3, JAR and HTR-8/SVneo trophoblast cell lines, we demonstrate that RVFV replicates to high titres and LRP1 expression varies across cell types. Competitive inhibition assays using the high-affinity LRP1 ligand murine RAP domain 3 and recombinant LRP1 fragments (cluster proteins) indicate that RVFV requires LRP1 for efficient trophoblast infection. Together, these findings implicate LRP1 as a determinant of RVFV infection at the maternal-fetal interface.
Thogotoviruses and quaranjaviruses are arthropod-borne orthomyxoviruses that circulate widely in wildlife and domestic animals and include several zoonotic members. Despite their close phylogenetic relationship to influenza viruses, the mechanisms underlying their replication remain poorly understood, and the host factors mediating viral entry are unknown. Here, we performed genome-wide loss-of-function CRISPR-Cas9 screens using replication-competent recombinant vesicular stomatitis viruses expressing thogoto- and quaranjavirus glycoproteins to identify cellular determinants of viral entry. These screens identified the glycosphingolipid (GSL) biosynthesis pathway as a key regulator of viral entry, with the upstream enzyme uridine diphosphate (UDP)-glucose ceramide glucosyltransferase (UGCG) emerging as a central host entry factor. Pharmacological inhibition of UGCG impaired thogoto- and quaranjavirus entry. The importance of GSL biosynthesis for thogotovirus replication was further validated using multiple thogotovirus isolates. Together, our findings establish GSLs as critical host entry factors for non-influenza orthomyxoviruses and identify UGCG as a potential target for antiviral intervention.
Acinetobacter baumannii has raised significant concern due to its high rates of morbidity and mortality, coupled with its increasing resistance to multiple antibiotics including carbapenems. In this current study, two new lytic phages Srynta and Nolivar were isolated from sewage water, characterized in vitro and their therapeutic potential was assessed using a mouse model of pneumonia. In vitro results showed that both phages show efficient adsorption to the propagating host, have good lytic activity, short latent period and high burst size. Sequence analysis showed that Srynta and Nolivar phages have linear dsDNA genomes of 45,218 and 41,230 base pairs with G+C content of 38 mol% and 37.9 mol% and contain 88 and 83 coding sequences, respectively. Transmission electron microscopy showed the icosahedral capsid and contractile tail of the phages and genomic analysis predicted that both phages have myovirus morphology and belong to the class Caudoviricetes. Genomic analysis suggested the therapeutic safety of these phages, as they do not have bacterial virulence factors, antimicrobial resistance genes or lysogenic genes, suggesting that they are potential candidates for phage therapy. The in vivo study demonstrated a high survival rate in the phage cocktail treatment group compared to the control group. Our findings support the potential application of phage therapy as an alternative therapeutic treatment against lung infections caused by carbapenem-resistant A. baumannii.
Herpes simplex virus type 1 (HSV-1) is the prototype of the α -herpesvirus family. To propagate within the host organism, HSV-1 has evolved several strategies to subvert the host immune response. Due to the pivotal role of dendritic cells (DCs), bridging innate and adaptive immunity and activating naïve T cells, they represent an attractive target for HSV-1-triggered immune regulation. Here, we report a novel HSV-1-mediated mechanism of signal transducer and activator of transcription 3 (STAT3) dysregulation in human monocyte-derived mature DCs (mDCs). Due to STAT3’s antiviral activity, STAT3 was shown to be modulated by viruses to efficiently replicate in several different cell types, however, not in DCs. We show that HSV-1 infection of mDCs leads to diminished total STAT3 expression and STAT3 phosphorylation (pSTAT3), as well as downregulation of STAT3 mRNA very early upon infection. Protein downregulation of STAT3 could be verified by label-free mass spectrometric analysis of HSV-1- and HSV-2-infected mDCs. We found that two viral proteins, the tegument protein virion host shutoff (vhs) and the immediate-early protein ICP27, contribute to STAT3 protein downregulation upon HSV-1 infection. However, HSV-1 Δvhs and ΔICP27 deletion strains still inhibited pSTAT3 in infected mDCs, suggesting that different viral proteins impair STAT3 phosphorylation and total STAT3. Moreover, STAT3 protein levels were degraded in a proteasome-dependent but apoptosis-independent manner. Taken together, we demonstrate that HSV-1 downregulates STAT3 in HSV-1-infected human mDCs at the transcript, protein and phosphorylation levels. Downregulation of STAT3 protein depended on vhs and the proteasome, very likely to shape DC function for immune evasion and modulation.
Formic acid treatment is widely used in diagnostic neuropathology to reduce the infectivity of prion-containing tissues; however, quantitative in vivo evidence supporting its effectiveness under routine laboratory conditions remains limited. Here, we assessed the impact of formalin fixation and formic acid treatment on the infectivity of type 1 sporadic Creutzfeldt-Jakob disease (sCJD) and variant CJD (vCJD) prions using highly sensitive transgenic mouse models overexpressing human-PrP M129 (Hu-Tg340) or bovine PrP (Bo-Tg110). Brain tissues were processed under conditions closely resembling standard histopathological workflows and tested as untreated, formalin-fixed or formalin-plus-formic-acid-treated inocula. Untreated samples produced short incubation times and full attack rates, whereas formalin fixation caused only a modest prolongation of incubation times. In contrast, formic acid treatment markedly extended incubation times and reduced attack rates for sCJD. Based on incubation-time interpolation, the estimated infectivity reductions were on the order of 4.4 log₁₀ for vCJD and 5 log₁₀ for sCJD. These estimates indicate a major reduction in infectious titre under the conditions tested, although residual infectivity was still detected. The findings support formic acid treatment as an important risk-reduction step in routine neuropathology workflows for the two prion strains examined.
Phosphatidylserine (PS) receptors facilitate entry of diverse enveloped viruses and have been implicated in enhancing coronavirus attachment, including for SARS-CoV-2, yet their precise contribution remains unclear. We examined this pathway using EjCoV-3, a recently identified bat merbecovirus. Exogenous PS, but not phosphatidylcholine, reduced viral infectivity in a concentration-dependent manner, suggesting competitive inhibition of receptor binding by virion-associated PS. T-cell immunoglobulin and mucin domain 1 (TIM-1) knockout (KO) in Vero/TMPRSS2 (transmembrane protease, serine 2) cells markedly reduced early viral infection. TIM-1 KO cells also showed markedly reduced virion attachment, indicating that TIM-1 acts as a PS-dependent attachment factor. Growth kinetics further revealed that TIM-1 contributes to efficient EjCoV-3 replication under conditions without exogenous protease and accelerates early viral growth in the presence of exogenous protease. A549 cells and A549/hACE2 cells are not susceptible to EjCoV-3 infection in the absence of exogenous proteases, and sustained viral replication was observed only when TIM-1 and TMPRSS2 were co-expressed, indicating that the two proteins synergistically promote infection. However, the expression of either factor alone was insufficient to sustain sustained viral replication. In contrast, in A549/EnACE2 cells, an EjCoV-3-susceptible cell line expressing a more compatible bat angiotensin-converting enzyme 2 (ACE2) orthologue, even in the absence of exogenous proteases, the expression of TMPRSS2 enhanced infection, whereas the expression of TIM-1 had no effect. These findings indicate that in environments where only low-affinity receptors are present, TIM-1 promotes EjCoV-3 infection primarily through PS-mediated attachment and functions in concert with TMPRSS2-dependent spike activation. This cooperative mechanism may reduce receptor barriers and enhance the potential for cross-species transmission of bat-derived coronaviruses, thereby contributing to their emergence as zoonotic pathogens.
Human cytomegalovirus (HCMV) infection is widespread within the global human population and is a major cause of human morbidity and mortality. As yet, there is no widely available anti-HCMV vaccine strategy. Therefore, treatment of HCMV disease relies on anti-HCMV drugs, principally the nucleoside analogue pro-drug ganciclovir (GCV). While it is over 40 years since the discovery of GCV as an anti-HCMV compound, the use of GCV in humans is still developing, for example in the treatment of congenital HCMV infection. Here, we provide an overview of often overlooked virological laboratory studies underpinning GCV development. We highlight long-standing, but rarely examined, data on adverse effects that is the basis for caution in using GCV during pregnancy or in children. This includes discussion of recent, contrasting data, suggesting that GCV can be further developed to treat congenital HCMV infection. To better understand how adverse effects may come about, we also review the viral and cellular factors required for GCV function in HCMV-infected and uninfected cells. This leads to a discussion of how adverse effects of GCV may or may not be avoided and future directions involving the use of GCV. Overall, these topics highlight how much molecular information remains to be understood about one of the oldest and most widely used anti-HCMV drugs in humans.
Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that poses significant economic threats to the swine industry and carries potential zoonotic risk. However, the interactions between PDCoV and host innate immunity, particularly those involving interferon-stimulated genes (ISGs), remain poorly understood. Porcine myxovirus resistance protein 1 (pMx1), a well-characterized ISG with broad-spectrum antiviral activity, has not been investigated in the context of PDCoV infection. This study demonstrates that PDCoV infection upregulates endogenous pMx1 expression in porcine intestinal epithelial cells (IPEC-J2 and IPI-2I) through a type I interferon alpha-dependent pathway. Functional analyses further revealed that pMx1 exerts potent antiviral activity against PDCoV. Mechanistically, we identified a direct interaction between the pMx1 protein and the PDCoV nucleocapsid (N) protein and found the GTPase domain of pMx1 as the critical binding region. This finding was strongly supported by a predicted interaction complex structural model generated using AlphaFold 3, and the GTPase domain was shown to be indispensable for the antiviral function of pMx1. Furthermore, we identified a critical synergistic interaction between the PDCoV N protein and the viral RNA-dependent RNA polymerase - nonstructural protein 12, a core component of the replication-transcription complex. Co-immunoprecipitation assays demonstrated that pMx1 effectively disrupts this interaction, suggesting that pMx1 may inhibit viral genome replication by impairing the formation or stability of the viral replication complex. Collectively, our study elucidates the mechanism by which the pMx1 protein, as a host antiviral factor, inhibits PDCoV infection. These findings provide novel insights into the innate immune defence against coronaviruses and highlight the pMx1 gene as a promising host-derived antiviral factor with potential applications in antiviral therapeutics and genetic improvement strategies for PDCoV control.
Geminiviruses are among the most threatening emerging insect-borne viruses and are responsible for serious outbreaks worldwide. Climate change (i.e. higher temperatures) could further exacerbate their impact on crops, highlighting the need for new diagnostic approaches to manage potentially dangerous situations. vector-enabled metagenomics (VEM) exploits the natural ability of highly mobile insects to accumulate viruses acquired from plants over time and space within an ecosystem; this approach is effective for monitoring the presence of new invasive or indigenous viruses in large areas. Geminiviruses have circular ssDNA genomes that can be readily targeted by rolling circle amplification (RCA). The combination of RCA and VEM largely increases the chances of detecting geminiviruses. This approach enabled us to identify the becurtovirus beet curly top Iran virus (BCTIV, Becurtovirus betae) in insects collected in Europe. BCTIV is a major pathogen of sugar beet but can also infect plants of other families; it is transmitted by cicadellids and has so far been detected only in Iran and Anatolia (Turkey). We also show that two cucurbit species, watermelon (Citrullus lanatus) and zucchini (Cucurbita pepo) are both natural and experimental hosts for BCTIV.
Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the USA. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication, facilitating high-throughput 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 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 titres in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against oseltamivir in vitro. Collectively, these results establish the H5N1 TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis and for use in serological and antiviral drug screens.
Antimicrobial resistance poses a critical global health threat, with enterococci among the leading contributors due to their intrinsic and acquired resistance to antibiotics. Clinically relevant species, including Enterococcus faecalis and Enterococcus faecium as well as the emerging poultry pathogen Enterococcus cecorum, highlight the need for alternative therapeutics across human and agricultural settings. Bacteriophages and their derived enzymes, particularly endolysins, offer promising antibacterial strategies but challenges such as phage resistance and limited lysin diversity hinder their application. In this study, we performed a large-scale analysis of prophage-encoded endolysins across these three enterococcal opportunistic pathogens, characterizing over 48,000 sequences. We identified 33 distinct domain architectures combining diverse catalytic and cell wall-binding domains (CBDs) including novel putative CBDs. These findings expand the known diversity of enterococcal lysins and provide a comprehensive resource for the rational design of stable, recombinant 'enzybiotics' to combat multidrug-resistant enterococcal infections.
Viral central nervous system (CNS) infections in adults frequently remain unresolved after routine diagnostic testing. We applied probe-based viral metagenomic next-generation sequencing (vmNGS) to cerebrospinal fluid samples from adults with suspected CNS infection and negative conventional diagnostics in a retrospective multicentre study conducted in Spain between 2022 and 2023. Among 40 idiopathic cases, vmNGS detected viral sequences in 6 patients without evidence of coinfection: human pegivirus (HPgV, n=3), Toscana virus (TOSV, n=1), herpes simplex virus type 1 (HSV-1, n=1) and varicella-zoster virus (VZV, n=1). Two HPgV-positive patients were transplant recipients, with neurological disease occurring more than 2 years after transplantation, compatible with possible long-term viral persistence in immunocompromised hosts. TOSV genotype B was identified in a patient residing in central Spain, supporting consideration of TOSV in selected cases of unexplained aseptic meningitis during the vector season, including outside traditionally recognized Mediterranean coastal regions. Furthermore, the failure of syndromic panel testing to detect HSV-1 and VZV highlights the need for complementary diagnostic strategies when clinical suspicion remains high. Overall, the detection of unexpected viral sequences, together with missed clinically actionable infections, supports the use of complementary molecular testing in selected cases of unexplained CNS syndromes when routine diagnostics are negative. These findings highlight the added diagnostic value of vmNGS and provide sequence-level data for future studies of viral diversity and molecular epidemiology in neurological disease.
