Interferon and TLR genes, but not endogenous bornavirus-like elements, limit BoDV1 replication after intracerebral infection

Borna disease virus 1 (BoDV1) is a neurotropic RNA virus, but the host factors limiting its replication in the brain are not fully understood. Endogenous bornavirus-like elements (EBLNs) have been proposed to suppress BoDV1 through piRNA-mediated mechanisms. This study aimed to evaluate the relative roles of interferon signaling, Toll-like receptor pathways, and EBLN-derived piRNAs in controlling BoDV1 replication in vivo. Neonatal mice were intracerebrally infected with a GFP-expressing recombinant BoDV1. Viral replication was analyzed in wild-type mice and knockout mice lacking the interferon-γ receptor (Ifngr1), Toll-like receptor 7 (Tlr7), or all piRNA-producing EBLNs. Viral RNA levels and infected brain cells were assessed by RT-qPCR and histological analyses. BoDV1 replication was significantly increased in Ifngr1- and Tlr7-deficient mice, indicating that interferon-γ signaling and TLR7-dependent innate immunity restrict viral growth in the brain. In contrast, deletion of EBLN-derived piRNAs had no effect on viral replication. These results demonstrate that classical immune pathways, rather than endogenous viral elements, are the primary factors limiting BoDV1 replication after intracerebral infection.
(MN)

A novel accessory gene product of tick-borne Dhori-Orthomyxovirus, encoded by overlooked spliced transcripts of RNA segment 6

This study identifies and characterizes a previously unrecognized accessory gene product encoded by RNA segment 6 of Dhori virus (DHOV), a tick-borne orthomyxovirus within the genus Thogotovirus. Contrary to the long-held assumption that Dhori-like viruses do not use RNA splicing, the authors demonstrate that DHOV segment 6 undergoes splicing in both mammalian and tick cells. This splicing event generates a novel protein, termed M2-248, which consists of the N-terminal portion of the canonical matrix protein fused to a unique C-terminal sequence translated from a −1 shifted reading frame. The splicing sites and the M2-specific sequence are conserved among Dhori-like viruses. M2-248 expression was confirmed by RT-PCR, mass spectrometry, and western blotting, and the protein was shown to be incorporated into released virions and to interact with the matrix protein. Using reverse genetics, the authors generated recombinant DHOV lacking M2 expression and demonstrated that loss of M2 does not impair viral replication, assembly, or particle morphology in cell culture. However, M2-deficient virus exhibited markedly reduced virulence in mice, with improved survival despite only modest reductions in viral replication and inflammatory cytokine induction. Functional analyses showed that, unlike the related Thogotovirus ML protein, M2-248 does not act as a type I interferon antagonist. These findings reveal an unexpected coding strategy in Dhori-like orthomyxoviruses and identify M2-248 as a novel virulence factor acting through an interferon-independent mechanism.
(TMR)

Comparison of the biological properties of bat-derived filovirus envelope glycoproteins

Several novel filoviruses have recently been identified in bats, including Lloviu, Bombali, Mengla, and Dehong viruses, but their biological properties and zoonotic potential remain unclear. This study aimed to compare the functional characteristics of envelope glycoproteins (GPs) from bat-derived filoviruses with those of pathogenic filoviruses such as Ebola and Marburg viruses. Using virus-like particles and vesicular stomatitis virus pseudotypes, the authors examined GP-mediated particle formation, antigenicity, cell entry mechanisms, and cell tropism. All bat-derived filovirus GPs supported the formation of filamentous particles and mediated cellular entry via known filovirus attachment factors, including TIM-1 and C-type lectins, although entry efficiency was generally lower than that of Ebola virus GP. Antigenic analyses showed limited cross-reactivity between bat-derived filovirus GPs and antibodies against Ebola or Marburg viruses, though partial neutralization was observed for some GPs. Infection assays revealed that bat-derived filovirus GPs could mediate entry into a broad range of mammalian cells, including human cells, while displaying virus- and host-specific differences.
(MN)

Experimental challenge of African green monkeys with contemporary Hendra virus isolates produces divergent clinical disease

Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes severe disease in humans and animals. While all confirmed human cases have been associated with genotype 1 (HeV-g1), a genetically distinct genotype 2 (HeV-g2) has recently been identified in bats and horses, raising questions about its pathogenic potential. This study aimed to compare the disease severity caused by contemporary HeV-g1 and HeV-g2 isolates using the African green monkey (AGM) model. AGMs were experimentally infected via intranasal and intratracheal routes with either HeV-g1 or HeV-g2 and monitored for clinical signs, survival, viral replication, pathology, and immune responses. HeV-g1 infection resulted in uniformly severe and lethal disease, characterized by acute respiratory distress, systemic viral dissemination, and extensive tissue pathology. In contrast, HeV-g2 infection caused markedly milder disease, with most animals surviving and showing limited clinical signs, lower viral loads, and minimal pathological changes. Importantly, HeV-g2–infected animals mounted robust neutralizing antibody responses without detectable infectious virus in tissues. These findings demonstrate that contemporary HeV isolates can cause divergent clinical outcomes and indicate that HeV-g2 has reduced pathogenicity in primates compared with HeV-g1, providing critical insights for risk assessment and preparedness strategies.
(MN)

Borna Disease Virus Infection Perturbs Energy Metabolites and Amino Acids in Cultured Human Oligodendroglia Cells

Borna disease virus (BDV) is an enveloped neurotropic RNA virus. It replicates in the nucleus of host cell instead of the cytoplasm as in the case of most viruses. This unusual mechanisms of protein synthesis has been associated with alterations of metabolism in the host cell without causing a cytolytic infection. To investigate how BDV alters host cell metabolism, human oligodendroglia cells were infected with human BDV Hu-H1 strain. Metabolic profiling of these cells was done using Nuclear Magnetic Resonance spectroscopy and direct comparison was done with uninfected control cells. Multivariate statistical analysis showed that twenty-three metabolites were significantly altered. Changes in key metabolites such as pyruvate, glucose, succinate and acetyl CoA suggested decrease in glycolysis and increase in citric acid cycle activity, mitochondrial respiration and lipid biosynthesis. In addition there were alterations in several amino acids accounting for disruptions in neurotransmitter pathways that are commonly observed in BDV infections. 
(SWM)

Development of a Double-Antigen Sandwich ELISA for Oz Virus and a Seroepidemiological Survey in Wild Boars in Miyazaki, Japan

This study reports the development and use of a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS ELISA) to study Oz virus infection in wild boars in Miyazaki Prefecture, Japan. Recombinant Oz virus nucleoprotein was produced in Escherichia coli and used as both the capture and detection antigen in the assay. The performance of the DAgS ELISA was assessed by comparison with an 80% plaque reduction neutralization test, and the cutoff value was determined using receiver operating characteristic analysis. In addition, viral RNA was examined by quantitative RT-PCR, and a partial glycoprotein gene sequence from a positive sample was analyzed phylogenetically. The DAgS ELISA showed a sensitivity of 72.2%, a specificity of 88.2%, and an overall agreement of 79.0% with the neutralization test, supporting its use for large-scale serological surveys. When applied to 1045 wild boar serum samples collected between 2022 and 2025, the assay identified an overall seroprevalence of 33.5%. Seropositivity did not differ by sex, age, or region, but clear seasonal differences were observed, with the highest prevalence in summer. Oz virus RNA was detected in only one sample (0.09%), and sequence analysis showed high similarity to a previously reported tick-derived strain. These results suggest that wild boars are frequently exposed to Oz virus, but active infection appears to be uncommon.
(TMR)

Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein

Rabies virus (RABV) phosphoprotein (P) is a multifunctional viral protein that plays essential roles in viral replication, transcription, and immune evasion. Although encoded by a single gene, P is expressed as multiple isoforms with distinct cellular functions, particularly the full-length P1 and the N-terminally truncated P3. The molecular basis for this functional diversification has remained unclear. This study investigates how conformational dynamics, RNA binding, and liquid–liquid phase separation (LLPS) regulate the multifunctionality of 　P. They show that P1 and P3 adopt distinct conformational states. P1 predominantly exhibits a compact intramolecular structure, whereas P3 displays greater conformational flexibility. Both isoforms are capable of undergoing LLPS in vitro, indicating that phase separation alone does not account for their functional differences. Instead, RNA binding emerges as a key determinant of isoform-specific activity. P3, but not P1, exhibits strong RNA-binding ability. RNA binding by P3 is shown to be critical for its selective localization to membrane-less organelles such as nucleoli and PML nuclear bodies, as well as for interactions with microtubules and immune-related cellular structures. Disruption of RNA binding impairs these P3-specific functions without abolishing phase separation. 
(MN)

Evaluation of Oz virus susceptibility in several vertebrate cell lines and its application in efficient isolation from field-collected ticks

This study evaluates the susceptibility of multiple vertebrate cell lines to Oz virus (OZV) and demonstrates an improved approach for isolating the virus from field-collected ticks. OZV, a tick-borne thogotovirus associated with the first fatal human case in Japan, was assessed for replication efficiency in eight vertebrate-derived cell lines. Among these, BHK-21, Vero, HuH-7, and Huh7.5.1–8 cells supported high viral titers (>107 PFU/mL), with BHK-21 identified as the most suitable medium for virus isolation. Using this optimized system, the authors successfully isolated a new OZV strain, designated 23AT5, from Amblyomma testudinarium ticks collected in the same area where the original strain EH8 was identified. Genomic and phylogenetic analyses revealed that the new isolate is highly conserved and closely related (99.75-100% similarity at nucleotide level) to the original strain EH8, indicating long-term stability of the local transmission cycle. Two to three nucleotide polymorphisms have been identified in all segments and formed a robust clade with strain EH8 during phylogenetic analyses. Overall, the study provides important methodological and epidemiological insights into OZV maintenance and surveillance.
(TMR)

Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a

Ebola virus causes a fatal hemorrhagic fever, which is zoonotic and endemic in some developing countries. Several deadly outbreaks have been recorded in the past, and timely diagnosis is always critical. Immunoassays that have been developed lack sufficient sensitivity and although PCR-based systems are the current gold standard, their use is limited by the need for high expertise, expensive reagents and sophisticated instrumentation. This creates need for a simple, cost effective and highly sensitive diagnostic method. In this study a technology based on the CRISPR-Cas13 enzyme is employed. The enzyme is bound to a guide RNA sequence that is complementary to the target viral RNA. When the target RNA binds, the enzyme is activated and begins to cleave the RNA molecules. A fluorescent RNA probe tagged with a fluorophore and a quencher is included, when the probe is cut the fluorophore is released and emits a detectable signal. The intensity of the signal is measured and quantified. Key advantages of this method are that it does not require RNA amplification and provides high sensitivity and specificity. In addition, the technology uses a microfluidic chip to automate reagent mixing and incubation, reducing the total detection time about five minutes. 
(SWM)

Negative-sense RNA virus nucleocapsid as a versatile platform for gene delivery, vaccine development, and antiviral screening

Negative-sense RNA viruses replicate their genomes as nucleocapsids composed of viral RNA tightly associated with nucleoproteins and polymerase complexes. In this study, the authors examined whether purified nucleocapsids can function as autonomous units capable of initiating viral gene expression and replication when directly delivered into host cells. Using vesicular stomatitis virus (VSV) as a model, purified NCs were isolated from infected cells and introduced directly into host cells by lipid-mediated transfection. Following transfection, robust expression of viral reporter genes and viral proteins was observed, confirming that the NCs retained transcriptional activity. Viral gene expression occurred independently of viral envelope proteins and cellular entry receptors. Even in cells with reduced expression of the VSV entry receptor, NC-mediated gene expression was efficiently induced, demonstrating that the NCs bypass receptor-dependent viral entry. NCs derived from wild-type VSV supported complete viral replication and led to the production of infectious progeny virus. In contrast, NCs derived from glycoprotein-deficient VSV enabled viral gene expression and genome replication but did not generate infectious particles, indicating a replication-competent yet non-infectious system. Additionally, nucleocapsid-driven viral replication was inhibited by antiviral compounds, supporting the applicability of this platform for post-entry antiviral drug screening. 
(MN)

Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines

Laboratory cell lines are essential for viral studies, providing also a cost effective alternative to animal models for propagating and characterizing viruses. Human parainfluenza viruses (HPIVs) are major causes of respiratory illnesses in pediatrics and geriatrics, making use of cells lines in vaccine development vital. However serial passage in standard cell lines often forces viruses to adapt to artificial environments. This selective pressure induces mutations that alter biological processes, yielding findings that do not accurately represent clinical behavior. While this phenomenon is well-documented in HPIV3, data for other HPIV types remain scarce. This study passaged HPIV1–4 five times in 3 cell lines; primary human bronchial/tracheal epithelial cells grown in an air-liquid interface (HBTEC-ALI), Vero cells and Vero cells expressing TMPRSS2. Findings revealed that the HBTEC-ALI, which mimic the human airway, maintained better genomic integrity with minimal mutations. In contrast, rapid and significant mutations, especially within the Hemagglutinin-neuraminidase (HN) and Large (L) protein genes were induced in Vero-based lines. Although HBTEC-ALI is the superior model, further validation requires more passages, comparison with other human respiratory cell lines and functional assays to determine how these mutations affect viral properties such as replication and drug resistance.
(SWM)