Development of a strand-specific RT-qPCR assay for detecting and quantifying Borna disease virus RNAs in infected cells

BoDV-1 (Borna disease virus 1) is a negative-sense RNA virus that establishes persistent infection in the mammalian central nervous system and has recently been associated with fatal encephalitis in humans. During replication, BoDV-1 produces three types of RNA: genomic RNA, antigenomic RNA, and mRNA. However, conventional RT-qPCR cannot distinguish these RNA species. This study aimed to develop a strand-specific RT-qPCR assay capable of detecting and quantifying each viral RNA individually. Standard RNAs were prepared using the 5′- and 3′-terminal 1,200-nucleotide regions of the viral genome, the full-length N mRNA, and the 3′ region of L mRNA. Strand specificity was achieved by using tagged RT primers and increasing the reverse transcription temperature to 57°C. Vero cells infected with BoDV-1 were monitored for one month. The assay specifically detected genomic and antigenomic RNAs, whereas N mRNA could not be completely distinguished from antigenomic RNA because of their highly similar sequences. Therefore, N mRNA levels were estimated by subtracting the antigenomic RNA signal. Viral RNA levels increased during infection, while the antigenome-to-genome ratio remained constant, suggesting stable viral replication. In contrast, the mRNA-to-genome ratio gradually decreased, indicating transcriptional suppression during persistent infection. Limitations of the study include the use of synthetic standard RNAs, experiments performed only in Vero cells, and the analysis of only N mRNA among the viral transcripts.
(AI)

The development of biologically contained Sudan virus as an alternative and safe tool for fundamental filovirus research

Filoviruses, including Ebola virus, Sudan virus, and Marburg virus, cause severe diseases with high fatality rates and require BSL-4 containment, which significantly restricts research. Although several BSL-2 surrogate systems have been developed, they often fail to fully reproduce viral replication and the complete life cycle. VP30 is a transcriptional activator essential for initiating viral mRNA synthesis in filoviruses and is functionally exchangeable among related viruses. VP30-deficient filovirus systems have been reported; however, a VP30-deficient Sudan virus (SUDV) system has not previously been established. In the reported study, a SUDV genome in which the VP30 gene was replaced with GFP was transfected into Vero cells expressing SUDV VP30. GFP fluorescence confirmed successful virus rescue. The rescued virus was passaged, and a VP30-deficient SUDV was isolated. Sequencing confirmed no unintended mutations, and no VP30 reversion was observed after 10 passages. Growth analysis showed replication only in VP30-expressing cells, while no replication occurred in VP30-negative cells. VP30 proteins from EBOV, RESTV, and SUDV showed high functional compatibility, whereas MARV VP30 showed little complementation. This system provides a platform that may reduce reliance on BSL-4 containment and facilitate safer studies of SUDV biology, antiviral development, and filovirus evolution.
(AI)

Development and Immunogenic Evaluation of a Recombinant Vesicular Stomatitis Virus Expressing Nipah Virus F and G Glycoproteins

Nipah virus is a highly pathogenic zoonotic virus that requires Biosafety Level 4 (BSL-4) containment. Because effective vaccines and antiviral drugs remain limited, understanding its infection mechanisms is essential. Viral entry is mainly mediated by the attachment glycoprotein (G) and fusion protein (F).
In this study, a replication-competent chimeric vesicular stomatitis virus (VSV) expressing Nipah virus G and F proteins was generated to enable the analysis of viral entry and neutralizing antibody responses outside BSL-4 facilities. Successful recovery of the recombinant virus was confirmed by GFP expression. Expression and incorporation of Nipah virus G and F proteins into viral particles were verified by Western blotting, immunofluorescence assays, and immunoelectron microscopy.
Although the recombinant virus showed lower growth kinetics than parental VSV, its infectivity depended on the expression of Nipah virus receptors, indicating that it utilized a receptor-dependent entry mechanism similar to that of wild-type Nipah virus. Furthermore, the recombinant virus was neutralized by anti-Nipah antibodies and induced neutralizing antibodies in hamsters. These results demonstrate that the chimeric VSV is a useful tool for studying Nipah virus biology under lower biosafety conditions.
(AI)

Application of the CPER reverse genetics system for genetic engineering of rabies virus

Rabies virus is a mononegavirus, and plasmid-based reverse genetics systems have been widely used for its manipulation. Reporter rabies viruses generated by reverse genetics are useful for visualization of neuronal circuits. However, construction of full-length genome plasmids requires considerable time and cost, and introduction of artificial mutations is difficult. Recently, CPER has been established as a novel reverse genetics method, although its application to mononegaviruses remains limited. In this study, a CPER-based reverse genetics system for rabies virus was established. Infectious viruses rescued by CPER showed growth kinetics similar to those of parental strain. Mutant viruses containing point mutations, reporter viruses expressing fluorescent proteins, and chimeric viruses with exchanged G proteins were successfully generated. Full-genome sequencing revealed relatively frequent mutations in CPER-derived viruses. In addition, some strains could not be rescued, and PCR amplification introduced mutations into the viral genome.
(AI)

RIG-I Activation by a Designer Short RNA Ligand Protects Human Immune Cells against Dengue Virus Infection without Causing Cytotoxicity

This study characterized a designer short hairpin RNA ligand, 3p10LG9, as a potent activator of the cytoplasmic RNA sensor RIG-I and evaluated its antiviral efficacy against dengue virus (DENV) infection in human cell lines and primary human skin antigen-presenting cells. The authors engineered structural modifications into a 5′-triphosphorylated double-stranded RNA hairpin and demonstrated that insertion of an additional guanine nucleotide created a kinked RNA structure that significantly enhanced type I interferon (IFN) induction compared with the parental construct 3p10L. Transfection of 3p10LG9 into U937-DC-SIGN monocytic cells and A549 epithelial cells induced strong IFN-stimulated signaling and inhibited DENV-2 infection in a dose-dependent manner. Mechanistic analyses showed that antiviral activity was highly dependent on RIG-I and downstream type I IFN signaling. RIG-I overexpression enhanced IFN activation, whereas MDA5 deficiency had minimal effect, confirming selective activation of the RIG-I pathway. In contrast, CRISPR-mediated RIG-I knockout abolished interferon-stimulated gene induction and antiviral activity, while IFNAR blockade reversed DENV inhibition. The study further demonstrated efficient uptake and innate immune activation of 3p10LG9 in ex vivo primary human skin dendritic cell subsets, including CD11c⁺ dermal dendritic cells, CD14⁺ dermal dendritic cells, and Langerhans cells. Transcriptomic analyses revealed robust upregulation of interferon-stimulated and antiviral response genes following 3p10LG9 treatment. Compared with poly(I·C), 3p10LG9 induced stronger antiviral transcriptional responses in several skin antigen-presenting cell populations. Functional assays showed that prophylactic treatment efficiently suppressed DENV replication with substantially lower EC50 values than the parental construct, whereas therapeutic administration after infection produced more modest effects. Overall, the study demonstrated that optimized minimal RIG-I agonist RNAs can induce potent antiviral innate immune responses with limited cytotoxicity, supporting their potential as prophylactic or therapeutic antiviral agents against dengue virus and related viral infections.
(TMR)

Amplification- free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs

This study developed an amplification-free CRISPR-Cas13 assay for detecting hantavirus and influenza A virus RNA. Cas13 was used to directly detect viral RNA without PCR amplification. Multiple crRNAs were designed to improve detection sensitivity. Both hantaviruses and influenza A virus were successfully detected. In hantaviruses, the use of multiple crRNAs increased detection sensitivity, whereas no significant improvement was observed for influenza A virus. The assay could detect viral RNA from infected cultured tissues and lung samples without amplification. In clinical samples, the positivity rate showed 85% agreement with RT-qPCR results, suggesting the potential utility of this method for rapid viral diagnostics and surveillance.
(AI)

Activation of the Beta Interferon Promoter by Unnatural Sendai Virus Infection Requires RIG-I and Is Inhibited by Viral C Proteins

This study investigated how unnatural infections with Sendai virus activate the host beta interferon (IFN-β) response and examined the respective roles of the viral C and V proteins in suppressing innate antiviral signaling. Using two engineered Sendai virus infection systems, one involving defective interfering (DI-H4) genomes that overproduce 5′-triphosphorylated trailer RNAs and another generating intracellular GFP-derived double-stranded RNA (dsRNA), the authors demonstrated that activation of the IFN-β promoter in mouse embryonic fibroblasts depended predominantly on the cytoplasmic RNA sensor RIG-I rather than mda-5. The study showed that both 5′-triphosphorylated single-stranded RNAs and dsRNAs acted as potent pathogen-associated molecular patterns capable of inducing IFN-β signaling through the RIG-I pathway. The authors further established that the Sendai virus C protein was the principal antagonist of RIG-I-mediated interferon induction, whereas the V protein played a comparatively limited role. Overexpression of the C protein strongly inhibited IFN-β activation induced by defective interfering virus infection, dsRNA formation, transfected poly(I-C), and synthetic 5′-triphosphorylated RNAs, with inhibitory activity comparable to dominant-negative RIG-I constructs and influenza virus NS1 protein. In contrast, the V protein only partially suppressed signaling and was ineffective against several dsRNA-mediated responses. Functional analyses using recombinant viruses lacking either the C or V protein confirmed that loss of the C protein resulted in strong enhancement of IFN-β activation and synergistic stimulation of antiviral signaling following RNA transfection, whereas V-deficient virus largely retained wild-type suppressive activity. The study also identified the C-terminal C24–204/Y1 interaction domain of the C protein as the major determinant responsible for inhibition of RIG-I-dependent signaling. Overall, this work demonstrated that Sendai virus employs the C protein as a major innate immune evasion factor to counteract RIG-I-mediated antiviral responses triggered by viral pppRNAs and dsRNAs, thereby providing important mechanistic insight into paramyxovirus interferon antagonism and host–virus interactions.
(TMR)