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)

Viral suppression of the interferon system

This review comprehensively examined the diverse strategies employed by viruses to evade and suppress the host type I interferon (IFN) system, a central component of innate antiviral immunity. The authors synthesized mechanistic evidence across a broad range of RNA and DNA viruses to illustrate how viral proteins interfere with interferon induction, signaling, and downstream antiviral effector functions at multiple levels of the host defense cascade. They described how viruses such as influenza virus, paramyxoviruses, hepatitis C virus, rabies virus, hantaviruses, and herpesviruses specifically target key innate immune sensors and signaling intermediates, including RIG-I, MDA5, IPS-1/MAVS, TBK1, IRF3, and NF-κB, thereby preventing IFN production. The review further highlighted viral antagonism of interferon-activated JAK–STAT signaling through degradation, sequestration, or inactivation of STAT proteins, as exemplified by the V and C proteins of paramyxoviruses, rabies virus phosphoprotein P, and Ebola virus VP24. In addition, the authors discussed how many viruses directly inhibit interferon-stimulated antiviral effectors such as PKR, RNase L, and the 2′–5′ oligoadenylate synthetase pathway through dsRNA sequestration, pseudosubstrate mimicry, translational shutoff, or manipulation of host regulatory factors. Importantly, the review emphasized that viruses have evolved both highly specialized interferon antagonists and multifunctional structural or replication-associated proteins with secondary immune evasion roles, reflecting strong evolutionary pressure to overcome host innate defenses. Overall, the article provides a broad conceptual framework for understanding viral interferon escape mechanisms and highlights how this knowledge can be leveraged for the development of attenuated vaccines and interferon-sensitive oncolytic virotherapies.
(TMR)

Evolutionary analysis of V protein pseudogenization in an RNA editing-deficient paramyxovirus

This study investigated the evolutionary consequences of RNA editing loss in human parainfluenza virus type 1 (HPIV-1) by analyzing the pseudogenization of the V protein–coding region within the P gene. The authors performed a comprehensive comparative genomic analysis using 240 full-length HPIV-1 P gene sequences and defined a pseudo-V reading frame by virtually inserting a nucleotide at the conserved RNA editing site, using Sendai virus as a reference. They observed a markedly elevated and non-random accumulation of stop codons within the 253-amino-acid pseudo-V region, with highly conserved positions across strains, indicating strong evolutionary fixation. Comparative analyses across other viral genes and with Sendai virus demonstrated that this enrichment was specific to the HPIV-1 P gene. Furthermore, in silico evolutionary simulations showed that such stop codon accumulation could not be explained solely by constraints acting on the primary open reading frame. Overall, the study demonstrates that the loss of RNA editing in HPIV-1 has driven lineage-specific pseudogenization of the V protein region, providing new insights into the evolution of overlapping gene architectures and functional gene loss in paramyxoviruses.
(TMR)