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)

Recovery of Infectious Oz Virus From Cloned cDNA

This study established a reverse genetics system for Oz virus (OZV), a recently identified tick-borne orthomyxovirus associated with a fatal human case in Japan. The authors constructed six plasmids encoding the full-length viral genome segments along with four helper plasmids expressing essential polymerase proteins (NP, PA, PB1, and PB2), and co-transfected them into BHK/T7-9 cells to recover infectious virus. They successfully generated recombinant OZV (rOZV), which induced cytopathic effects in cocultured cells and produced high viral titers comparable to wild-type virus. Growth kinetics analysis in Vero cells showed that rOZV replicated similarly to the wild-type strain, confirming the functional integrity of the rescued virus. The study also demonstrated that all genome segments and polymerase components are required for virus recovery, and that the chosen cell system significantly improves efficiency. Overall, this work provides a robust platform for studying OZV replication, pathogenicity, and for future development of antiviral strategies.
(TMR)

Heterozygous and generalist MxA super-restrictors overcome breadth-specificity trade-offs in antiviral restriction

This study investigated how the antiviral protein MxA evolves to restrict different viruses and overcome limitations in its activity. The authors used combinatorial mutagenesis of the MxA L4 loop to generate variants and tested their ability to block H5N1 influenza and Thogoto virus (THOV). They found that some mutations created “super-restrictor” variants with much stronger antiviral activity than the normal (wild-type) MxA. However, most of these variants showed a trade-off, meaning improved restriction of one virus reduced activity against another. A key finding was that a single amino acid (position 561) largely determines this trade-off: different residues favor restriction of different viruses. Despite this limitation, rare “generalist” variants were identified that can effectively restrict both viruses. Importantly, the study showed two ways to overcome this trade-off: either evolving generalist variants or combining different specialist variants in heterozygous form, which together provide broad antiviral protection. Overall, this work reveals how host antiviral proteins adapt and suggests strategies to enhance broad-spectrum viral restriction.
(TMR)

Sialic acids are a barrier to the entry of non-influenza orthomyxoviruses

This study explored how cell surface sialic acids (SAs) influence the entry of thogoto- and quaranjaviruses, a group of non-influenza orthomyxoviruses. Using pseudotyped vesicular stomatitis virus (VSV) and lentiviral systems expressing viral glycoproteins, alongside authentic Thogoto (THOV) and Dhori (DHOV) viruses, the authors evaluated viral entry under conditions of altered SA availability. Enzymatic removal of SAs or their masking with lectins consistently enhanced viral entry, in sharp contrast to influenza viruses, where SAs serve as essential receptors. Further analyses across multiple cell lines revealed an inverse relationship between SA abundance and viral entry efficiency, indicating that SAs act as inhibitory factors rather than facilitators. These findings were corroborated in primary human airway epithelial cells and with infectious virus assays, where depletion of SAs significantly increased viral replication. In addition, adaptive mutations identified through experimental evolution partially mitigated this restriction, suggesting that viral glycoproteins can evolve to counteract SA-mediated inhibition. Overall, the study uncovers a distinct entry mechanism for these viruses and highlights sialic acids as a natural barrier to infection, offering new insights into host restriction, viral adaptation, and transmission dynamics.
(TMR)