Jul 2026· npj Antimicrobials and Resistance· 0 citations
TL;DR
A programmable RNA-targeting antiviral platform based on RfxCas13d is developed and its activity in avian cells is evaluated, establishing RfxCas13d as a versatile RNA-guided antiviral platform and providing a foundation for cross-strain influenza control through conserved RNA targeting.
Abstract
Highly pathogenic avian influenza viruses (HPAIVs) continue to cause substantial disease in birds and mammals, with repeated H5N1 spillovers highlighting the need for broadly protective antiviral strategies. Here, we develop a programmable RNA-targeting antiviral platform based on RfxCas13d and evaluate its activity in avian cells. Screening of five Cas13 orthologs in chicken DF1 fibroblasts revealed RfxCas13d as the most potent and well tolerated effector. Virus-specific CRISPR RNAs (crRNAs) targeting conserved regions of positive- and negative-sense influenza RNA were tested against A/WSN/033 (H1N1) and multiple HPAIV isolates, including a member of clade 2.3.4.4b H5N1. Targeting positive-sense RNA conferred superior influenza inhibitory activity and was further enhanced by multiplexed crRNA expression. These findings establish RfxCas13d as a versatile RNA-guided antiviral platform and provide a foundation for cross-strain influenza control through conserved RNA targeting.
This review discusses selected RNA viruses, focusing on the structure of their RNA polymerases and interactions with host factors during the different stages of the viral lifecycle, as well as the traditional antivirals targeting these structures and pathways.
Hana Krnjić, Adna Hrapović, Aiša Galijatović et al.· Viruses· 0 citations
SUMMARY Highly pathogenic avian influenza (HPAI) viruses, especially subtype H5N1, have caused major outbreaks in poultry and serious human infections since the first description of “fowl plague” in 1878. Over time, these viruses have expanded their host range, causing huge losses in domestic poultry, spreading among wild bird populations, and occasionally infecting humans. Understanding how influenza viruses adapt and cross species barriers depends on analyzing their fundamental molecular characteristics. Key features such as the polybasic cleavage site within the hemagglutinin protein and the distinctive “1+7” ribonucleoprotein complex, which consists of a single polymerase core surrounded by seven RNA segments, enhance viral replication and broaden host range. Despite decades of intervention, including lessons from the 1997 Hong Kong outbreak and the continued global circulation of clade 2.3.4.4b, HPAI remains difficult to control. A major shift occurred in 2024, when H5N1 was detected in U.S. dairy cattle. This was the first confirmed instance of viral shedding into milk from a mammalian host, suggesting a new potential route of transmission beyond the traditional avian reservoirs. This review unites historical milestones, structural insights, epidemiological data, and recent cross-species findings to better define the current landscape of risk and transmission. We further discuss economic, public health, and agricultural impacts of these developments, particularly the 2024 cattle cases, and underscore the urgent need for an integrated One Health approach to better manage the growing risks posed by HPAI viruses.
Leila Wogick, S. Goyal· Clinical Microbiology Review...· 0 citations
Summary Influenza A virus (IAV) remains a major threat to human and animal health, while the emergence of drug-resistant strains necessitates new antiviral strategies. Here, we developed an integrative host-directed drug discovery framework combining functional genomics and pharmacotranscriptomics. By aggregating published genome-wide screens, we assigned host genes functional scores reflecting their effects on IAV replication and used these scores to estimate the antiviral status of host cells. Screening nearly 20,000 drug-induced transcriptional signatures identified compounds that shift host gene expression toward an antiviral state. Among 54 selected hits, 18 showed anti-IAV activity. Notably, lithocholic acid and ALW-II-49-7 inhibited viral replication in vitro and protected mice from lethal infection in vivo. This host-targeted framework provides a systematic and scalable strategy for discovering antivirals that are less susceptible to resistance and potentially applicable to other rapidly evolving pathogens.
Jianfa Qiu, Xuecong Xing, Jing-Feng Wang et al.· iScience· 0 citations
Findings reveal a viral trade-off: the mutation attenuates epithelial replication and immunopathology but enhances DC uptake and disables mucosal immune functions, a strategy that likely contributed to the global predominance of PA-X N193S mutation in epidemic H5 subtype viruses.
X. Miao, Xing Xu, Yixuan Han et al.· Emerging Microbes and Infect...· 0 citations
Avian influenza A virus (IAV) is a major global health threat. Not only does it cause significant economic losses to the agricultural industry, but it also presents a risk of spillover into the human population. However, several obstacles must be overcome for a successful cross-species transmission event to occur. One of these obstacles is the innate immunity of the host. While the innate immune response to IAV in humans has been well-studied, the innate immune response to IAV in birds is not as well understood. Here, we explore the interferon-induced immune response by identifying interferon-stimulated genes (ISGs) in chicken, quail, and duck cells, and find that they are largely species-specific. Using this data, we performed a CRISPR knockout ISG screen against two strains of IAV, the mouse-adapted PR8, and the low pathogenicity avian IAV isolate WF10. We find that for PR8, IFITM3 is the dominant restrictor of viral replication with several additional hits with modest effects, consistent with previous work in mammals. For avian WF10, IFITM3 was also a hit however there were two additional hits absent for PR8, the HSP70 co-chaperone protein BAG5 and deubiquitinase OTUD4. Overall, these findings confirm the divergence of interferon-mediated immunity between species and establish a screening system that can be used to identify ISGs important for viral restriction in avian species. IMPORTANCE Wild birds, especially waterfowl and shore birds, are the primary natural reservoir for influenza A viruses. These birds can transmit IAV to domestic poultry, where the spillover risk to humans increases. Chickens are the most common species of domestic poultry world-wide and are frequently infected with avian influenza viruses. Despite this, relatively little is known about how their immune system responds to influenza infection. This lack of knowledge creates a barrier to finding ways of preventing or mitigating IAV infection in chicken flocks. In this study, we characterized the interferon-induced immune response to IAV in chickens and found that only a small subset of ISGs were responsible for restricting IAV in chicken cells.
Alison J. Barkhymer, Jordan T. Becker, Frances K. Shepherd et al.· bioRxiv· 0 citations
Current evidence on cross-reactive and cross-protective immunity to H5N1 in humans, focusing primarily on humoral immune responses is summarized, and particular attention is given to antibodies targeting conserved regions of hemagglutinin (HA) as well as neuraminidase (NA), which may provide heterosubtypic protection.
Iván Sanz-Muñoz, Carlos J. Ciria-Gil, Marta Hernández et al.· Journal of Infection· 0 citations
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