RNA splicing is a fundamental driver of eukaryotic transcriptomic and proteomic diversity. Constrained by compact genomes, diverse DNA and RNA viruses, including adenovirus, HIV-1, and influenza virus, have evolved to hijack the host splicing machinery. This exploitation not only maximizes viral coding capacity but also ensures the precise spatiotemporal regulation of viral infection. In this review, we summarize current advances in the molecular mechanisms of viral RNA splicing, illustrating how viruses co-opt the host spliceosome and reprogram global alternative splicing landscapes to support their infection cycle. Through representative viral models, we detail the convergent strategies of alternative splice site selection and the dynamic interplay between viral RNA elements and host trans-acting factors. Furthermore, we spotlight the emerging frontier of viral circular RNAs (vcircRNAs), highlighting their biogenesis via non-canonical back-splicing and their versatile roles in immune evasion. Finally, we summarize recent methodological breakthroughs, particularly long-read sequencing and single-cell analyses, that are rapidly charting the complex splicing landscape. Together, this review provides an integrated perspective on the virus-host splicing interface, exposing critical vulnerabilities that offer promising avenues for next-generation, broad-spectrum antiviral interventions.

H9N2 avian influenza viruses (AIVs), which are enzootic in poultry and possess spillover potential to humans, represent a considerable global public health threat. Since 2021, we have observed an unprecedented decline in the ability of H9N2 viruses to agglutinate chicken red blood cells (CRBCs), a change that impairs effective detection in routine surveillance. Substituting CRBCs with turkey or guinea pig erythrocytes in hemagglutination assays effectively restored virus detectability. Through integrated bioinformatic and biological approaches, we identified that this phenotype is associated with specific amino acid substitutions at residues 131 and 132 of the 130-loop in the hemagglutinin (HA) protein. The HA motif 131/132-NT emerged in 2015 and rapidly increased in frequency, becoming the dominant pattern since 2021 in chickens and humans. Critically, the 131/132-NT motif attenuated viral binding affinity to short-chain α2,6-linked sialic acid receptors, explaining the loss of agglutination with CRBCs. Animal experiments further demonstrated that viruses carrying the HA 131/132-NT mutations maintained strong infectivity in chickens but altered tissue tropism in mice, showing reduced replication in the lungs. Collectively, our findings reveal a potential surveillance gap caused by reduced hemagglutination activity of H9N2 viruses, which may compromise the sensitivity of CRBC-based detection and potentially lead to under-detection of circulating viruses. Incorporating turkey or guinea pig red blood cells into surveillance protocols is therefore recommended to enhance detection sensitivity.

Introduction:Duck hepatitis A virus type 3 (DHAV-3) causes acute fatal hepatitis in ducklings. Methods:This study compared host transcriptomic responses in duck livers following infection with virulent (HB) or attenuated (HB80) strains. Results:RNA sequencing (RNA-Seq) revealed that the virulent HB strain induced 2,355 differentially expressed genes (DEGs) at 2 days post-infection (dpi), compared to only 322 DEGs triggered by the attenuated HB80 strain. Functional analysis showed that the HB strain robustly activated immune pathways, particularly Toll-like receptor (TLR) and RIG-I-like receptor (RLR) signaling, leading to a potent type I interferon (IFN-I) response and marked chemokine upregulation. In contrast, the HB80 strain elicited a markedly milder immune reaction. Among the DEGs, 77 immune-related genes were identified, with significant enrichment in the IFN-I signaling pathway, suggesting their critical role in initiating an interferon storm and subsequent chemokine upregulation. Selected key genes (IFN-α2, RSAD2, RIG-I, MDA5, TBK1, TLR7) were validated by RT-qPCR and ELISA for targeted protein confirmation. Discussion:These findings delineate divergent host transcriptomic responses to virulent vs. attenuated DHAV-3 and highlight IFN-I signaling as a central axis in antiviral immunity.

Glucose metabolism impacts the innate immune response against viral infection. However, the key enzymes or the natural products and mechanisms involved are not well elucidated. Here, we found that arrestin domain containing 4 (ARRDC4), a critical regulator of glucose metabolism, senses influenza A virus (IAV) infection by interacting with viral PA protein. Upregulated ARRDC4 increases the enzymatic activity of phosphofructokinase, muscle type (PFKM) via binding its His298 site to promote the production of the metabolite fructose-1,6-bisphosphate (FBP). Consequently, FBP inhibits the K48-linked ubiquitination degradation of HSP90β, subsequently enhances its interaction with IKKβ and IKKε, and enhances NF-κB- and IRF7-mediated antiviral innate immunity, respectively. Importantly, FBP supplementation enhanced IFN-β-mediated antiviral innate immunity in vitro and in vivo. Our findings highlight a unique immunometabolic regulatory mechanism in which ARRDC4 senses IAV infection and regulates antiviral innate immunity through the PFKM-FBP metabolic axis and provide a strategy for manipulating FBP-related metabolism to treat viral infection.

H9N2 avian influenza virus (AIV) is endemic in poultry worldwide and increasingly zoonotic. Despite the long-term widespread use of inactivated vaccines, H9N2 AIVs remain dominant in chicken flocks. We demonstrated that inactivated vaccines did not prevent the replication of H9N2 AIVs in the upper airway of vaccinated chickens. Viral transmission was enhanced during sequential passage in vaccinated chickens, which was attributed to the restricted production of defective interfering particles and the introduction of stable mutations (NP-N417D, M1-V219I, and NS1-R140W) which enhanced viral replication. Notably, the genetic diversity of H9N2 AIVs was greater and included more potential mammal/human-adapted mutations after passage through vaccinated chickens than through naïve chickens, which might facilitate the emergence of mammal-adapted strains. By contrast, vaccines inducing cellular/mucosal immunity in the upper respiratory tract effectively limit H9N2 AIV. These findings highlight the limitations of inactivated vaccines and the need for revised vaccination strategies to control H9N2 AIV.

Global public health faces substantial challenges from malignant tumors and infectious diseases. Vaccination provides an approach for treating and preventing these diseases. Oral vaccinations are particularly advantageous in disease treatment and prevention due to their non-invasive nature, high patient compliance, convenience, cost-effectiveness, and capacity to stimulate comprehensive and adaptive immune responses. However, the overwhelming majority of oral vaccines remain in experimental development, struggling with clinical and commercial translation due to their suboptimal efficacy. Thus, enhancing scientists’ understanding of the interaction between vaccines and gastrointestinal immune system, creating antigen delivery systems suitable for the gut mucosal environment, developing more potent antigenic epitopes, and using personalized combination therapies are critical for advancing the next generation of oral vaccines. This article explores the fundamental principles and applications of current oral anti-tumor and anti-infective vaccines and discusses considerations necessary for designing future oral vaccines.

Since 2021, an epidemic disease characterized with hydrosalpinx fluid syndrome (HFS) has been circulating in the laying Sheldrake ducks in China, which seriously endangers the healthy development of the duck industry. The causative agent of this disease has been traced to avian metapneumovirus subtype C (aMPV/C), known to cause acute upper respiratory tract infections and egg-drop in poultry. To date, no reports have been made to isolate and characterize aMPV/C infection in Sheldrake ducks in China. Here, a strain of virus, designated aMPV-FJ21, was successfully isolated from the diseased ducks exhibiting HFS. Transmission electron microscopy revealed that the virus is an enveloped particle exhibiting a spherical or pleomorphic morphology. Indirect immunofluorescence assays demonstrated that the aMPV-FJ21 strain had an obvious reactive activity with the ploy-antibody against aMPV/C F protein. The complete genome of aMPV-FJ21 was determined to be 14,149 nucleotides in length. Notably, the amino acid sequence of the G protein was only 55.6%-78.7% identical to those of other aMPV/C reference strains. Phylogenetic analysis indicated that aMPV-FJ21 forms a distinct lineage within the aMPV/C group and is genetically distant from the North American and Eurasian lineages, suggesting that it may represent a novel genetic lineage. In challenge experiments, laying Sheldrake ducks with aMPV-FJ21 reproduced the typical clinical symptoms and pathological lesions observed in the field cases. Altogether, we had isolated a novel aMPV/C variant from Sheldrake ducks exhibiting HFS, distinct from previously reported strains, and provided the first evidence confirming its role as the causative agent of HFS in ducks.

Infectious bursal disease (IBD) remains one of the most important immunosuppressive diseases in poultry and has been endemic in China for over four decades. In recent years, outbreaks characterized by markedly reduced mortality have emerged. To investigate the current prevalence and pathogenicity of infectious bursal disease virus (IBDV), strains were isolated from vaccinated poultry farms in central and eastern China, and representative isolates of distinct genotypes were evaluated for pathogenicity. Phylogenetic analysis of segment A (HVR) and segment B (B-marker) classified segment A into eight genogroups (A1-A8), with A2 further divided into A2.1 and A2.2 and A3 into six subtypes (A3.1-A3.6). Segment B was grouped into four genogroups (B1-B4), with B3 and B4 subdivided into B3.1-B3.4 and B4.1-B4.2, respectively. The 23 isolates obtained were grouped into three genotypes: A2.2B1 (14/23, 60.9 %), A3.2B3.2 (8/23, 34.8 %), and A3.5B3.3 (1/23, 4.3 %). The A2.2B1 and A3.5B3.3 strains showed only minor amino acid substitutions relative to reference strains, whereas the newly emerged A3.2B3.2 genotype displayed substantial divergence. Pathogenicity evaluation in SPF chickens revealed no mortality after infection with SD/23 (A3.2B3.2), SHX/24 (A3.5B3.3), or WD/22 (A2.2B1). However, all three strains significantly reduced the bursa-to-body weight index (BBIX) to below 0.7 at 5 days post-inoculation and caused severe bursal atrophy. Histopathological examination showed medullary necrosis and lymphocyte depletion in the bursa following infection with SD/23 and SHX/24, closely resembling the lesions induced by WD/22. Moreover, SD/23 and SHX/24 displayed replication dynamics similar to those of the reference strain LX (A3.1B2), with elevated viral detection rates and high viral loads in multiple organs. In summary, multiple IBDV genotypes are co-circulating in China. Despite the reduced pathogenicity of circulating strains, diminished attention to subclinical infections may facilitate viral transmission and cause considerable economic losses.

Wild birds are key natural reservoirs and play a central role in the global spread of avian influenza viruses (AIVs). However, the absence of a standardized global list of wild bird hosts has limited comprehensive AIV risk monitoring and assessment within the One Health framework. Here, we generate a taxonomically harmonized dataset of AIV wild bird hosts, derived from 23,358 viral isolates of wild bird origin reported in the GISAID EpiFluTM database from 1973 to 2023. Host names were systematically extracted, validated, and harmonized to resolve reporting inconsistencies and unify taxonomy across records. The dataset comprises 394 wild bird species spanning 26 orders, with Anseriformes and Charadriiformes representing a substantial share of host diversity. By clarifying the global spectrum of wild bird hosts for AIVs, this dataset provides a foundation for host identification, phylogenetic annotation, and ecological trait-based analysis. Structured in machine-readable formats, it enables reproducible and large-scale, species-level studies spanning virology, epidemiology, and biodiversity.

African swine fever (ASF) has caused a devastating pandemic among domestic and wild swine, leading to significant economic losses in the global swine industry. Recombinant live-attenuated vaccines are a potential option for the control of ASF. However, safe and effective vaccines against the ASF virus (ASFV) are not yet commercially available, and thus, additional vaccine candidates still need to be developed. In this study, we demonstrate that the simultaneous deletion of the ASFV genes CD2v and A137R from the highly virulent isolate ASFV HuB/HH/2019 substantially attenuated the virulence in swine. All pigs (4/4) infected with 105 TCID50 doses of the double gene deletion virus HuB/HH/2019ΔCD2vΔA137R (HuBΔCD2vΔA137R) remained healthy during a 27-day observation period, with no fevers (>40.5°C) observed. Cases of viremia were mild and cleared by 24 days post-inoculation (dpi). Antibodies against ASFV p30 were induced, and the inoculated pigs were well-protected against homologous challenges. All inoculated pigs survived after being challenged with 102 HAD50 ASFV HuB/HH/2019, and no fever or clinical symptoms developed during the 22-day monitoring period. In contrast, all control group pigs died before 11 dpi (2/2). Cytokine assays and RNA sequencing revealed mild responses elicited by HuBΔCD2vΔA137R. Successful innate and passive immune responses were also triggered. The findings suggest that deletion of the CD2v and A137R genes can attenuate ASFV, and the deletion mutant virus HuBΔCD2vΔA137R is a promising vaccine candidate.IMPORTANCEThe emergence of ASF has caused substantial economic losses in the global pig industry. In light of this, the development of a safe and effective vaccine is crucial to control the spread of ASF. In this study, a live-attenuated ASFV strain was developed by simultaneously knocking out the viral genes CD2v and A137R. The mutant virus exhibited weakened virulence in pigs, elicited robust humoral and cellular immunity, and conferred protection upon challenge with high doses of the parental virus. Notably, no fever or clinical symptoms were observed during a 27-day monitoring period. Consequently, our research presents a highly promising candidate strain for a live-attenuated vaccine, offering a significant step forward in the quest for effective prevention measures. Additionally, the identification of these specific gene targets opens up avenues for further exploration and refinement of strategies aimed at combating ASF outbreaks.

Influenza A virus (IAV) infection causes significantly greater morbidity and mortality in the elderly population, but the molecular mechanisms in the aging process responsible for severe infection remain unclear. In this study, we found that increased severity in IAV infection and reduced innate immune response correlated with extensive mitophagy in senescent human cells and in the lung of aged mice. Apolipoprotein D (ApoD) was identified as strongly elevated in the lungs and sera of aged human (>65 y old) and mouse (>21 mo old). ApoD was able to localize to mitochondria and interact, through its WXXI motif in the LC3B-Interacting Region domain, with LC3B to trigger mitophagy during IAV infection, in a PINK1 pathway independent manner, which attenuated type I interferon response and promoted virus replication. ApoD deficiency, on the other hand, protected older mice from severe influenza and improved survival. Likewise, depletion of senescent cells by ABT-263, a senolytic compound, in aged mice lowered ApoD level and restored innate immune antiviral response, limiting virus propagation and associated pulmonary damage. Thus, age-induced ApoD drives IAV-mediated mitophagy, and promotes virus replication and infection severity, and is therefore a promising target for inhibition to improve disease outcome in older patients.

Nationwide surveillance of avian influenza viruses (AIVs) in live poultry markets across China has occurred since 2014, providing a resource for AIV prevalence and genetic diversity studies. Here we report that 3,237 of 18,425 samples from poultry were AIV positive (17.57%) between 2019 and 2023, with H9N2 being the dominant subtype. We developed an automated phylogeny-based nomenclature system to classify genetic clades of the dominant H9N2 lineage, the BJ94 lineage. Using this model, we found that ten haemagglutinin (HA) sub-subclades cocirculated in poultry and showed antigenic variation. In addition, 99.46% and 96.17% of H9N2 AIVs in 2021-2023 possessed human-receptor binding-related HA-L226 and human MxA-resistance-related NP-N52 mutations, respectively. H9N2 strains with these two mutations preferred human-type receptors and increased replication in human cells in vitro, regardless of the presence of PB2-V/K/E627. Moreover, H9N2 AIVs containing HA-L226, PB2-V/K627 and NP-N52 were transmitted from infected to naive guinea pigs and ferrets through direct contact and respiratory droplet. This highlights the potential zoonotic risks of H9N2 AIVs.

The influenza A viruses (IAV) are the principal pathogens for annual (seasonal) influenza, which cause world-wide outbreaks in poultry and pose a persistent threat to public health. The Hemagglutinin protein (HA) of IAV promotes virus infection by binding the host membrane receptor and mediating virus-host membrane fusion. Immunoprecipitation-mass spectrometry (IP-MS) provides global insights into IAV HA-host protein interactions. However, various experimental conditions might affect the identification of interactions. Here, we performed a serial IP-MS to compare interactors of IAV HA in accidental host human, chicken and reservoir host duck cells. We found that the positive ratio of interactors identified by the IP-MS was improved when the transfected HA plasmid had a similar expression level to HA proteins found in IAV virus infection. Comparing interactors in human, chicken and duck cells, we found that HA-interacting host factors might play a role in the susceptibility of accidental hosts (human and chicken) to IAV infection compared to reservoir hosts (duck). We then focused on the function of two heat shock proteins (HSPA5 and HSPA8), which interacted with IAV HA proteins in all three species (human, chicken and duck). We found that both HSPA5 and HSPA8 promoted the IAV replication by enhancing the viral attachment and internalization. These findings extend our knowledge about the mechanisms of IAV entry to host cells and provide target genes to create chickens resistant to avian influenza.

The pathogenicity of emerging early zoonotic avian H7N9 and H3N8 low-pathogenic avian influenza viruses (LPAIVs) in humans is significantly enhanced compared to that of their internal gene providers, H9N2 avian influenza viruses (AIVs), suggesting a pivotal role for surface HA or NA. Here, we generated several reassortant AIVs that combined HA, NA, or HA + NA from emerging zoonotic H7N9 or H3N8 LPAIV with internal H9N2 AIV genes and investigated the impact of HA and NA on the replication and pathogenicity of reassortants in human cells and mice. The crucial affected phase was determined by analyzing the receptor binding, viral adsorption, HA cleavage efficiency, viral endocytosis, and budding. We found that mice infected with the virus containing the early zoonotic H7N9 LPAIV HA, but not NA, exhibited high mortality, weight loss, severe lung damage, and increased viral load in the lungs. We found that HA substitution enhanced viral replication in human A549 cells and displayed dual sialic acid receptor binding ability. This substitution also facilitated viral attachment to mammalian cells and promoted endocytosis by enhancing HA0 cleavage efficiency; however budding was not affected. Additionally, HA from emerging zoonotic H3N8 LPAIVs elevate the pathogenicity of reassortants in mice. Together, our study revealed that HA of emerging zoonotic LPAIVs contributes to high pathogenicity in mammals by augmenting viral entry and causing lung injury, thereby highlighting HA with double receptor binding properties and HA cleavage efficiency as new markers for risk assessment of emerging zoonotic AIVs in the future.

AbstractTo bolster the capacity for managing potential infectious diseases in the future, it is critical to develop specific antiviral drugs that can be rapidly designed and delivered precisely. Herein, a CRISPR/Cas13d system for broad‐spectrum targeting of influenza A virus (IAV) from human, avian, and swine sources is designed, incorporating Cas13d mRNA and a tandem CRISPR RNA (crRNA) specific for the highly conserved regions of viral polymerase acidic (PA), nucleoprotein (NP), and matrix (M) gene segments, respectively. Given that the virus targets cells with specific receptors but is not limited to a single organ, a Susceptible Cell Selective Delivery (SCSD) system is developed by modifying a lipid nanoparticle with a peptide mimicking the function of the hemagglutinin of influenza virus to target sialic acid receptors. The SCSD system can precisely deliver an all‐RNA‐based CRISPR/Cas13d system into potentially infected cells. This drug is shown to reduce the viral load in the lungs by 2.37 log10 TCID50 mL−1 and protect 100% of mice from lethal influenza infection. The SCSD‐based CRISPR/Cas13d system shows promise for the flexible and efficient therapy of infections caused by rapidly evolving and novel viruses.

Canine health is consistently affected by the circulation of the H3N2 strain of canine influenza virus (CIV). Prior research has indicated that the isolation rate of H3N2 CIVs in dogs has gradually increased in China, and these viruses have progressively adapted to humans over the course of their evolution within canine hosts, posing a significant public health threat. However, the key factors influencing the spread of CIVs remain elusive. From January 2020 to December 2022, during the COVID-19 pandemic, strict epidemic prevention policies were implemented in China. Thus, this measure provides an ideal model for studying factors influencing the prevalence of CIVs. In this study, we continuously monitored the prevalence of CIVs in China before and after the COVID-19 pandemic. We found that the pathogen detection rate and seropositivity rate of domestic CIVs significantly declined after the implementation of epidemic control measures. However, after restrictions on human movement were lifted in 2023, the circulation of CIVs gradually increased. Our results demonstrate that restricting human activity directly impacts the epidemic caused by CIVs and provide a theoretical basis for the implementation of control measures during outbreaks of zoonotic diseases in human companion animals.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Influenza A/H9 viruses circulate worldwide in wild and domestic avian species, continuing to evolve and posing a zoonotic risk. A substantial increase in human infections with A/H9N2 subtype avian influenza viruses (AIVs) and the emergence of novel reassortants carrying A/H9N2-origin internal genes has occurred in recent years. Different names have been used to describe the circulating and emerging A/H9 lineages. To address this issue, an international group of experts from animal and public health laboratories, endorsed by the WOAH/FAO Network of Expertise on Animal Influenza, has created a practical lineage classification and nomenclature system based on the analysis of 10,638 hemagglutinin sequences from A/H9 AIVs sampled worldwide. This system incorporates phylogenetic relationships and epidemiologic characteristics designed to trace emerging and circulating lineages and clades. To aid in lineage and clade assignment, an online tool has been created. This proposed classification enables rapid comprehension of the global spread and evolution of A/H9 AIVs.

The H9N2 subtype of avian influenza viruses (AIVs) is widely prevalent in poultry and wild birds globally, with occasional transmission to humans. In comparison to other H9N2 lineages, the BJ/94 lineage has raised more public health concerns; however, its evolutionary dynamics and transmission patterns remain poorly understood. In this study, we demonstrate that over three decades (1994-2023), BJ/94 lineage has undergone substantial expansion in its geographical distribution, interspecies transmission, and viral reassortment with other AIV subtypes, increasing associated public health risks. These changes were primarily driven by the emergence of a dominant genotype G57. In the first decade, G57 emerged in East China and rapidly adapted to chickens and spread across China. Since 2013, the G57 genotype has expanded beyond China into eight other countries and reassorted with various AIV subtypes to form new zoonotic reassortants. Chickens have played a key role in the generation and circulation of the G57 viruses, with ducks and other poultry species likely assuming an increasingly importantly role. Over the past decade, G57 has been more frequently detected in wild birds, mammals, and humans. Additionally, Vietnam has emerged as a new hotspot for the international spread of G57. Our results suggest that the BJ/94 lineage H9N2 virus may continue to overcome geographical and species barriers, with potentially more severe consequences.