Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

The continued diversification of the H9N2 avian influenza virus (AIV) into multiple antigenically and phylogenetically distinct lineages is promoting the emergence of strains with pandemic potential. Constant monitoring of the genetic evolution and changes in biological characteristics of the H9N2 viruses is therefore essential. In this study, we analyzed the genetic evolution of the H9N2 viruses isolated from poultry farms between 2022 and 2023 and evaluated their pathogenicity in chickens and mice. The HA genes of all ten isolates belonged to the h9.4.2.5 lineage, which is currently the predominant evolutionary lineage in China. Yet, their HA genes further divided into distinct subbranches within the h9.4.2.5 lineage. The NA genes of these viruses shared high homology with the prevalent H9N2 AIVs in recent years. However, these viruses were located in different evolutionary groups. Notably, the internal genes showed close relationships with those of recent H3, H6, and H9 subtype AIVs, suggesting active reassortment events among co-circulating viruses. Pathogenicity assessment in mice and chickens demonstrated divergent virulence between two representative isolates, FS22 and JM14, which clustered into different h9.4.2.5 subbranches. FS22 exhibited more efficient and prolonged replication in the lungs and turbinates of mice compared to JM14. Both viruses replicated efficiently in the lungs, kidneys, and trachea of chickens at 3 days post-infection (DPI), but differed in their horizontal transmission potential. Particularly, inoculated and contacted chickens all produced high antibody levels from the 5 DPI until the end of the experiment, and peak antibody titers for both viruses occurred at 7 DPI. These findings underscored the continuous evolution ofH9N2 AIV enhanced its genetic and phenotypic diversity, leading it to pose a threat to public health. Thus, continuous surveillance in poultry farms is necessary.

Goose parvovirus (GPV), duck enteritis virus (DEV), Muscovy duck parvovirus (MDPV), duck hepatitis A virus type 1 (DHAV-1), duck hepatitis A virus type 3 (DHAV-3), duck Tembusu virus (DTMUV), and novel duck reovirus (NDRV) are significant pathogens that spread extensively among waterfowl populations, causing economic losses for the waterfowl industry. In order to detect seven pathogens simultaneously, a visual gene chip for the detection of multiple waterfowl disease pathogens was developed in this study. The gene chip was capable of specifically amplifying GPV, DEV, MDPV, DHAV-1, –DHAV-3, DTMUV, and NDRV. The sensitivity results showed that the lowest detection limit of the gene chip was 1 copy/μL for single and mixed samples. The reproducibility and stability tests demonstrated that the gene chip developed in this experiment exhibited not only excellent reproducibility but also remarkable stability, remaining functional for a minimum of 180 days. Compared to qPCR methods, the results showed that the sensitivity of the gene chip was slightly better than that of the qPCR method in detecting both single and mixed pathogens of the seven viruses. In this study, a total of 210 clinical samples were detected by the gene chip and qPCR, respectively, and the results of the two methods had a concordance rate of 98.1~100%, with a kappa value of 0.952, indicating that the consistency of the two detection methods was good.

African horse sickness (AHS) is an arthropod-borne, severe equid disease caused by African horse sickness virus (AHSV). AHSV has high mortality and is endemic to sub-Saharan Africa. It has been classified into nine distinct serotypes (AHSV-1 to AHSV-9) based on VP2 immunogenicity. The AHS outbreak in Thailand in 2020, caused by AHSV-1, marked the first occurrence of this disease in Southeast Asia. It posed a substantial threat to the security of the equine industry in the nations across the region. To ensure the emergency reserve for AHS prevention and control, the AHSV strain imported to China from abroad over 60 years ago was characterized in this study. The strain was passaged in mice and then blind-passaged in Vero cells. The plaque purification method was then used to purify the strain and obtain its cell-adapted version, named AHSV/C. Neutralization tests confirmed that the virus belongs to AHSV-1. Whole-genome sequencing revealed that AHSV/C was highly homologous to AHSV-1 isolate 1180, with over 95% homology of major antigenic protein VP2, as compared to other AHSV-1 strains, including the prevalent strain in Thailand. In the mouse models, AHSV/C exhibited no clinical signs or histopathological lesions, suggesting low virulence and safety. This research for the first time characterized the in vitro growth characteristics and viral subtypes of the AHSV in China, determined its complete whole-genome sequence, and evaluated its safety using a mouse model. It provides crucial experimental materials and scientific foundations for the development of diagnostic methods and vaccines against AHSV-1.

H6 subtype avian influenza viruses (AIVs) have a broader host range and circulate globally in wild birds, domestic ducks, geese, and terrestrial poultry all over the world. Their demonstrated capacity to adapt receptor-binding affinity for mammalian species constitutes a persistent concern for zoonotic transmission and public health. In this study, a novel triple-reassortant H6N8 AIV strain was isolated from a chicken farm in southern China and designated as A/chicken/Guangdong/JM642/2023 (H6N8). The complete genome of the virus was sequenced using Next-Generation Sequencing. Phylogenetic analysis indicated that the HA gene of the isolate clustered into the Group III/HN573-like, which encompasses H6 subtype viruses bearing various NA genes and belonging to the Eurasian gene pool. The NA gene showed the closest genetic relationship with viruses originating from North America. All six internal genes were derived from H9N2 AIVs. The virus possesses several key molecular determinants known to contribute to an expanded host range and increased virulence. Animal infection studies demonstrated that the virus was capable of infecting mice without prior adaptation. It replicated efficiently in the lungs and nasal turbinates, followed by systemic dissemination resulting in lethal outcomes. Inoculated chickens remained asymptomatic; however, the virus replicated efficiently in multiple organs, with high viral loads detected particularly in the lungs and kidneys. Viral shedding occurred via both the respiratory and digestive tracts, and horizontal transmission was observed among chickens. Notably, infected and contacted chickens developed high levels of antibodies from 8 days post-inoculation (DPI) to the end of observation period. This study enhances our understanding of the genetic and biological characteristics of the novel reassortant H6N8 AIVs and underscores their potential risk to public health.

Avian influenza, a disease caused by avian influenza virus (AIV), mainly infects birds but can also infect mammals, which poses a serious threat to public health. Therefore, thorough understanding of its pathogenic mechanism and the identification of antiviral targets are essential for the prevention, control, and treatment of AIV. The polymerase acidic protein (PA) is a core component of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription through its cap-snatching activity during early infection. We employed a multi-omics approach combining transcriptome analysis with PA interaction proteomics to characterize host responses during AIV infection and explore the PA–host interaction network. Transcriptomics revealed a polarized host response marked by activated translation-related processes, mitochondrial energy metabolism, and innate immune signaling, alongside broad suppression of nuclear transcriptional regulation and cell cycle pathways. Immunoprecipitation–mass spectrometry identified host proteins associated with PA that were enriched in RNA metabolism, ribosome biogenesis, and protein homeostasis. Integrative analysis of transcriptomic and interactome data, along with protein–protein interaction network analysis, prioritized a subset of high-confidence PA-interacting host factors. Among these, ribosomal protein RPS27A was validated to interact with PA and to support viral replication during early infection in this study.