Shunlin Hu

H7N9 avian influenza viruses have caused repeated human infections since 2013, but the effects of naturally occurring PB2 substitution combinations on polymerase activity remain poorly understood. We analyzed 899 avian-derived and 1087 human-derived H7N9 PB2 sequences collected between 2013 and 2025. Six PB2 positions (109, 122, 191, 340, 559, and 640) showed distinct frequency changes across epidemic waves and host origins. Pairwise analysis identified five representative combinations with different temporal patterns. In minigenome assays, four paired substitutions reduced polymerase activity in human 293T cells at both 33 °C and 37 °C, whereas none of the five significantly altered activity in chicken DF1 cells at 37 °C or 41 °C. Several paired substitutions showed polymerase activity profiles different from those of the corresponding single substitutions in the tested cellular assays. ANP32 overexpression experiments showed different polymerase activity responses across the tested cellular conditions. Under chicken ANP32A expression, none of the paired-substitution polymerases differed significantly from the wild type, whereas human ANP32A and ANP32B overexpression did not enhance their activity in DF1 cells. Co-immunoprecipitation confirmed detectable interactions between PB2 variants and human or chicken ANP32 proteins. Structural modeling further revealed differences in predicted PB2–ANP32A interfaces among the tested variants. Together, these results show that the tested PB2 substitution combinations produced different polymerase activity profiles in human 293T and chicken DF1 cells and exhibited different responses to ANP32 overexpression under the examined conditions. The findings provide insight into the functional consequences of PB2 evolution during H7N9 circulation.

Newcastle disease virus (NDV) incurs substantial economic losses within the global poultry industry. The matrix (M) protein of NDV is integral to viral replication. In this study, we examined the interaction between the NDV M protein and annexin A6 (AnxA6) in DF-1 cells. Our findings revealed that AnxA6 associates with the C-terminus of the NDV M protein, predominantly within the cytoplasm. Functional analyses indicated that the knockdown of AnxA6 expression significantly enhanced NDV replication, whereas the overexpression of AnxA6 inhibited viral replication. Taken together, these findings suggest that AnxA6 may function as a negative regulator of NDV replication in this cell culture model, potentially through its interaction with the M protein. This study offers new insights into the intricate relationship between viral proteins and host factors in NDV infection and suggests that AnxA6 may serve as a potential target for further investigation. Further research is necessary to elucidate the detailed mechanisms governing the AnxA6-M protein interaction.

Accumulating studies have identified the pivotal role of long non-coding RNAs (lncRNAs) in participating in host–virus interactions during virus infections. However, the regulatory roles of lncRNAs in influenza A virus (IAV) infection are still not fully elucidated. In this study, using high-throughput sequencing, we comprehensively compared the expression profiles of lncRNAs and mRNAs in mouse lungs infected either with the nonpathogenic parental (SDL124) H7N9 virus or its moderately pathogenic mouse-adapted (S8) variant. A total of 7636 significantly differentially expressed (SDE) lncRNAs were obtained in the S8-infected group compared to the mock group. As for the SDL124 group, 1042 SDE lncRNAs were identified. Subsequently, the mRNAs co-expressed with SDE lncRNAs were subjected to functional annotation and pathway enrichment analysis. The results indicated that the target mRNAs regulated by the S8 virus were mainly enriched in various immunological processes and exhibited a strong correlation with inflammatory-related signaling pathways. Moreover, 12 lncRNAs and 10 mRNAs co-expressed with SDE lncRNAs were selected and successfully verified by RT-qPCR. Among these lncRNAs, NONMMUG032982.2 and NONMMUG032328.2 exhibited strong antiviral activity against IAV. Additionally, these two lncRNAs were chosen for further in-depth bioinformatics analysis, including transcription factor prediction, coding capacity assessment, genomic location, construction of secondary structure, and prediction of potential interacting proteins. Taken together, these findings provide a cluster of lncRNAs probably associated with the virulence of IAV in mice and shed light on the anti-IAV effects of two functional lncRNAs, establishing a molecular foundation for further exploring the regulatory mechanisms of lncRNAs in IAV infection.

Infection with genotype VII Newcastle disease virus (NDV) poses a substantial threat to poultry health and is characterized by severe damage to immune organs, with effects such as lymphocyte depletion. However, the precise mechanisms underlying this phenomenon remain unclear. Our present investigation focused on the dynamic changes in macrophages, T cells, and B cells in the spleens of chickens infected with genotype VII NDV by using multicolour flow cytometry. We found that NDV selectively infected chicken splenic macrophages and significantly increased the number of macrophages at 4 days post-infection. In contrast, T and B cells became progressively depleted. In vitro experiments revealed that following genotype VII NDV infection, T cells underwent apoptosis more potently when they were cocultured with macrophages than when they were not. Overall, our findings highlight the changes in chicken splenic immune cell populations triggered by genotype VII NDV and illuminate the role of macrophages in T-cell depletion.

The endemic status of goose parvovirus (GPV) continues to devastate the poultry industry in China. Novel GPV (NGPV) and Mutated GPV (MGPV) represent the predominant lineages. However, the comparative pathogenicity between these viruses remains poorly understood. Herein, we selected representative NGPV and MGPV strains as model viruses to assess their pathogenic potential both in vitro and in vivo. In vitro cellular and embryo assays demonstrated that both NGPV and MGPV were capable of replicating in DEF and GEF cells, leading to pronounced cytopathic effects. However, these viruses exhibited distinct levels of intra-embryonic replication capabilities. Furthermore, we conducted in vivo infection experiments and systematically evaluated the pathogenic differences between NGPV and MGPV by examining various indicators, including growth, clinical signs, gross pathology, skeletal development, viral load, and humoral response in the infected animals. The results showed that both NGPV and MGPV inhibited weight gain in goslings and ducklings, with NGPV exerting a more significant suppressive impact. MGPV induced classical gosling plague pathology in goslings, while NGPV led to short beak and dwarfism syndrome in ducklings, notably disrupting skeletal development. Moreover, MGPV and NGPV exhibited diverse host tropisms, with MGPV being more pathogenic to goslings and NGPV to ducklings. Both viruses elicited specific antibody responses, with MGPV being more effective in goslings and NGPV in ducklings. Additionally, MGPV exhibited stronger humoral response compared to NGPV. These findings enhance our understanding of the pathogenicity of prevalent GPV strains in waterfowl, offering a critical theoretical foundation for devising strategies to prevent GPV infections.

The novel H10N3 avian influenza virus (AIV) has infected four individuals since 2021 and caused severe respiratory damage, posing a significant threat to public health. However, its pathogenic mechanisms remain poorly understood. Our findings revealed that H10N3 infection induces severe lung damage and causes death in mice, even at low doses. The elevated levels of multiple pro-inflammatory factors in the bronchoalveolar lavage fluid were significantly increased during infection, displaying hallmarks of a cytokine storm. Transcriptome sequencing further revealed systematic activation of inflammation-related pathways, predicting that viral infection induces multiple forms of programmed cell death, including apoptosis, pyroptosis, and necroptosis. Protein-level validation showed that the activation of key cell death markers, including Caspase-3, GSDMD, and MLKL, significantly increased as the infection progressed, with their dynamic changes correlating strongly with the expression pattern of viral proteins. This study elucidates the central role of the synergistic effect between the cytokine storm and multiple cell death pathways in H10N3 pathogenesis. These findings not only advance our understanding of the pathogenic mechanisms of AIVs but also provide a critical theoretical basis for the development of targeted therapeutic strategies.

Several viruses, including influenza A virus (IAV), encode viral factors to hijack cellular RNA biogenesis processes to direct the degradation of host mRNAs, termed "host shutoff." Host shutoff enables viruses to simultaneously reduce antiviral responses and provides preferential access for viral mRNAs to cellular translation machinery. IAV PA-X is one of these factors that selectively shuts off the global host genes. However, the specific role of PA-X host shutoff activity in viral fitness of IAV remains poorly understood. Herein, we successfully mapped PA-X 100 V as a novel site important for host shutoff of the H7N9 and H5N1 viruses. By analysing the polymorphism of this residue in various subtype viruses, we found that PA-X 100 was highly variable in H7N9 viruses. Structural analysis revealed that 100 V was generally close to the PA-X endonuclease active site, which may account for its host shutoff activity. By generating the corresponding mutant viruses derived from the parental H7N9 virus and the PA-X-deficient H7N9 virus, we determined that PA-X 100 V significantly enhanced viral fitness in mice while diminishing viral virulence in chickens. Mechanistically, PA-X 100 V significantly increased viral polymerase activity and viral replication in mammalian cells. Furthermore, PA-X 100 V highly blunted the global host response in 293T cells, particularly restraining genes involved in energy metabolism and inflammatory response. Collectively, our data provided information about the intricate role of the PA-X host shutoff site in regulating the viral fitness of the H7N9 influenza virus, which furthers our understanding of the complicated pathogenesis of the influenza A virus.

The ongoing global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates the continuous development of innovative vaccine strategies, especially in light of emerging viral variants that could undermine the effectiveness of existing vaccines. In this study, we developed a recombinant virus-like particle (VLP) vaccine based on the Newcastle Disease Virus (NDV) platform, displaying a stabilized prefusion form of the SARS-CoV-2 spike (S) protein. This engineered S protein includes two proline substitutions (K986P, V987P) and a mutation at the cleavage site (RRAR to QQAQ), aimed at enhancing both its stability and immunogenicity. Using a prime-boost regimen, we administered NDV-VLP-S-3Q2P intramuscularly at different doses (2, 10, and 20 µg) to BALB/c mice. Robust humoral responses were observed, with high titers of S-protein-specific IgG and neutralizing antibodies against SARS-CoV-2 pseudovirus, reaching titers of 1:2200–1:2560 post-boost. The vaccine also induced balanced Th1/Th2 immune responses, evidenced by significant upregulation of cytokines (IFN-γ, IL-2, and IL-4) and S-protein-specific IgG1 and IgG2a. Furthermore, strong activation of CD4+ and CD8+ T cells in the spleen and lungs confirmed the vaccine’s ability to promote cellular immunity. These findings demonstrate that NDV-S3Q2P-VLP is a potent immunogen capable of eliciting robust humoral and cellular immune responses, highlighting its potential as a promising candidate for further clinical development in combating COVID-19.

Pigeon Newcastle disease (ND) is the most common viral infectious disease in the pigeon industry, caused by pigeon paramyxovirus type 1 (PPMV-1), a variant of chicken-origin Newcastle disease virus (NDV). Previous studies have identified significant amino acid differences between PPMV-1 and chicken-origin NDV at positions 347 and 349 in the hemagglutinin–neuraminidase (HN) protein, with PPMV-1 predominantly exhibiting glycine (G) at position 347 and glutamic acid (E) at position 349, while most chicken-origin NDVs show E at position 347 and aspartic acid (D) at position 349. However, the impact of these amino acid substitutions remains unclear. In this study, we generated a recombinant virus, NT-10-G347E/E349D, by introducing the G347E and E349D dual mutations into a PPMV-1 strain NT-10 using reverse genetics. The biological characteristics of NT-10 and NT-10-G347E/E349D were compared both in vitro and in vivo. In vitro, the G347E and E349D dual mutations reduce NT-10′s replication and neuraminidase activity in pigeon embryo fibroblast (PEF) cells while enhancing both in chicken embryo fibroblast (CEF) cells. Additionally, these mutations decrease NT-10′s binding affinity to the α-2,6 sialic acid receptor while significantly increasing its affinity for the α-2,3 receptor. In vivo, NT-10-G347E/E349D exhibited reduced pathogenicity in pigeons but increased pathogenicity in chickens compared to the parental NT-10 strain. The mutations also reduced the pigeon-to-pigeon transmission of NT-10 but enhanced its transmission from pigeons to chickens. Notably, significant antigenic differences were observed between NT-10 and NT-10-G347E/E349D, as an inactivated vaccine based on NT-10 provided full protection against NT-10 challenge in immunized pigeons but only 67% mortality protection against NT-10-G347E/E349D. Overall, these findings underscore the critical role of amino acids at positions 347 and 349 in PPMV-1 infection, pathogenicity, and transmission, providing a theoretical foundation for the scientific prevention and control of PPMV-1.

Newcastle disease virus (NDV), known as avian paramyxovirus-1, poses a significant threat to poultry production worldwide. Vaccination currently stands as the most effective strategy for Newcastle disease control. However, the mesogenic vaccine strain Mukteswar has been observed to evolve into a velogenic variant JS/7/05/Ch during poultry immunization. Here, we aimed to explore the mechanisms underlying virulence enhancement of the two viruses. Pathogenically, JS/7/05/Ch mediated stronger virulence and pathogenicity in vivo compared to Mukteswar. Comparative transcriptome analysis revealed 834 differentially expressed genes (DEGs), comprising 339 up-regulated and 495 down-regulated genes, in the spleen, and 716 DEGs, with 313 up-regulated and 403 down-regulated genes, in the thymus. Gene Ontology (GO) analysis indicated that these candidate targets primarily participated in cell and biological development, extracellular part and membrane composition, as well as receptor and binding activity. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis unveiled a substantial portion of candidate genes predominantly involved in cellular processes, environmental information processing, metabolism, and organismal systems. Additionally, five DEGs (TRAT1, JUP, LPAR4, CYB561A3, and CXCR5) were randomly identified through RNA-seq analysis and subsequently confirmed via quantitative real-time polymerase chain reaction (qRT-PCR). The findings revealed a marked up-regulation in the expression levels of these DEGs induced by JS/7/05/Ch compared to Mukteswar, with CYB561A3 and CXCR5 exhibiting significant increases. The findings corroborated the sequencing accuracy, offering promising research directions. Taken together, we comprehensively evaluated transcriptomic alterations in chicken immune organs infected by NDV strains of diverse virulence. This study establishes a basis and direction for NDV virulence research.