Replication of influenza A virus (IAV) is highly reliant on host cell function, and to identify the key host factor required in the influenza A virus life cycle, a genome-wide CRISPR/Cas9 knockout (KO) screen was conducted in A549 cells infected by H1N1 influenza virus. The results showed that glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) plays a crucial role in the replication of influenza A virus, and knockout of the GNE significantly reduced the replication of multiple subtype influenza A viruses in A549 cells, and restoration of the GNE expression in the GNE-knockout cells partially recovered IAV infection. Additionally, overexpression of the GNE in wild-type (WT) cells promoted IAV infection to a certain extent. Further study showed that the GNE is involved in α-2,3- and α-2,6-linked sialic acid (Sia) synthesis, which is the major receptor of the influenza A virus, and the GNE knockout downregulated the α-2,3- and α-2,6-linked sialic acid expression. In summary, the knockout of the GNE inhibits adsorption and endocytosis of IAV. This study provides a new approach to elucidate the replication mechanism of IAV and identifies GNE as a potential target for anti-influenza drug development. IMPORTANCE: Influenza A virus infection requires the assistance of the host proteins. Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) was identified as an important host factor in influenza A virus infection by genome-wide CRISPR screening. GNE knockout (KO) extensively inhibited the replication of multiple subtype influenza A viruses. It indirectly participated in IAV adsorption and endocytosis by regulating the expression of sialic acid (Sia), the main receptor of IAV. This finding provides novel insights into the replication mechanism of IAV.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses to the poultry industry in China since 2010. However, the potential threat of TMUV to mammals has not been well studied. In this study, a TMUV HB strain isolated from diseased ducks showed high virulence in BALB/c mice inoculated intranasally compared with the reference duck TMUV strain. Further studies revealed that the olfactory epithelium is one pathway for the TMUV HB strain to invade the central nervous system of mice. Genetic analysis revealed that the TMUV HB virus contains two unique residues in E and NS3 proteins (326K and 519T) compared with duck TMUV reference strains. K326E substitution weakens the neuroinvasiveness and neurovirulence of TMUV HB in mice. Remarkably, the TMUV HB strain induced significantly higher levels of IL-1β, IL-6, IL-8, and interferon (IFN)-α/β than mutant virus with K326E substitution in the brain tissue of the infected mice, which suggested that TMUV HB caused more severe inflammation in the mouse brains. Moreover, application of IFN-β to infected mouse brain exacerbated the disease, indicating that overstimulated IFN response in the brain is harmful to mice upon TMUV infection. Further studies showed that TMUV HB upregulated RIG-I and IRF7 more significantly than mutant virus containing the K326E mutation in mouse brain, which suggested that HB stimulated the IFN response through the RIG-I-IRF7 pathway. Our findings provide insights into the pathogenesis and potential risk of TMUV to mammals.

Avian viral arthritis (AVA), caused by avian reovirus (ARV), is a viral disease in chickens that has led to significant economic losses in the poultry industry. Recent studies have shown that traditional ARV vaccines based on the S1133 strain fail to protect against emerging ARV variants. In this study, we isolated and characterized three ARV strains (G4, YV, WF) from immunized chicken flocks with respiratory and arthritic symptoms. Genomic analysis revealed that the σC genes of G4, YV, and WF shared only 55.5%, 55.7%, and 58.7% sequence homology, respectively, with the S1133 strain. Phylogenetic analysis placed them in different branches, indicating they are variant strains. YV and WF belong to genotype III, and G4 falls into genotype VI. Whole genome analysis revealed gene segment reassortment among the variants. Pathogenicity testing in three-week-old SPF chickens showed that G4 (genotype VI) caused swelling of footpads, whereas WF (genotype III) did not. G4-infected chickens exhibited significantly higher viral loads in the thymus, lungs, spleen, and bursa of Fabricius than those in the WF-infected chickens, indicating viruses from different genotypes showed various pathogenesis. These results suggested an urgent need for new updates of vaccines against the variant ARVs, especially the genotype VI virus.

Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China, 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants, possessing internal genes of identical origins (PB2, PB1, PA, NP, M: pdm09/H1N1 origin, NS: North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mice. Taken together, our study indicates that the H1 subtype SIVs recently circulating in China pose a potential threat to human health and emphasizes the importance of continuing to closely monitor their evolution and spread.