Influenza H9N2 subtype viruses and their reassortants (such as H7N9) are posing increasing threats to birds and humans in China. During 2009–2013, multiple novel subtype viruses with H9N2 original genes emerged in China. Yet, the genetic evolution of H9N2 viruses in various host organisms in China has not been systematically investigated since 2009. In the present study, we performed large-scale sequence analysis of H9N2 viral genomes from public databases, representing the spectrum of viruses isolated from birds, mammals and humans in China from 1994 to 2013, and updated the clade classification for each segment. We identified 117 distinct genotypes in 730 H9N2 viruses. We analyzed the sequences of all eight segments in each virus and found three important time points: the years 2000, 2006 and 2010. In the periods divided by these years, genotypic diversity, geographic distribution and host range changed considerably. Genotypic diversity fluctuated greatly in 2000 and 2006. Since 2010, a single genotype became predominant in poultry throughout China, and the eastern coastal region became the newly identified epidemic center. Throughout their 20-year prevalence in China, H9N2 influenza viruses have emerged and adapted from aquatic birds to chickens. The minor avian species and wild birds exacerbated H9N2 genotypes by providing diversified genes, and chickens were the most prevalent vector in which the viruses evolved and expanded their prevalence. It is the necessity for surveillance and disease control on live-bird markets, poultry farms and wild-bird habitats in China.

Chicken nephropathogenic infectious bronchitis (IB) was prevalent in the most chicken farms during recent years, although the IB vaccination program has been widely performed in China. To characterize the S1 protein of infectious bronchitis virus (IBV) from China, five representative nephropathogenic IB viruses isolated from chickens in different provinces were genetically and phylogenetically analyzed. The results showed that the length of the S1 genes of the isolates were quite different (1,617, 1,620, 1,623, 1,629, and 1,632 nucleotides, respectively). The homology of the nucleotides and amino acids among the five isolates were 76.7%-92.1% and 73.9%-89.5%, respectively, indicating a great variation in S1 genes of the isolates. The variation in S1 genes might affect the antigenicity and pathogenicity of the viruses. Genetically, point mutations, insertions, and deletions in the S1 protein can be observed at many positions, especially at the first 150 amino acids in the N-terminal of the S1 protein. Two motif cleavage sites (R-R-X-R-R/S, H-R-R-R-R/S) were observed in the five sequenced strains. Phylogenetic analysis suggested that they belonged to different lineages. These findings indicated that different genotypes of nephropathogenic IB viruses were co-circulating in the chicken population in China.

Previous studies have identified a functional role of PA-X for influenza viruses in mice and avian species; however, its role in swine remains unknown. Toward this, we constructed PA-X deficient virus (Sw-FS) in the background of a Triple-reassortment (TR) H1N2 swine influenza virus (SIV) to assess the impact of PA-X in viral virulence in pigs. Expression of PA-X in TR H1N2 SIV enhanced viral replication and host protein synthesis shutoff, and inhibited the mRNA levels of type I IFNs and proinflammatory cytokines in porcine cells. A delay of proinflammatory responses was observed in lungs of pigs infected by wild type SIV (Sw-WT) compared to Sw-FS. Furthermore, Sw-WT virus replicated and transmitted more efficiently than Sw-FS in pigs. These results highlight the importance of PA-X in the moderation of virulence and immune responses of TR SIV in swine, which indicated that PA-X is a pro-virulence factor in TR SIV in pigs.

During 2012–2017, we collected throat swabs from dogs in China to characterize canine influenza virus (CIV) A(H3N2) isolates. A new antigenically and genetically distinct CIV H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and replaced previously circulating strains. This clade probably poses a risk for zoonotic infection.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.