The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.

The S2 subunit of infectious bronchitis virus (IBV) plays a critical role in the process of IBV infection. A comparison between the S2 subunit sequence of chicken embryo kidney cell (CEK) adapted virulent QX-like IBV strain SczyC30 (hereafter referred to as zy30) and its CEK-attenuated strain, SczyC100, revealed an N1038S substitution in S2 subunit and a 1154EQTRPKKSV1162 residue deletion in the C-terminus of the S2 subunit. In order to explore whether these two mutations are related to changes in the biological characteristics of IBV, we firstly constructed an infectious clone of zy30 using a bacterial artificial chromosome (BAC), which combines the transcription of infectious IBV genomic RNA in non-susceptible BHK-21 cells with the amplification of rescued virus rzy30 in CEK cells. Then, three recombinant viruses, including an rzy30S2-N1038S strain that contained the N1038S substitution, an rzy30S2-CT9△ strain that contained the 1154EQTRPKKSV1162 deletion, and an rzy30S2-N1038S-CT9△ strain that contained both mutations, were constructed using rescued virus rzy30 as the backbone. The results showed that each mutation did not significantly affect the replication titer in CEK cells but reduced pathogenicity in chickens, while in combination, the N1038S substitution and 1154EQTRPKKSV1162 deletion improved the proliferation efficiency in CEK cells and reduced pathogenicity, compared to rzy30 strain. The contribution made by the 1154EQTRPKKSV1162 deletion in reducing pathogenicity was higher than that of N1038S substitution. Our results revealed that the N1038S substitution and 1154EQTRPKKSV1162 deletion in S2 subunit were deeply involved in the replication efficiency of IBV and contributed to reduction of viral pathogenicity.

The Chinese poultry industry has experienced outbreaks of Newcastle disease (ND) dating back to the 1920s. However, the epidemic has exhibited a downtrend in recent years. In this study, both observational and genetic data [fusion (F) and haemagglutinin-neuraminidase genes (HN)] were analyzed, and phylogeographic analysis based on prevalent genotypes of Newcastle disease virus (NDV) was conducted for better understanding of the evolution and spatiotemporal dynamics of ND in China. In line with the observed trend of epidemic outbreaks, the effective population size of F and HN genes of circulating NDV is no longer growing since 2000, which is supported by 95% highest posterior diversity (HPD) intervals. Phylogeographic analysis indicated that the two eastern coastal provinces, Shandong and Jiangsu were the most relevant hubs for NDV migration, and the geographical regions with active NDV diffusion seemed to be constrained to southern and eastern China. The live poultry trade may play an important role in viral spread. Interestingly, no migration links from wild birds to poultry received Bayes factor support (BF > 3), while the migration links from poultry to wild birds accounted for 64% in all effective migrations. This may indicate that the sporadic cases of ND in wild bird likely spillover events from poultry. These findings contribute to predictive models of NDV transmission, and potentially help in the prevention of future outbreaks.

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