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Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HAA180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 (F/98, A180), SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus. [ABSTRACT FROM AUTHOR]

H9N2 virus has become the most widespread subtype of avian influenza in Chinese poultry. Although many studies have been published on this disease, the pathogenesis of the H9N2 virus remains to be fully understood. In our previous work, we identified 44 viral strains with 67–76 amino acid deletions in the neuraminidase protein (NA ∆67–76) from trachea and lung tissues after 20 successive generations in vaccinated chickens. Interestingly, these 10 amino acid deletions are located in the stalk of the NA protein, and all mutations were unique to the viruses under the selection pressure of vaccine antibodies. To investigate the effect of NA ∆67–76 on the H9N2 virus, the NA ∆67–76 deletion mutant (rF/NA Δ67–76) was constructed in the H9N2 virus A/Chicken/Shanghai/F/98 (F/98) to assess the phenotypic changes between the parental and mutant strains. The results showed that the recombinant virus rF/NA Δ67–76 had no significantly effect on the antigenicity of the virus or on the infectivity of the host cells, but it significantly inhibited the release of virions from host cells. In addition, rF/NA Δ67–76 efficiently enhanced the neuraminidase activity and improved the receptor binding ability of the virus, indicating that the influence of receptor binding ability on the rF/NA Δ67–76 virus is much greater than that of neuraminidase activity. Furthermore, this study revealed that rF/NA Δ67–76 reduced the viral replication ability at 6 and 12 h post-infection, but improved it at 24, 48, and 72 h post-infection. Chicken experiments showed that rF/NA Δ67–76 exhibits a much higher tissue tropism for the trachea rather than lung tissue. rF/NA Δ67–76 still had the ability to infect the upper respiratory tract through aerosol, but its cloaca replication capacity was significantly reduced. Both in vivo and in vitro experiments confirmed that rF/NA Δ67–76 could produce a stronger innate immune response after infecting cells and chickens, especially significantly enhancing the transcription levels of TLR3, TLR4, TLR7, TLR21, MDA5, and NLRP3. Altogether, the results of this study propose that antibody selection pressure plays an important role in the evolution of H9N2 avian influenza virus. • Deletion of 67-76 amino acids in NA protein were unique to the viruses with the selection pressure of vaccine antibodies. • rF/NA Δ67-76 efficiently enhanced the neuraminidase activity and improved the receptor binding avidity. • rF/NA Δ67-76 exhibits a much higher tissue tropism for trachea rather than lung tissue. • rF/NA Δ67-76 still had the ability to infect the trachea by aerosol, but its cloaca replication capacity was reduced. • rF/NA Δ67-76 could produce stronger innate immune response after infecting cells and chickens. [ABSTRACT FROM AUTHOR]