Loss of amino acids 67-76 in the neuraminidase protein under antibody selection pressure alters the tropism, transmissibility and innate immune response of H9N2 avian influenza virus in chickens.

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) 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 on the H9N2 virus, the NA deletion mutant (rF/NA) 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 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 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 virus is much greater than that of neuraminidase activity. Furthermore, this study revealed that rF/NA 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 exhibits a much higher tissue tropism for the trachea rather than lung tissue. rF/NA 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 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.