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There are three conserved N-linked glycosites, namely, Asn10, Asn23 and Asn286, in the stem region of haemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/ Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF cells in comparison with that of the WT virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role in HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128) were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.