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.

The pathogenicity and transmissibility of H9N2 influenza viruses has been widely investigated; however, few studies comparing the biological characteristics of H9N2 viruses isolated from different hosts have been performed. In this study, eight H9N2 viruses, isolated from chickens (Ck/F98, Ck/AH and Ck/TX), pigeons (Pg/XZ), quail/(Ql/A39), ducks (Dk/Y33) and swine (Sw/YZ and Sw/TZ) were selected, and their biological characteristics were determined. The results showed that all H9N2 viruses maintained a preference for both the avian- and human-type receptors, except for Sw/TZ, which had exclusive preference for the human-type receptor. The viruses replicated well in DF-1 and MDCK cells, whereas only three isolates, Ck/F98, Ck/TX and Sw/TZ, could replicate in A549 cells and also replicated in mouse lungs, resulting in body weight loss in mice. All H9N2 viruses were nonpathogenic to chickens and were detected in the trachea and lung tissues. The viruses were shed primarily by the oropharynx and were transmitted efficiently to naïve contact chickens. Our findings suggest that all H9N2 viruses from different hosts exhibit efficient replication and contact-transmission among chickens, and chickens serve as a good reservoir for the persistence and interspecies transmission of H9N2 influenza viruses.

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.

The location and number of glycosylation in HA proteins exhibit large variations among H5 subtype avian influenza viruses (AIVs). To investigate the effect of glycosylation in the globular head of HA on the pathogenicity and antigenicity of H5N1 AIVs, seven rescued AIVs differing in their glycosylation patterns (144N, 158N and 169N) within the HA globular head of A/Mallard/Huadong/S/2005 were generated using site directed mutagenesis. Results showed that loss of glycosylation 158N was the prerequisite for H5 AIV binding to the α2,6-linked receptor. Only in conjunction with the removal of the 158N glycosylation, the H5 AIVs harboring both 144N and 169N glycosylations obtained an optimal binding preference to the α2,6-linked receptor. Compared with the wild-type virus, growth of viruses lacking glycosylation at either 158N or 169N was significantly reduced both in MDCK and A549 cells, while replication of viruses with additional glycosylation 144N was significantly promoted. Mutant viruses with loss of 158N or 169N glycosylation sites showed increased pathogenicity, systemic spread and pulmonary inflammation in mice compared to the wild-type H5N1 virus. In addition, chicken studies demonstrated that inactivated de-glycosylation 169N mutant induced cross-reaction HI and neutralization antibody against various clades of H5N1 AIVs. Moreover, this type of glycan pattern vaccine virus provided better cross-protection in chickens compared to wild-type vaccine virus. Thus, the glycosylation alteration of HA should be considered in the global surveillance and vaccine design of H5 subtype AIVs.

The present invention relates to H9N2 avian influenza virus NS1 gene deletion candidate live attenuated vaccine strain rTX-NS1-128 constructing method and application. The H9N2 subtype avian influenza virus NS1 gene deletion candidate live attenuated vaccine strain rTX-NS1-128, is A / chicken / Taixing / 10 / 2010TX female parent strain, the length of the NS1 gene is deleted, such that the NS1 protein retaining only shown in SEQ ID NO.15 as 128 amino acids. The vaccine candidate can be copied in the trachea, contact transmission occurs, safety. The candidate vaccine strain can be used alone as an effective tool H9 subtypes of avian influenza, can also be used in combination with inactivated vaccine, provide a more exact immune protection. It will play a very important role in H9 subtype of avian influenza prevention and control.

H9N2 avian influenza virus (AIV) evolves rapidly in both genovariation and antigenicity. It is essential to monitor the change of antigenicity, in particular in the hemagglutinin (HA) protein. Here we reported the selection of antigenic variants from A/Chicken/Shanghai/F/98 (H9N2) and A/chicken/Taixing/10/2010 (H9N2) viruses using HA-specific monoclonal antibodies (MAbs). Based on the reactivity of these variant and wild-type strains with the MAbs, we identified 6 critical amino acid positions (92, 145, 166, 167, 168, and 197) in the H9 antigenic sites, including the position 92 that has never been reported. Among AIVs originated from chicken in mainland China, the rates of Gly and Arg at position 92 within BJ/94-like (A/chicken/Beijing/1/1994) lineage viruses were 62.2% (28/45) and 37.8% (17/45), respectively; whereas the rates of Gly and Arg at position 92 within Y280-like (A/duck/Hong Kong/Y280/97) lineage viruses were 0.3% (2/670) and 99.1% (673/679), respectively. Our study suggests that G92R mutation together with other identified antigenic sites may serve as molecular markers for H9N2 virus evolution, and may aid improving AIV vaccine effectiveness.