Antigenic Evolution Of H9N2 Chicken Influenza Viruses And Cross-protection Of Cold Adapted Vaccine

To control H9N2 influenza virus in chickens, a widespread vaccination program has been implemented in mainland China. However, H9N2 viruses were still widespread and caused a country-wide outbreak during 2010-2013. Previous research demonstrated that H9N2 AIVs isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E). However, the antigenic evolution of prevailing H9N2 viruses isolated since 2009 is not well known. These facts emphasize that understanding the antigenicity of circulating viruses and updating matched vaccine strains are extremely urgent actions.The HI and neutralization assay was used to antigenically characterize the H9N2 viruses isolated in China from 2009 to 2013. Among the 26 tested viruses,2 belonged to the previously identified antigenic groups D and E, and all others formed a novel antigenic group, F. The group F viruses had relatively different antigenicities and can be categorized into 3 subgroups:Fl, F2, and F3. Furthermore,2009-2013 viruses are antigenically distinct from the commercial vaccine strains showed low reactivity to vaccine antisera, which may caused country-wide outbreak in vaccinated farms.To investigate the molecular characteristics of the HA genes of H9N2 influenza viruses in China, the deduced amino acid sequences were aligned by using all available HA sequences from 1994 through 2013. Fifteen mutations were identified as predominant in the 2009-2013 viruses, which might be responsible for the novel antigenicity of group F.. Nine of the mutations predominant in the 2009-2013 viruses can be mapped to known antigenic sites. Combining the results of HI and neutralization assays shows that Ck/HeB/YT/10(YT) had the best cross-reactive response to all of the prevalent H9N2 viruses. Furthermore, the YT bears subsititutions of HA gene predominant in 2009-2013 viruses. Therefore, we selected YT as an vaccine candidate and evaluated the efficacy of YT inactived vaccine against viruses from different antigenic groups. Upon challenge in the groups with low antibody levels(HI titer=26-29), immunization with Freund’s adjuvantea inactivated vaccine Ck/HeB/YT/10 provided complete protection against homologous virus, C group virus, E group virus and partial protection against other viruses, as shown by lower isolation rate in trachea and cloaca of inoculated chickens, decreased viral titers at 3 dpi and decreased virus isolation rate in contact group. However, in the high antibody level groups, vaccination elicited increased protection efficacy, and none of the tested viruses were detected in both inoculated and contact groups. In summary, these results demonstrated that vaccination with vaccine Ck/HeB/YT/10 efficiently inhibited the replication and transmission of H9N2 AIVs from different antigenic groups, especially in vaccinated chickens with high antibody levels. Therefore, it is of importance to analyse antigenic, genetic, and epidemiological data of circulating H9N2 viruses and monitor the efficacy of vaccine against epidemic strains in order to aid the prevention and control of H9N2 virus.The antigenic variation of avain influenza viruses make the efficacy of inactived vaccine is limited in the prevention of virus. Live attenuated influenza vaccine (LAIV) can induce influenza virus-specific antibody and T-cell responses and may be effective against antigenic drift/heterologous strains. Previous research demonstrated that live attenuated vaccine provide solid protection against heterologous virus challenge in mice. However, the role of live attenuated influenza vaccine (LAIV) in heterologous protection against influenza virus in chickens was not known. Novel reassortant H5N2 viruses have emerged and have been circulating in both domestic and wild birds in China since 2010, resulted in great economic losses. In the present study, we evaluated the cross protection of LAIV againt H5N2 AIV in chickens. In the H9N2 LAIV vaccinated group, the titers of challenge virus in the lungs, tracheae, kidneys were all significantly lower than those in the control mice. The survival rate of H9N2 LAIV vaccinated group is 100%, while that of control group is 0. H9N2 LAIV could induce significant IFN-7+CD4+ T cells and IFN-y+CD8+ T cells to heterologous H5N2 influenza viruses. When vaccinated chickens were challenged with H5N2 viruses, a significant increase in percentage of IFN-γ+CD4+ T cells and IFN-y+CD8+ T to heterologous H5N2 influenza viruses was observed starting on 4dpi compared with PBS-treated controls. This increase in virus specific T cells correlates with the period of time during which virus clearance in the lung of vaccinated chickens challenged with H5N2 influenza viruses. It suggested that cellular immunity may play an important role in chickens vaccinated with LAIV in terms of cross-protection against the H5N2 influenza viruses. Although neutralizing antibody and hemagglutination inhibition antibody against H5N2 influenza virus were not detectable in H9N2 LAIV vaccinated serum, we detected antibodies in H9N2 LAIV vaccinated serum capable of binding H5N2 influenza virus via ELISA assay. Therefore, heterologous protection induced by H9N2 LAIV can be attributed by T-cell response and cross-reactive IgG antibodies bing H5N2 viruses.To evaluate whether the antibodies produced by the H9N2 LAIV could confer heterologous protection, we passive transferred LAIV immune serum into SPF chickens 24 h before intranasal infection with lethal H5N2 virus. The survival percentage of LAIV-immune serum recipients was 100% by day 14 after challenge, and that of negative control serum recipients was 0. The results indicated that the LAIV vaccinated serum are capable to confer heterologous protection. Furthermore, we evaluated the protective efficacy against H5N2 viruses and NP-specific IgG titer of LAIV immune serum and homogenous inactived vaccine immune serum. The survival percentage of LAIV-immune serum recipients was 100%, while that of inactived vaccine immune serum recipients was 0. The NP specific IgG titer of LAIV-immune serum is higher than that of inactived vaccine immune serum. When passive transfer the combination of inactived vaccine immune serum and NP specific immune serum to reach the same IgG titer with LAIV-immune serum, the survival percentage of vaccinated chicken increased to 40%. The result showed that H9N2 LAIV immune serum could induce heterologous protection against H5N2 viruses which is related to NP-specific IgG titer. Our finding enhanced the understanding of the heterologous protection of LAIV.In summary, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak. LAIV could induce heterologous protection against H5N2 viruses which can be attributed by T-cell response and cross-reactive IgG antibodies bing H5N2 viruses. The study provides the theoretical foundation for the molecular epidemiology of H9N2 viruses and important guidance to the prevention of AIVs.