Study On The Antigenic Variation Of H9N2 Avian Influenza Viruses

H9N2 avian influenza is prevalent in the world,which not only causes serious damage to the poultry industry,but also poses a potential threat to human beings.Due to immune pressure and the variability of avian influenza viruses,the antigenic variation of H9N2 avian influenza viruses often happen,which results in the failure in vaccine immunization in the end.Therefore,it is of great significance to explore the regularity of antigenic variation of H9N2 avian influenza viruses.Comparing with the earlier H9N2 avian influenza isolates,the antigenicity of the viruses isolated in2009 showed significant changes.Therefore,the HA gene of A/chicken/Shanghai/441/2009?H9N2??SH441?was selected to construct eukaryotic expression plasmid pCAGGS-H9N1.To develop the hybridoma cells,the mice were immunized with the plasmid for 3 times,and they were injected intramuscularly with SH441 virus.After the mouse with the highest HI titer was selected to be boosted,the mouse spleen cells were isolated and fused with SP2/0 cells.The hybridoma cells were screened by ELISA and IFA,and 20 of these cells secreting monoclonal antibodies against H9 were cloned.According to HI test,15 monoclonal antibodies had HI activity,of which 4E7 and 10G8 had the highest titer of 2¹⁰,while 4B7 had the lowest titer of 2⁴.Except 3C8 and 8F11,the other monoclonal antibodies had neutralization activity,and 3F7 had a high neutralization titer of 1:128 in micro-neutralization test on cells.The neutralizing activity and the HI activity of monoclonal antibodies were inconsistent,which indicated that some monoclonal antibodies with neutralization activities did not bind to the amino acids related the hemagglutination in HA.Western blot showed that 14 monoclonal antibodies bound to a linear conformation of polypeptides of HA,and that 6 monoclonal antibodies bound to a spatial conformation of polypeptides of HA.These 20 monoclonal antibodies belonged to?subtype,and 19 of them belonged to the IgG subtype and one of them?8F11?belonged to IgM.And all of 20 monoclonal antibodies showed binding activities to the HA1 region of H9.In order to explore the antigenic variation of H9N2 avian influenza virus in China in recent years,18 strains isolated during 1998 to 2015 were selected according to the isolation time and place to be tested by HI and IFA respectively with these 20 monoclonal antibodies.The results showed that the viruses isolateed before 2009 were less reactive with the monoclonal antibodies obtained in this study.There were no HI activities of all monoclonal antibodies to F strain,and the F strain only reacted with 12 monoclonal antibodies by IFA which the fluorescence was weak.It was found that 9 relatively broad-spectrum monoclonal antibodies could react with the isolates from 2009-2015,while the other monoclonal antibodies showed significantly different to different strains by HI and IFA.The results were consistent with the antigen variation of H9N2 avian influenza viruses reported previously,indicating these monoclonal antibodies could be used for the antigenic analysis of H9N2 avian influenza viruses.In order to explore the molecular mechanism of the antigenic variation between SH441 and F strain,the plasmids expressing HA protein containing chimeric HA1 of F and SH441 were constructed.The IFA results showed that the antigenic variation between SH441 and F was mainly due to the amino acid difference in the region of 120 to 240 on HA1.There were 7 amino acid changes at positions 127,145,168,180,181,216 and 234 between SH441 and F by sequence analysis.The mutant viruses of these seven sites of F and SH441 were rescued by reverse genetics.The results of HI and IFA showed that the168th and 234th amino acids of HA gene resulted in antigen difference between SH441 and F strain.In summary,20 monoclonal antibodies against HA protein of H9N2 avian influenza virus were prepared and applied preliminarily in the study of antigenic variation of the virus.The findings would provide materials in the development of H9N2 avian influenza virus antigen analysis and analysis of antigen maps.