Development Of H9N2 Avian Influenza Virus Monoclonal Antibodies And Their Application In Antigenic Variation

H9N2 subtype avian influenza viruses (AIV) have widely circulated in the world since its first detection and have made huge economic losses for poultry industries and have significant impacts on human health.Vaccination is used as an important strategy in the control of AIV in poultry. Because the viral RNA polymerase has no proof-reading function, the RNA genome of AIV is not stable.Thus, AIV alters its antigenic properties frequently. However, the vaccine antibody pressure makes AIV easier to alter its antigenic properties, contributing to the failure of immune and the emergence of new pandemic.In this study, Five monoclonal antibodies which against hemagglutitin of H9N2 subtype of AIV were generated from the BALB/C mice immused with A/Chicken/Shanghai/F/98 (H9N2) AIV.The antigenic variation of the viruses with/without the homologous vaccine antibody pressure were tested using the five monocloneal antibodies, which we made. As the passage number increased, the haemagglutination inhibition (HI) titers of passaged viruses decreased. Further more, the HI titers of the viruses with the homologous vaccine antibody pressure decreased earlier than those of the viruses without the homologous vaccine antibody pressure,indicating that the homologous vaccine antibody pressure could accelerate the virus to alter its antigenic properties. Further studies showed that the mutation at the position G181E of HA gene in passaged virus might be recognized by one of the monoclonal antibody. The results in this study may provide a useful tool for diagnosis, the epidemiological surey of avain influenza virus.1. Developmemt of monoclonal antibodies againat the hemagglutinin of H9N2 subtype of avain influenza virusA/Chicken/Shanghai/F/98 (H9N2) AIV was propagated in the allantoic cavities of 10-day-old embryonated chicken eggs and purified by differential gradient centrifugation. Then immunized eight-week-old BABL/c mice. Monoclonal antibodies were generated through the fusion of myeloma SP2/0 cells with splenocytes. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) and positive subclones were obtained by limiting dilution method. All these were repeated three times. Five hybridoma cell lines of A/Chicken/Shanghai/F/98 H9N2 subtype AIV were obtained, named 1D6,3G8,4A5,6B11 and 6C12 strains. Their HI titer of the ascites were 29,212,27,214 and 29 respectively. The ELISA titers of the ascites were 1.0×105,1.6×106,3.2×106,3.2×106 and 2.0×105 respectively.By haemagglutination inhibition assay, the five monoclonal antibodies were specific to H9 subtype AIV,but not reacted with H5 subtype AIV, Newcastle disease vius, avian infectious bronchitis virus and avian egg drop syndrome virus. Five monoclonal antibodies could react with 75kD HA protein of H9N2 subtype AIV confirmed by Western blot method. All the results confirmed that the five monoclonal antibodies were specific to the hemagglutinin of H9N2 subtype of avain influenza virus. H9N2 subtype avain influenza virus antigen could be detected with the five monoclonal antibodies by immunohistochemistry in the trachea of SPF chickens inoculated by A/Chicken/Shanghai/F/98 (H9N2) and mF47 AIV.2. Application of monoclonal antibodies in antigenic variationIn the prvious study, we obtained the viruses from the passage 15,30,47,50 and 52 after continuous passaging in the allantoic cavities of 10-day-old embryonated chicken eggs with/without the homologous vaccine antibody pressure. In order to find out the relationship between the homologous vaccine antibody pressure and the antigenic variation, we compared the HI titers of the virus with/without the homologous vaccine antibody pressure by using five monoclonal antibodies. We found that as the passage number increased, the haemagglutination inhibition (HI) titers of the passaged viruses decreased. Further more, the HI titers of the viruses with the homologous vaccine antibody pressure decreased earlier than the the viruses without the homologous vaccine antibody pressure, indicating that the homologous vaccine antibody pressure could accelerate the virus to alter its antigenic properties.In the prvious study, we discovered that there were four amino acids changed in the HA gene of antigenic variation viruses. To find out the relationship between the amino acid change in the HA gene and the antigenic variation, we constructed the four mutations in HA, called rFHA1,which included mutations of K131R, S145N,G181E and A198V,the three mutations in HA, called rFHA2,which included mutations of K131R, G181E and A198V, and the single mutation in HA, called rFHA181, which included mutation ofG181E. The HA genes were each cloned into a pCAGGS expression plasmid and transfected into 293T cells.Cell-based ELISA was done using the five monoclonal antibodies.The results indicated that the monoclonal antibody 1D6 reacted well with the HA of A/Chicken/Shanghai/F/98 H9N2 subtype AIV,but didi not react with pCA-rFHA1,pCA-rFHA2 and pCA-rFHA181,while the other four monoclonal antibodies reacted well with pCA-rFHAl, pCA-rFHA2 and pCA-rFHA181.Thus, we supposed that the mutation at the position G181E might be recognized by the 1D6 monoclonal antibody, and might be critical to the antigenic variation. Because we did not have plenty of monoclonal antibodies, further studies should be taken in order to make clear of the other three mutation positions.