Analysis Of Representative Strains And Preparationy Pathogenic Avian Influenza Virus Of Monoclonal Antibodies Of H7N9 Highl

After the first detection of human infection with the new avian influenza virus(H7N9)in March 2013,in China,a total of 1568 laboratory-confirmed human cases and at least 615 related deaths have been reported,as of April 3,2019.The fifth and largest wave of LPAI H7N9 occurred in southern China in the 2016-2017 influenza season,during which HPAI H7N9 viruses emerged and infected humans,suggesting that the H7N9 AIV has became high-pathogenic.Therefore,the development of HA specific high-pathogenic H7N9 AIV monoclonal antibody is of great application value.In this study,we first analyzed the genetic evolution of 8 H7N9 subtype avian influenza viruses isolated and identified in our laboratory by referring to 80 representative strains of H7N9 subtype influenza viruses selected from Gen Bank and GISAID databases among all H7N9 subtype influenza viruses since 2013.According to the results of genetic evolution analysis,the representative strain HZ-3 was screened from 8 strains of H7N9 subtype avian influenza virus as immunogen to produce monoclonal antibodies.After three times of subcloning,six hybridoma cell stains 2E2C5,3C6D3,5D2C9,8H5A9,9F8E3 and21B6F9 were developed,which were determined by immunoperoxidase monolayer assay(IPMA).The results of antibody subtype identification showed that all the six Mc Abs were Ig G.The specificity identification results showed that none of the 6 monoclonal antibodies cross-reacted with other subtypes of avian influenza.The broad-spectrum identification results showed that 2E2C5,3C6D3,8H5A9,9F8E3 and 21B6F9 had better broad-spectrum characteristics,which could react with other H7N9 avian influenza virus.IPMA titers analysis show that 2E2C5,3C6D3 got highest IPMA titers.Western-blot analysis showed that 2E2C5,3C6D3,9F8E3 and 21B6F9 all targeted HA protein of H7N9 subtype avian influenza virus.Hemagglutination inhibition test showed that only 2E2C5,3C6D3,9F8E3 and 21B6F9 had hemagglutination inhibition activity.Neutralization test showed that the above four Mc Abs also had broad-spectrum neutralization.This study successfully screened the specific Mc Abs of H7N9 AIV HA,which laid a foundation for the later establishment serological detection method of H7N9 AIV.