Construction Of H13N8 Wild Bird Influenza Replication Deficient Virus And Stability Analysis Of Its HA Protein

Avian influenza virus(AIV)can be divided into 16 HA subtypes and 9 NA subtypes according to the difference between hemagglutinin(HA)and neuraminidase(NA).Wild birds are the natural host of AIV and can infect all subtypes of AIV.The monitoring and evaluation of wild bird influenza virus can provide a certain theoretical basis for the early warning and prevention and control of new influenza virus.H13 subtype wild bird influenza virus has been found in Qinghai Province,Shandong Province and Liaoning Province.After analysis,it is found that some strains recombine with duck AIV,which has the risk of cross host transmission.HA protein is an important structural protein of influenza virus.The stability of HA protein can affect the infection activity and invasion ability of the virus.Therefore,it is of great significance to study the HA protein of H13 wild bird influenza virus.When studying the molecular mechanism of the effect of HA protein on the biological characteristics of virus,it is necessary to recombine or change the HA gene of virus,which may lead to biosafety problems.Replication deficient viruses lack some genes related to virus replication and can only replicate in cells or animals stably expressing foreign proteins,which ensures the biosafety of influenza virus research.Therefore,in this study,the H13N8 parent strain was rescued by reverse genetic technology,and then most of the coding region of PB2 gene of H13N8 subtype wild bird influenza virus was replaced with enhanced green fluorescence reporter gene EGFP gene,and the replication defective virus H13N8-PB2-KO with fluorescence was rescued.H13N8-PB2-KO virus can only replicate in cells expressing PB2 protein,but can not replicate in normal cells and chicken embryos,which improves the biosafety of the virus.Studies have shown that the replication ability and other biological characteristics of H13N8-PB2-KO virus and its parent strain are similar,and can be stably subcultured for 6 times.In this study,the HA protein of Liaoning isolate A/Eurasian curlew/Liaoning/ZH-385/2014(H13N8)was selected as the research object for stability analysis.H9 subtype AIV and H13 subtype wild bird influenza virus belong to low pathogenic avian influenza virus,and their host range and epidemic range are wide.Therefore,the HA protein of Liaoning isolate A/chicken/Liaoning/07/2016(H9N2)was selected as the control.Firstly,the HA protein acid stability test and thermal stability test were carried out.The results showed that the HA protein acid stability and thermal stability of H13N8 were significantly stronger than that of H9N2.The results of HA protein mediated cell fusion test and trypsin sensitivity test showed that the HA protein of H13N8 could be cleaved by TPCK trypsin,and its membrane fusion p H was 5.0,which was lower than that of H9N2.In this study,H13N8 subtype wild bird influenza replication deficient virus was constructed,which provides a biosafety guarantee for the further study of the biological characteristics of AIV subtypes,especially the pathogenicity and transmission of highly pathogenic virus strains.The study on the HA protein stability of H13N8 subtype wild bird influenza virus found that H13N8 subtype wild bird influenza virus has stronger stability than H9N2 subtype AIV,which lays a foundation for further exploring the host barrier of H13N8 subtype wild bird influenza virus.