Screening And Identification Of High Yield Influenza Virus Strains Adapted To MDCK Cells

Influenza B Virus（IBV）is a respiratory pathogen.Seasonal IBV can cause epidemics in varying degrees,which has a serious impact on health worldwide every year,and is an important cause of death in the elderly and children.At present,annual influenza vaccination is the most effective measure to prevent and control influenza epidemics.Studies have shown that during the transmission process of influenza viruses,the glycoprotein hemagglutinin（HA）on the surface of the virus continues to mutate,enabling the influenza virus to evade immune recognition in the human body.Therefore,it is necessary to receive vaccines annually and update the vaccine components.This study aims to select IBV strains with stable passage and high replication ability in MDCK（Madin Darby Canine Kidney）suspension cells as vaccine skeleton strains,providing technical support for the development of seasonal influenza vaccines.The specific experimental content is as follows:Firstly,five strains of IBV,B/Phuket/3073/2013,B/Maryland/15/2016,B/Brisbane/9/2014,B/Darwin/7/2019,and B/Singapore/INFTT-16-0610/2016,were adaptively passaged in MDCK suspension cells.The hemagglutination titer,viral titer,and passability stability of the five influenza virus strains were compared through hemagglutination testing and CCID₅₀ testing.After evaluation,B/Singapore/INFTT-16-0610/2016 had the best subculture stability and high virus titer.At the same time,the virus titer was detected using the plaque method to verify the above results,and the conclusion is consistent.The results of electron microscopy showed that the morphology of the virus did not change and the number of virions increased during passage.Immunize mice with the selected high-yield strain and preliminarily evaluate the immunogenicity of the high-yield strain in mice through immune serum antibody detection.Secondly,the infectious cloning plasmids was constructed from the skeleton fragments PA,PB1,PB2,NP,NS,M,and HA,NA fragments from B/Singapore/INFTT-16-0610/2016,as well as HA and NA fragments from B/Phuket/3073/2013,to obtain the recombinant IBV through transfection experiments.In summary,we successfully screened a highly productive and stable IBV vaccine skeleton strain,which has been confirmed to have significant immunogenicity in animal experiments.We successfully rescued the recombinant IBV through reverse genetic manipulation technology.The screened B/Singapore/INFTT-16-0610/2016 skeleton strain may lay the foundation for the development and production of MDCK cell matrix influenza vaccine.