Study On Construction Of Reverse Genetic System Of Influenza D Virus And Immunogenicity Of HE Gene

Influenza D virus is a newly described influenza type of the virus influenza family that was first isolated from diseased swine in 2011 and has subsequently been detected in cattle around the world in 2014,the full range of susceptible hosts for this novel virus includes swine,bovine and small ruminants.In addition,serological evidence for IDV infection in humans has been recently established.At the same time,influenza D virus is also considered to be closely related to the respiratory diseases of cattle.The HEF protein is the only glycoprotein on the surface of influenza D virus,which determines the characteristics of infection.Therefore,the studies on the genetic characteristics of influenza D virus and the immunogenicity of HEF protein have played an important role in the prevention of influenza D virus.For the first time,we attempted to construct a reverse genetic manipulation platform for influenza D virus and construct an effective diagnostic antigen against influenza HEF protein using the baculovirus expression vector system,meanwhile through the nucleic acid to immune the mice,antiserum the HEF protein was prepared.The experiment was mainly divided into two parts.The first part: By the pHH21 and pCAGGS vectors of the thirteen plasmids reverse genetic system which was used to rescue nfluenza A virus,the twelve plasmids reverse genetic manipulation platform of influenza D virus was constructed by molecular cloning and other means.At the same time,based on the pBD vector that widely used in the eight-plasmid reverse genetic system of influenza A virus,the seven-plasmid reverse genetic manipulation system for influenza D virus was constructed.Then the recombinant plasmids from two systems were co-transfected into 293 T cells,respectively,and the transfected 293 T cell supernatants were infected in cells and guinea pigs,meanwhile,rescued viruses were identified by Real-time RT-PCR and electron microscopy.The second part: the HEF protein was expressed by using baculovirus expression vector system,the immunogenicity of HEF protein was tested by blood coagulation and Western blotting.The pCAGGS vector was used to construct a recombinant plasmid for eukaryotic expression of HEF protein.which was used to immune the mice with CpG-ODN,and the antibody titer tested by hemagglutination inhibition.Results:(1)The twelve and seven plasmids genetic systems of influenza D virus was successfully constructed;(2)The virions from that the seven plasmid reverse genetic system co-transfeced in the 293 T cell was successfully observed;(3)The rescued virus could not infect on A549,MDCK,ST cells,but a small amount of copy could be detected in the upper respiratory tract of guinea pigs,but the infection effect is still low;(4)The recombinant baculovirus system that can be used as a diagnostic antigen for influenza D virus was successfully constructed;(5)Antiserum of HEF protein with higher hemagglutination inhibitory titer was prepared by immunizing mice with CpG-ODN molecular adjuvant.