Construction And Application Of Recombinant Turkey Herpesvirus Expressing H7N9 Subtype HPAIV Hemagglutinin

Herpesvirus of turkey(HVT)is a double-stranded DNA virus with a genome of about160?180 kb.The large genome provides multiple insertion sites for foreign genes.HVT,a cell-associated herpesvirus,can be propagated in birds carrying high levels of maternal antibodies,it can maintain long-lasting immunity and mainly induce the production of cellular immunity and humoral immunity in the host.Based on the above characteristics,the recombinant HVT vector vaccine has the potential to become a multivalent vaccine.The highly pathogenic H7N9 subtype avain influenza virus(AIV),which were emerged in 2016,could result in acute death of poultry and infection of people,and cause serious economic losses and public health security.The transmission of AIV was reduced by vaccine strategy in poultry,thereby promoting the development of poultry industry and public health safety.AI vaccine which is a recombinant HVT vector vaccine is a new type of vaccine,could overcome the disadavantages of traditional inactivated vaccine,overcome parental antibody interference and long-lasting immunity.Alternatively,the commercial products have been listed abroad.Firstly,the method of constructing recombinant HVT was established in this study.The recombinant herpesvirus of turkey(r HVT-EGFP)expressing enhanced green fluorescent protein(EGFP)was obtained by inserting the EGFP-labeled gene expression cassette(MCMV+EGFP+SV40 poly A)into between the HVT genome HVT065 and HVT066 by using traditional homologous recombination technique.The efficiency of primary cell transfection was enhanced by means of electrotransfection,so that improving the efficiency of homologous recombination of transferred plasmid and virus genome.Screening and purifying cell-associated herpesvirus was tackled through using limited dilution method with SPGA as a protective agent.r HVT-EGFP was successful probably by PCR identifying and observating on fluorescence microscopy and the recombinant HVT method was successfully established.A recombinant Turkey herpesvirus(r HVT-H7HA)expressing H7 subtype avian influenza virus HA protein was constructed by inserting the gene expression box(MCMV+H7HA+SV40poly A)which expressing high pathogenic H7 subtype avain influenza virus HA protein into the HVT genome between HVT065 and HVT066 by using the established method above.Chicken red blood cells can be agglutinate by the HA protein expressed by r HVT-H7HA;protein expression can be detected intracellular by western blotting while CEF(Chick embryo fibroblast)were infected with r HVT-H7HA virus,and with the prolongation of the culture time of r HVT-H7HA,the quantity of HA protein were increased and then stabilized.The results the growth kinetics of r HVT-H7HA in CEF showed that with the extension of incubation time the virus titer of r HVT-H7HA was increased,reached the peak at 96 h and then decreased rapidly.The proliferation ability of r HVT-H7HA on CEF was lower than that parental strains.Passaging stability analysis of r HVT-H7HA on CEF stated clearly that r HVT-H7HA can stably propagate for at least 15 generations in vitro.SPF chicken were immuned with r HVT-H7HA and the average antibody titer was about 3log2 in fifth week.Subsequently,using 10⁵EID₅₀ highly pathogenic H7N9 avian influenza virus(A/Chicken/Huizhou/HZ-3/2016),the challenge experiment was carried out by intranasal administration in fifth week.Within 14 days after challenged,all group were observed.Indeed,after challenged,protection rate of immune group were reached 90%at large.