Construction And Protective Efficacy Of Recombinant Herpesvirus Of Turkey Vaccine Strains Expressing Hemagglutinin Of Subtype H9 Avian Influenza Virus

H9N2 subtype of avian influenza virus(AIV)has been widespread in China since it’s first discovered and reported in 1992,which has seriously endangered the development of poultry industry in China.Studies have shown that H9N2 subtype of AIV can infect humans and mammals without adaptation,and it can provide partial or the whole cassette of internal genes to other influenza viruses which can infect people,so that it has a significance in sanitary science.In China,the unique geographical environment,breeding mode and the existence of live poultry market provide favorable conditions for the occurrence and spread of H9N2 subtype of AIV,and immune pressure is the important factor of recombination and variation of H9N2 subtype of AIV.The sequence analysis of hemagglutinin(HA)gene of H9N2 subtype of AIVs obtained from 2010 to 2015 shows that most of the epidemic strains isolated in our country belonged to H9.4.2.5.However,the strain of conventional inactivated vaccine is not belong to this clade,and inactivated virus vaccine can not effectively induce cellular immune response and mucosal immune response,so the immune protection rate of inactivated vaccines is not high sometimes.Herpesvirus of turkey(HVT)is the usual vaccine to prevent chicken Marek’s disease,it has more non-essential regions for replication that can be used to insert the exogenous genes.So,it is widely used in the construction of recombinant vaccine.In this study,HVT was used as the vector to construct a recombinant virus rHVT-H9HA expressing HA of H9N2 subtype of AIV.In this study,an expression cassette include the HA gene of the H9 subtype of AIV strain(YZ1406)that located on the h9.4.2.5 branch was amplified by PCR,to constitute an expression cassette with the human cytomegalovirus early promoter(CMV)and TK polyA.And inserting expression cassette into the US2 region from the turkey herpes virus(HVT)FC126 vaccine,to yield the transfer vector pHVT-H9HA.pHVT-H9HA and the genome of recombinant virus rHVT-EGFP which was expressing EGFP were co-transfected into chicken embryo fibroblasts(CEF),The recombinant virus rHVT-H9HA was obtained by replacing the EGFP gene of rHVT-EGFP with HA gene of H9 subtype of AIV by homologous recombination.The recombinant virus rHVT-H9HA was identified by PCR,Southern-blot,Western-blot and indirect immunofluorescence assay,and its growth characteristics on CEF were detected.The results showed that HA gene expression cassette was correctly inserted into the US2 non-essential region for replication and the target protein HA could be expressed at about 85kD in rHVT-H9HA.At the same time,rHVT-H9HA showed the same replication level on CEF as the HVT FC126 vaccine strain.This study preliminarily evaluated the immunogenicity of recombinant virus rHVT-H9HA.Eighty 1-day-old SPF chickens were randomly divided into four groups:rHVT-H9HA immunized group,inactivated vaccine immunized group,non-vaccinated but challenged group and blank control group.Each bird of the recombinant vaccine immunized group was subcutaneously inoculated with 5000 plaque forming unit(PFU)of rHVT-H9HA,inactivated vaccine immunized group was inoculated with 0.25 mL of avian influenza virus H9 subtype of inactivated vaccine each feather at 14 days of age through intramuscular route.The antibody titers of H9 subtype of AIV were measured by hemagglutination inhibition test(HI)at 7,14,21 and 28 days of age.In addition to the blank control group,the other groups were challenged via eye intranasal method with 108 EID50 H9 subtype of AIV dose at 28 days of age.At 3 and 5 days after the challenge swab samples of throat and cloacal were collected to test the rate of virus shedding.The results showed that,the antibody level of rHVT-H9HA immunized group continued to rise after vaccination and on the fifth day after the challenge,the virus isolation rate was significantly reduced,while the samples from non-vaccinated but challenged group were still positive.The results indicated that the recombinant virus rHVT-H9HA had a good protective effect on the attack of H9 subtype AIV.