Construction And Immunogenicity Assessment Of Recombinant Fowlpox Virus Expressing HA Gene Of H9 Subtype Avian Influenza Virus

Avian influenza(AI)is an infectious disease syndrome caused by type A influenza virus, which including highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). In recent years,the widespread H9 subtye avian influenza virus has caused great economic loss in poultry industry[0] in China.The recombinant virus is a potential vaccine to control avian influenza ,several different recombinant fowlpox virus(rFPV) expressing AIV HA gene demonstrated effective protection against homologous AIV challenge in chickens.In this study fowlpox virus transfer vector pSY681- gfp-gpt with two reported genes(gfp and gpt) and two promoters ,P7.5(early) and P11(Later) in a back to back style was constructed. P11 initiated the expression of gfp gene and gpt gene, P7.5 was used for intiating exogenous gene. Two restriction sites NotI and Af1 II were inserted into downsteam of the early promoter P7.5, which were used for insertion of exogenous gene. HA gene of H9 subtype avian Influenza virus was inserted into designed site, then the recombinant vector pSY681-gfp-gpt-HA was transfected into CEF cells which infected with S-FPV-017 previously . Fowlpox virus recombinants were selected by gfp and gpt following several cycles purification.The results of PCR and Western-blot indicated that recombinant FPV could eficiently express HA gene of AIV in CEF.Twenty-one-days old SPF chickens were inoculated with the recombinant virus and challenged three weeks later with 106EID50 of LPAIV isolate per chicken.In order to evaluate the protection efficacy, throat and cloacal swabs were detected for virus existing in 3,5,7 days after challenge.HI test was also conducted before and after challenge.HI antibody could be dectected one week after inoculation and reached the level of 7log2 in three weeks. There was low virus shedding rate in the inoculation groups(15%) while 100% in control group. The high antibody level(HI test) and low virus shedding rate reflected that the recombinant fowlpox virus could produce satisfactary immune protection effect.The successfully construction of the universal transfer vector pSY681- gfp-gpt with dual selection system(gpt and gfp) would greatly improve the development of live vector vaccine for AI.The recombinant virus rFPV -HA9 also could be a candidate vaccine for the control of Avian influenza.