Construction And Immunogenicity Assessment Of Recombinant Fowl Pox Virus Expressing HA Gene Of H7 Subtype Avian Influenza Virus

Avian influenza(AI)is an infection and/or disease syndrome caused by type A influenza virus. It is widespread in many countries and areas throughout the world, and of great economic losses in poultry industry. The great characteristic of the highly pathogenic avian influenza (HPAI) is high mortality, and it is classified A virulent infectious disease by O.I.E. In China,.H5N1 subtype of AIV infected human directly, especially caused them dead, which was the first time that AIV overacrossed interspecies barrier and infected human. H5 subtypes highly pathogenic avian influenza virus (HPAIV) becomes the big issue of the current research. There are a few of reports about H7 subtype HPAIV and have not enough biology products referred to them. Although the H7subtype have not widespread in recently years, we can't forecast which substype of HPAVI will widespread. The H7 subtype recombinant fowlpox virus constructed in this study would provide the storage of vaccine and technology for preventing H7 subtype HPAI.In this study,the HA gene of A/Chicken/Hebei/1/02(H7N2) which was the only isolation from China was cloned into vector pSY538.Then the fragment LacZ which includes promoter P11 was cloned into the SmalI site in pSY538.At last, the fragment consist of HA gene and LacZ gene was sub-cloned into the NotI site in vector pSY681 and successfully generated the transferring vector pSY681-HA7-LacZ. Chick embryo fibroblast cell (CEF) was co-transfected with plasmid pSY681-HA7-LacZ and s-FPV-017 virus for homologous recombinant. Recombinant virus rFPV- H7HA was screened in the presence of X-gal to purify positive recombinant virus. The genome DNA of the recombinant fowl pox virus was used as template for PCR and 1.7 Kb HA gene fragments was obtained.This result indicated that the target gene was inserted into the genome DNA successfully. The results of Western-blot demonstrated that hemagglutinin glycoprotein of AIV was expressed efficiently in CEF infected with the recombinant pox virus in vitro.Four- week–old SPF chickens were immunized with different doses of 106,104,102 PFU rFPV- H7HA and challenged with 100LD50 of HPAIV A/FPV/Rostock/34 (H7N1) through nostril .The throat and cloacal chamber swabs were collected 3,5,7 days after challenging. And chickens were observed daily for disease signs and deaths for 2 weeks. Sera were collected every week after vaccination for detecting the HI and AGP antibodies. Results shown that all chickens vaccinated with different doses of rFPV-H7HB generated high HI antibodies and were completely protected from virus challenge (no disease signs, no virus shedding and no deaths), but the control group died within eight days and can be detected high titers of H7 HPAIV from throat and cloacal chamber swabs. The results implied that the recombinant virus rFPV-H7HA that expressing HA gene of A/Chicken/Hebei/1/02(H7N2) would be the storage of vaccine and technology for preventing H7 subtypes HPAIV.