Construction And Immunity Assessment Of Recombinant Fowlpox Virus Expressing Ha Protein Of H5Subtype Avian Inlfuenza Virus

Highly pathogenic avian influenza (HPAI) caused by the H5N1subtype avian influenza virus (AIV)has posed severe threat to the poultry industry and human health. A culling combined with vaccinationstrategy has been adopted in china for the control of HPAI. Currently, inactivated vaccine and livevirus-vectored vaccine have been used in the vaccination. Disturbing to the AIV surveillance andepidemiolog ical investigation might be produced by using of inactivated vaccine.So the study of livevirus-vectored vaccine is of great significance.In this study, HA genes from A/Duck/Anhui/1/2006（DK/AH/1/2006) andA/Duck/Guangdong/S1322/2010(DK/GD/S1322/2010) were recombined with FPV（CVCCAV1003) byinfection-transfection method in Chicken embryo fibroblasts (CEF). DK/AH/1/2006andDK/GD/S1322/2010belonged to Clade2.3.4and Clade2.3.2.c respectively. They were both therepresentative strain that now circulating in waterfowl in southern china. After the construction ofrFPV-AHHA and rFPV-GD1322HA, they were indentified by western blot. The result show that HAproteins were correctly expressed by them.The immune effects of rFPV-AHHA and rFPV-GD1322were assessment in SPF chickens.Three-week-old chickens were randomly divided into twelve groups (Eight chickens of each group).Three groups were vaccinated with106PFU rFPV-AHHA, five groups with106PFU FPV-GD1322HA,and the rest four groups with FPV (Control). Serum for HI antibody detection was collected every weekafter vaccination, and HI antibodies were detected by using different antigens. Six weeks aftervaccination, groups which vaccinated with FPV or rFPV-GD1322HA were challenged with105EID50HPAIV A/Duck/Hubei/S1513/2010(DK/HuB/S1513/2010), A/Duck/Fujian/31/2007(DK/FJ/31/2007),A/Chicken/Shandong/A-10/2011(CK/SD/A-10/2011) and DK/GD/S1322/2010, which belonged toClade2.3.2.b, Clade2.3.4, Clade7.2and Clade2.3.2.c respectively. The fifth group which also vaccinatedwith rFPV-GD1322was treated as a HI duration group. The rFPV-AHHA vaccinated groups werechallenged with these four viruses, except for DK/GD/S1322/2010. The immune effect was evaluatedby HI antibody variation, virus excretion and protection efficiency.rFPV-AHHA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010, andCK/SD/A-10/2011antigens, HI angtibodies were4.00log2,3.38log2and4.25log2respectively. Afterchallenge with viruses, HI antibodies reached4.14log2,7.33log2and8.83log2respectively.100%protection was provided by rFPV-AHHA when challenged with DK/FJ/31/2007. In all seven chickens,no chicken was found dead or virus shedding; After challenged with DK/HuB/S1513/2010, twochichkens were found died and one chicken excreted virus. The protection efficiency was75%(6/8); Inthe other group, when challenged with CK/SD/A-10/2011, there were two chickens dead and onechicken shed virus. So the protection efficiency was85%(6/7); All chickens were died in three days incontrol groups. Those results show that rFPV-AHHA can fully protect chickens from homologous viruschalleng e. But, partially protection was provided by rFPV-AHHA when challenged with heterologousviruses. rFPV-GD1322HA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010,CK/SD/A-10/2011and DK/GD/S1322/2010antigens, HI antibodies were0.00log2,4.29log2,6.71log2and3.43log2respectively. After challenge, HI antibodies reached to1.75log2,6.00log2,8.75log2and8.14log2respectively. rFPV-GD1322HA could protect chickens at leve l of100%, when challenged withDK/FJ/2007and DK/GD/S1322/2010; However, when challenged with DK/HuB/S1513/2010, one deadand one virus shedding were observed in the group; In CK/SD/A-10/2011challenge group, two chickenswere found dead and one chicken excreted virus, the protection efficiency was66.7%(4/6).In this study, two recombinant viruses rFPV-AHHA and rFPV-GD1322HA were constructedsuccessfully. Their protection efficiency in SPF chickens show that the HI antibody could be differentwhen detected with different antigens. But rFPV-AHHA and rFPV-GD1322HA could protect chickensfrom death when challenged with homologous virus as well as some heterologous viruses. All thoseresults will provide basic data for the further study of immune effect.