| Subtype H5 highly pathogenic avian influenza (Avian Influenza, AI) is a kind of acute and highly contagious infectious disease caused by influenza A virus. The mutation of HA gene rate of H5 subtype avian influenza virus is rapid, and many branchs have been identified. In recent years, Clade 2.3.2.1, Clade 7.2 and Clade 2.3.4.4 are the main epidemic branchs in our country, there is an urgent need to develop a new type of vaccine for prevention and control of subtype H5 avain inflenza. HVT (Herpesvirus of turkey, HVT) is a viral vector having a broad application prospects, since HVT has the advantages of can be lyophilized, easy to use, have no pathogenic effects for chickens, and does not affect its performance, therefore, is widely used as carrier in constructing a recombinant virus.The study was conducted under the homologous recombination principle, and we used turkey herpes virus (HVT) FC 126 vaccine virus as vector and its non-essential region for replication US2 as insertion site, to construct the transfer vector EGFP. Then, transfer vector EGFP and HVT genome were co-transfected CEF, in this way, we obtained recombinant virus rHVT-EGFP which marked by EGFP gene.In this study, we chosed three subtype H5 AIV strains,which belong to the branches of Clade 2.3.2.1, Clade 7.2 and Clade 2.3.4.4, and constructed transfer vector pFR-HA-R6, pFR-HA-R7, pFR-HA-R8 contain HA gene by using CMV as promoter. The transfer vector and recombinant virus rHVT-EGFP genome are co-transfected CEF, with the HA gene expression cassette replace the EGFP marked gene in US2 region, in this way to rescue recombinant virus rHVT-HA-R6, rHVT-HA-R7 and rHVT-HA-R8 contain HA gene expression cassette. By PCR identification, three recombinant virus strains had been correctly inserted into the genome of HVT, and purified completely. By using Western-blot test, the foreign genes of the three recombinant viruses obtain stable expression. The in vitro growth detection results of recombinant virus showed that the recombinant virus had the same replication capacity compared to parental virus.In this study, we evaluated the protection efficacy of recombinant (rHVT-HA-R6, rHVT-HA-R7 and rHVT-HA-R8) against subtype H5 Aâ…£ by vaccinating 1-day-old SPF chickens. Two hundred 1-day-old SPF chickens were randomly divided into 8 groups,and named as rHVT-HA-R6 immune group, rHVT-HA-R7 immune group, rHVT-HA-R8 immune group, H5 subtype Aâ…£ trivalent inactivated vaccine group, Re-6 attack poison control group, Re-7 attack poison control group, Re-8 attack poison control group and blank control group. H5 subtype Aâ…£ trivalent inactivated vaccine group was 60 SPF chickens and the other groups were 20 SPF chickens. The first three groups inoculated 5000 PFU vaccine in the neck subcutaneous at 1 days of age; the inactivated vaccine group conducted 0.25 mL/feather muscle injection of H5 subtype Aâ…£ inactivated vaccine at 14 days of age. Then using hemagglutination inhibition test (HI) to detect the antibody titer of H5 subtype Aâ…£ at 7,14,21,28,35 and 38 days of age. Except control group, other groups inoculated H5 subtype Aâ…£ with dose of 105 EID50 via eye intranasal method at 28 days of age. The rHVT-HA-R6 immune group, Re-6 attack poison control group, and 20 SPF chickens of H5 subtype Aâ…£ trivalent inactivated vaccine group was challenged with HJ-14 strain AIV. The rHVT-HA-R7 immune group, Re-7 attack poison control group, and 20 SPF chickens of H5 subtype Aâ…£ trivalent inactivated vaccine group was attacked with JA-14 strain Aâ…£.The rHVT-HA-R8 immunization group,Re-8 attack poison control group,and 20 SPF chickens of H5 subtype Aâ…£ trivalent inactivated vaccine group were infected with YZ-14 strain Aâ…£. After challenged with Aâ…£, we observed for 7 days and sta statistically treated to analyze the clinical symptoms, incidence, mortality, antibody growth and decline and detoxification law of the test group. The results showed that in the whole test period, the antibody level of three recombinant vaccine group chickens showed rising trend, while the attack control group was not detected antibody before challenged with AIV. After challenged the H5 subtype of Aâ…£ virulent virus, compared with the control group, the antibody level of three recombinant vaccine group chickens increased rapidly. The protective rate of rHVT-HA-R6, rHVT-HA-R7, and rHVT-HA-R8 immune group was 100%. All the results showed that the recombinant virus vaccine can provide complete immunoprotection. |
