Construction And Comparison Of Recombination Fowlpox Viruses Expressing HA Gene Of H5 Subtype AIV In Different Insertion Sites

H5 subtype avian influenza (AI) caused serious economic losses to the poulty industry in our country. Currently, vaccine immunization is the most important methods for prevention and control of H5 subtype AI. The hemagglutinin (HA) of avian influenza virus (AIV) is the major protective antigen for inducing host to produce neutralization antibody. However, the antigen drift is easy to occur in the HA gene and results in many different clades in H5 subtype AIV. Therfore, the development of multivalent AI vaccines has important economic significance for prevention and control of H5 AI. Fowlpox virus (FPV) has many nonessential regions, so it is an ideal vector to construct recombinant vaccines expressing HA gene of different H5 clades. Although recombinant fowlpox virus vaccines that multiple HA genes were inserted in one nonessential region have reported, but their immune efficacies against H5 AIVs were limited.We can also insert different HA genes into different nonessential regions to construct multivalent vaccine, while the selection of nonessential regions may be a key factor for immune efficacy of FPV vaccines. In this study, we constructed recombinant fowlpox virus expressing HA gene in nonessential region (FPV34), which was further compaired with recombinant fowlpox virus expressing same HA gene in another nonessential region (FPV 12) on growth characteristics, HA transcription level, protein expression level and immune efficacy. This study laid the foundation for the development of multivalent recombinant fowlpox vaccine against AIV.1 Construction of recombinant fowlpox virus (rFPV) expressing HA gene of H5 subtype AIVThe FPV nonessential region fragments FPV3 and FPV4 gene were PCR amplified and cloned into vector pBluescript SK(-) to construct the recombinant plasmid pSKP3P4. Then expression cassette of P11-LacZ reporter gene was cutted from plasmid pFG1175-1 and promoter Ps was cutted from plasmid P12LS, and then both were cloned into pSKP3P4 to construct the recombinant plasimd p34xLs. The HA genes were inserted into transfer vector p34xLs under the control of promoter Ps. Finally, we obtained transfer vector p34xLs-SYHA and p34xLs-W6-7HA.The transfer vectors were used to transfect chicken embryo fibroblast (CEF) infected with FPV by PolyJet reagent.The rFPV34-SYHA and rFPV34-W6-7HA were obtained by screening of blue-white plaque.The DNA of recombinant fowlpox viruses was extracted and sudjected to PCR for confirming the insertion of HA genes in the chromosomal DNA of FPV. The results of indirect immunofluorescent assay (IFA) indicated that HA genes were expressed correctly in rFPVs.2 Comparison of biological characteristics, transcription and protein expression of recombinant fowlpox viruses with HA insertion in different nonessential regionsAfter the recombinant fowlpox viruses rFPV12-SYHA and rFPV34-SYHA infected CEF cells, the viral titers and growth characteristics were determined. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze and compare the HA expression levels of rFPV34-SYHA and rFPV12-SYHA at the mRNA level and the protein level, respectively. The results of growth curve showed that recombinant viruses rFPV12-SYHA and rFPV34-SYHA had similar growth rate. There was no significant difference in HA transcription between the two recombinant viruses detected by Quantitative PCR. There was no significant difference in HA expression between the two recombinant viruses detected by ELISA.3 Immune efficacies of recombinant fowlpox viruses expressing HA gene of H5 subtype AIV in chickensTen-day-old SPF chickens were grouped randomly, and chickens were immunized with 0.2mL inoculum containing 105 PFU of rFPVi2-SYHA、rFPV34-SYHA、rFPV34-W6-7HA, and wt-FPV at a dosage of respectively. Chickens were also vaccinated with 0.2mL oil adjuvant inactivated vaccines of AIV SY strain (clade2.3.4) and AIV W6-7 strain (clade7.2) as positive control, and 0.2mL PBS as negative control. On days 7,10,14,17, and 21 post immunization, the HI titers of antibody were determined. The results showed that the titers of HI antibody induced by rFPVs in chickens were significantly lower than that by oil adjuvant inactivated vaccines (P< 0.05). Then these chickens were challenged with H5 subtype AIV with 105.5 EIDso/chickens at 21 d after immunization.The results showed that the protection rate against SY induced by rFPV12-SYHA and rFPV34-SYHA was 85%, and the protection rate against SY induced by oil adjuvant inactivated vaccines of AIV SY was 95%. Protection rate against W6-7 induced by rFPV34-W6-7HA was 90%, which was same as that induced by the oil adjuvant inactivated vaccines of AIV W6-7.In summary, the growth characteristics,gene transcription level,protein expression level and the protective efficacy of recombinant fowlpox virus expressing HA gene of H5 AIV in different nonessential regions (FPV12 and FPV34) were similar, indicating that these two nonessential regions (FPV12 and FPV34) could be the insertion region for exogenous fragment, which laid the foundation for the development of multivalent H5 AIV vaccine.