Protection Of Chickens Against Highly Pathogenic Avian Influenza Virus By Recombinant Fowpox Virus Expressing Avian Influenza Hemagglutinin Gene

Full-length eDNA of haemagglutinin (HA) gene of A/Goose/Guangdong/3/96 (H5N 1) (GD3) was amplified using reverse transcription-polymerase chain reaction (RT-PCR). After identification with neucleotide restriction enzymes,the cDNA was cloned into Sma I site of pUC 18 plasmid and then sequenced. The result of sequencing shows that the eDNA contains whole open reading frame of HA gene, initiation codon and termination eodon. The results of sequence analysis indicated that the cDNA had 11 nucleotide differences and 99.4% of homology in comparison with A/Goose/Guangdong/1/96(H5NI) (GD1), 25 nucleotide differences and 98.6% of homology with AIHongKongIl56I97(H5NI) (HK156), and 30 nucleotide differences and 98.3% of homology with AlChicken/HongKongI2SB/97(1-15N 1) (HK258). Furthermore, The HA of GD3 had the homology of 99.2% with CDI, 98.6% with HK156, and 98.1% with HK258 at amino acid level. They shared the same basic amino acid insert at the cleavage site, and the amino acid sequence is -P-Q-R-E-R-R-R-K-K-R-G-LP-,which indicates that all 4 influenza virus isolates are probably evolved from same ancestor and have similar virulence and biological propertiesIn order to develop a recombinant fowlpox virus (FPV) expressing avian influenza virus (AX) haemagglutinin, the HA cDNA of GD3 and LacZ reporter gene were cloned into pSY538 plasmid and then subcloned into Not I site of pSY68 I plasmid. After construction of transfer vector, it was transfected on CEF cells with FPV F-0l 7. Following 6 cycle screenings, donning and purefieation of blue plaque ,the pure HA-fowlpox virus reeombinants were obtained, and identified by restriction enzyme analysis and PCR. The results showed that the recombinant fowlpox virus contained HA gene of GD3 and had the stable genetic properties.The result of Dot-ELISA showed that the recombinant fowlpox virus expressed the hemagglutinin glycoprotein of AIV efficiently, and the expressing products possessed good antigenicity reacting with antibodies specifically. The result of Western-blotting demonstrated that hemagglutinin glycoprotein expressed by this recombinant virus existed with two cleavaged products of 44kDa and 23kDa representing HAl and L-1A2,there was no evidence of existence of HA0 with 63kDa.Sixty-day-old SPF chickens were immunized and challenged three weeks later. After challenged with 50 X LD50 of CDT isolate per chicken, all chickens vaccinated with the recombinant fowlpox virus and inactivated oil-emusion vaccine prepared with CDI isolate were survived, the control chickens and chickens immunized with fowlpox virus F-0l7 were died. The chickens vaccinated with recombinant fowlpox virus had HI antibody titer of 5.91og2 at 7 days postvaccination, and HI antibody titer rised to peak at 14 days postvaccination. HI antibody ~vas detaetable at 14 days postvaccination, and I-Il antibody titer increased to top at 35 days postvaccination in all chickens immunized with inactivated oilemusion vaccine. None I-Il antibody were detected in the control or fowlpox virus immunized chickens before or after immunization or challenge. Challenge virus was not reisolated from chickens vaccinated with the recombinant fowlpox virus and inactivated oil-emusion vaccine, and detected in everyone of all control or fowlpox virus immunized chickens.Virus was detected more frequently in long of control chickens.Determination of T lymphocytes indicated that vaccination of recombinant fowlpox virus and inactivated oil-emusion vaccine stimulated effectively cell-mediated immune response of chickens and activated I lymphocytes.The numbers of CD4-,CD8- and TCR 1-expressing cells was increased or did not changed in peripheral blood, thymus and spleen in chickens vaccinated with i~combinant fowlpox virus or inactivated oil-emttsion vaccine postchallenge, and decreased sharp in all control and fowlpox virus immunized chickens afler challenge.