| Highly pathogenic avian influenza (HPAI) is caused by special some HA subtypes of influenza A virus. It is a poultry disease with high morbidity and mortality. It has been classified as one of the list A animal diseases by the Office International des Epizooties (OIE) for many years. In order to protect poultry against HPAI, inactivated whole viral vaccines in oil emulsion have been developed and proved to be effective in the control and stamping out of the disease. However, the use of inactivated vaccines was restricted because of the disadvantages they inherited, including higher cost and interference in serological surveillance. Sometimes its preparation and administration run high risks to disseminate HPAIV and may result in serious consequences. The genetic engineering vaccines are safe, convenient for use and highly protective. We constructed a recombinant fowlpox virus with the highly expressed HA gene of H5N1 subtype avian influenza virus and its protective efficacy in both SPF and commercial chickens were evaluated in this study.1. Construction of a recombinant fowlpox virus with highly expressed HA gene of H5N1 subtype avian influenza virusWe amplified the HA gene by RT-PCR, and the HA gene was cloned into pGEM-T easy vector (pT-H5A) and sequenced. Plasmid pT-H5A was digested with BamHI and HA fragment was isolated, purified and directly inserted into the plasmid pP12LS in which the HA gene was under the control of synthesized strong promoter Ps, resulting in transferring vector pP12LS-HA. Then the vector was transfected into the CEF infected with parent 282E4 strain of fowlpox virus. The recombinant fowlpox virus was obtained and purified by blue plaque selection. HA expression was detected by the indirect immunofluorescence array. The rFPV-12LS-HA could express HA gene.2. Construction of recombinant fowlpox viruses expressing partial HAgene in secretion and chimeric gene HAA and LLO respectively2.1 The antigen part of HA gene was amplified by PCR, cloned into pCR2.1 vector (pCR2.1-HAA) and sequenced. We used the same method as above-mentioned and obtained the recombinant fowlpox virus expressing antigen part of HA gene, rFPV-12LS-HAA.2.2 The chimeric genes consisting of HAA gene and LLO gene was amplified by megaprimer method of mutagenesis, cloned into pCR2.1 vector (pCR2.1-HAB, pCR2.1-HAC) and sequenced. The chimeric genes HAB, HAC was removed by digesting with BamHI, and purified and directly inserted into the plasmid pP12LS. Recombinant FPVs were generated as previously described.3. Protective efficacy of recombinant fowlpox viruses rFPV-12LS-HA, rFPV-12LS-HAA, rFPV-12LS-HAB, rFPV-12LS-HAC against HPAIV challenge in chickensDifferent groups of SPF chickens, commercial chickens with maternal antibodies were vaccinated subcutaneously and challenged with AIV at 21 days post immunization. Protective efficacies were evaluated by HI antibody titer, morbidity and mortality. These vaccination trials were carried out to determine the efficacy and effective dosage of recombinant viruses, to elucidate the influence of maternal antibodies on active immune response, to examine whether the different localization of the HA antigens induced different HI antibodies and to assess the potential use of LLO expressed as an adjuvant.3.1 Protective efficacy of the rFPVs in SPF chickensGroups of SPF chickens vaccinated with different dosages of rFPV-12LS-HA ranging from 103PFU to 106PFU, and rFPV-12LS-HA, rFPV-12LS-HAA groups all survived from HPAIV challenge. The protective efficacy reaches 100%. In contrast to vaccinated chickens, all the chickens in control groups developed typical signs of HPAI and all died within 7days post challenge. The results indicated that vaccination with the rFPV-12LS-HA of 103PFU could provide adequate protection from illness and death, although HI antibodies in vaccinated chickens were very low or negative.3.2 Influence of the maternal Abs on immune response to recombinant virusesGroups of 10-day-old commercial chickens with 4 log2 HI titer of H5 AIV maternalAbs were v... |
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