Development Of Recombinant Vaccines Against H5 And/H9 Subtype AI With Different Fowlpox Virus Insertion Vectors And Their Protective Efficacies

Avian Influenza(AI) and fowlpox are two important diseases which have made greateconomic losses in poultry industry in recent years. Conventional AI vaccines areinactivated ones made of whole virus preparations which have been proved to provideadequate protection against the disease. However, there are several disadvantages forthese vaccines. For example, the antibodies induced by vaccination interfere theserological surveillance because they can not be differentiated from those generatedfrom natural infections. In addition these killed vaccines are usually rather expensiveand inconvenient for use in mass vaccination. Although recombinant fowlpox virus(rFPV)-based AI vaccines appear to be promising, yet their efficacies in commercialchickens with maternal antibodies are usually not high. The purpose of this study isdevelope better recombinant FPV vaccines against H5 and/or H9 subtype AI, in terms ofthe ability to resist the interference of maternal antibodies(MAbs). We constructedrecombinant viruses expressing HA gene of H5 or H9 subtype and coexpressing HA-NAgenes of H5N1 subtype ATV or two HA genes of H5 and H9 subtype AIVs with twodifferent FPV insertion vectors and two different wild type FPVs. Vaccinationexperiments in both SPF and Mab-positive commercial chickens were conducted toevaluate the abilities of various rFPVs generated with different insertion vectors anddifferent wild type FPVs in resisting the interference of maternal antibody. The resultsshowed that insertion vector pl2LS is much better than pi IS in terms of generatingrFPVs against AI with higher ability to resist the interference of Mabs. We successfullydeveloped rFPV vaccines rFPVLP-12LSH9A, rFPVLP-12LSH5NA and rFPVLP-12LSH5H9 against H5 and/or H9 subtype AI with promising efficacies in commercialchickens with high titers of Mabs.1. Construction of recombinant fowlpox viruses expressing the HA gene of H5 subtype AIV.The hemagglutinin (HA) gene of H5 subtype ATV was amplified by PCR, and then cloned into pGEM-T easy vector, and the resulting plasmid pTH5A was identified by sequencing. The HA(H5) gene removed from pTH5A was directionally inserted into the insertion vector p11S or p12LS to form transfer plasmid p11SH5A or pl2LSH5A respectively. Chicken embryo fibroblast (CEF) cultures pre-infected with wt-FPVLP or wt-FPV₂82E4, was transfected with p11SH5A or pl2LSH5A. Three recombinant viruses expressing HA(H5), namely rFPVLP-11SH5A, rFPVLP-12LSH5A and rFPV282E4 -12LSH5A were obtained by blue plaque selection. PCR assays of these rFPVs indicated that the HA(H5) gene had been inserted into the genomic DNA of FPV correctly. Subsequently, HA(H5) expressed in CEF infected with rFPVs were confirmed by indirect immunofluorescence assay(IFA).2. Construction of different recombinant fowlpox viruses coexpressing the HA gene and NA gene of H5 subtype AIV.The genomic RNA of AIV strain G5 was prepared by phenol-chloroform extraction and used as the template for RT-PCR to obtain the neuraminidase (NA) gene. The amplified NA gene was cloned into pGEM-T easy vector to form pTNA and its sequence was determined. In order to construct transfer plasmid p11SH5NA or p12LSH5NA, the NA gene with the promoter PE/L was subcloned into the plasmid PUC18, resulting in the plasmid pPPELNA. Then the NA gene with the promoter removed from pPPELNA was directionally inserted into the plasmid p11SH5A or pl2LSH5A in opposite direction to HA(H5), resulting in transfer vector p11SH5NA or pl2LSH5NA respectively. Three rFPVs designated as rFPVLP-11SH5NA, rFPVLP -12LSH5NA and rFPV282E4-12LSH5NA were generated with the transfection and selection techniques.3. Construction of different recombinant fowlpox viruses coexpressing the HA genes of H5 and H9 subtype ATVs.The HA gene of H9 subtype AIV was amplified by PCR and inserted into pGEM-T easy vector, resulting in the plasmid pTH9A, and its sequence was determined.The HA(H9) gene with the promoter PE/L was subcloned into the plasmid PUC18,resulting in the plasmid pPPELH9A. Then the HA(H9) gene with the promoterremoved from...