Different Nonessential Regions For Insertion Of Foreign Gene Influence The Ability Of Recombinant Fowlpox Viruses To Resist The Interference Of Maternal Antibodies

Recombinant fowlpox viruses (rFPVs) have been well documented and many protective antigens of different viruses have been successfully expressed by using FPV vectors since late 1980s. However, the rFPV based vaccines have not been widely used in the field because maternal antibodies present in commercial chickens usually exert substantial interference to the efficacy of rFPVs. Selecting suitable FPV nonessential regions for insertion of foreign gene and generation of recombinant viruses might be an effective way to solve this problem. We constructed a panel of rFPVs with vectors based on the four selected nonessential regions. Protective efficacies of rFPVs were determined in specific-pathogen-free (SPF) chickens and commercial chickens with different levels of maternal antibodies.1. Directional cloning of nonessential regionsEight pairs of primers were designed according to the published complete genomic sequence of a pathogenic FPV to amplify the flanking regions of 4 supposed nonessential regions (FPV 1-2, FPV3-4, FPV5-6 and FPV7-8) by PCR. A series of vectors pP12L, pP34L, pP56L and pP78L which contained corresponding nonessential region and the expression cassette of Pll-ZacZ reporter gene were constructed. Four vectors were transfected into chicken embryo fibroblast (CEF) pre-infected with 282E4 strain of FPV respectively. rFPVs were obtained and purified by blue plaque selection. The results indicated that all four putative nonessential regions used in vectors were genuine nonessential regions of FPV. LacZ was expressed stably after 10 successive passages of rFPVs in CEF.2. Construction of transfer vectors expressing F or HN gene of Newcastle disease virus (NDV)The multiple cloning site (MCS) was inserted into the vector p1 IS to generate vector pN11S , which was more convenient for the insertion of foreign genes. The expression vectors pl2LS, P34LS, p56LS and p78LS were developed by inserting promoter Ps into vector pP12L, pP34L, pP56L and pP78L respectively, and then the F gene of NDV was inserted into pN11S and these 4 expression vectors to form the transfer vectors pN11SF, pl2LSF, p34LSF, p56LSF and p78LSF respectively. HN gene of NDV was inserted into pN11S and pl2LS to form the transfer vectors pN11SHN and pl2LSHN respectively. These 7 transfer vectors were transfected into CEF pre-infected with 282E4 strain and Large plaque strain of FPV respectively. A total of 14 rFPVs (rN11SF282, rl2LSF282, r34LSF282, r56LSF282, r78LSF282, rN11SHN282, rl2LSHN282, rNllSFLP, rl2LSFLP, r34LSFLP, r56LSFLP, r78LSFLP, rN11SHNLP and r12LSHNLp) were successfully generated and purified by blue plaque selection.3. Vaccination trials for evaluating protective efficacies of rFPVs against virulent NDV challenge 3.1 Protective efficacies of rFPVs in SPF chickens (experiment I)Eighteen groups of 5-day-old SPF chickens were immunized by the subcutaneous route with inoculum containing 104PFU of one of the rFPVs (rN11SF282, rN11SFLP., rl2LSF282, rl2LSFLP, r34LSF282, r34LSFLP, r56LSF282, r56LSFLP, r78LSF282, r78LSFLP, rNllSHN282, rN11SHNLP, r12LSHN282 and r12LSHNLP), of the wild type FPVs (FPV282 or FPV LP), or containing the killed vaccine of NDV or PBS (0.2ml/bird), respectively. At 21 days post-immunization, chickens were challenged with 105 ELD50 of virulent NDV strain F48E8 by nasal inoculation. Challenged chickens were observed for 14 days for clinical signs and mortality.Chickens inoculated with rFPVs or the killed vaccine had no signs of ND, and they provided 100% protective efficacy against virulent NDV challenge while those inoculated with the wild type FPVs or PBS experienced 100% mortality. ELISA antibody titers agaist NDV were detected at 7, 10, 14, 17, 21 days post-immunization. The P/N values increased gradually in chickens of rFPVs and the killed vaccine inoculated groups after immunization, while the values remained unchanged for chichens in wild type FPVs or PBS inoculated groups. The P/N values of rFPVs expressing HN gene increased more rapidly and were little higher than those of corresponding rFPVs expressing F ge...