Construction Of Recombinant Fowlpox Viruses Co-expressing Avian Influenza Virus HA (H5) Gene And Newcastle Disease Virus HN Gene Or Newcastle Disease Virus F Gene And HN Gene And Their Protective Efficacies

A recombinant fowlpox virus rFPV-12LSH5AHN co-expressing the HA gene from SY strain of H5 subtype avian influenza virus (AIV) and the HN gene from ZJ1 strain of Newcastle disease virus (NDV), and another recombinant fowlpox virus rFPV-12LSFHN co-expressing F gene and HN gene from ZJ1 stain of NDV were developed, and their protective efficacies against AIV and/or NDV challenge in both SPF and commercial chinckens were evaluated.1. Construction of recombinant fowlpox viurs (rFPV) co-expressing the HA gene of H5 subtype AIV and the HN gene of NDVThe transfer vector pP12LSH5AHN was constructed by inserting the HA gene of H5 subtype AIV , the HN gene of NDV and their promoters into the plasmid pP12LS which contains the genomic DNA fragments of fowlpox virus that were non-essential for replication. Chicken embyo fibroblast (CEF) monolayers pre-infected with wild type fowlpox virus(LP) were transfected with pP12LSH5AHN to generate the recombinant fowlpox virus co-expressing HA and HN (rFPV-12LSH5AHN). By series blue plaque screening on the CEF, rFPV-12LSH5AHN was obtained and purified. The presence of the HA and HN genes was confirmed by Polymeras Chain Reaction (PCR). The expression of HA and HN proteins in rFPV-12LSFHN were characterized by indirect-immunofluorescence test. Compared to the parental virus, rFPV-12LSH5AHN showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken CEF.2.Construction of recombinant fowlpox viurs (rFPV) co-expressing the F gene and HN gene of NDVBoth the HN gene and F gene from velogenic NDV strain ZJ1 were inserted into the transfer vector pP12LS, resulting in the recombinant plasmid pP12LSFHN. The F gene and HN gene were under the control of promoter Ps and PE/L, respectively. Then pP12LSFHN was transfected into the chicken embryo fibroblasts (CEF), which were pre-infected with wild type fowlpox virus (LP) to generate the recombinant fowlpox virus co-expressing F and HN (rFPV-12LSFHN). By series blue plaque screening on the CEF, rFPV-12LSFHN was obtained and purified. The presence of the F and HN genes was confirmed by Polymeras Chain Reaction (PCR). The expression of F and HN proteins in rFPV-12LSFHN was characterized by indirect-immunofluorescence test. Compared to the parental virus, recombinant virus produce plaque with obvious syncytia and higher growth rate3 . Vaccination trials for evaluating protective efficacies of rFPV-12LSH5AHN and rFPV-12LSFHNTo evaluate the protective efficacies of both rFPV-12LSH5AHN and rFPV-12LSFHN, vaccination trials were carried out in SPF and commercial chickens, respectively. For rFPV-12LSH5AHN, two groups of SPF chickens were challenged at 21 days post-immunization by nasal inoculation, one group with 5×104 ELD50 of H5 AIV strain SY and the other with 5×107 ELD50 of virulent NDV strain F48E8. The Protection index (PI) induced by 2×104 PFU rFPV against H5 AIV or NDV challenge was 100. And then the PI against H5 AIV induced by 2×105 PFU rFPV-12LSH5AHN was 75 in comercial chickens with high level of maternal antibodies. For rFPV-12LSFHN, SPF chickens immunizad with 2×104 PFU rFPVs were provided 100% protective efficacy against virulent NDV strain F48E8 when challenged with 5×107 ELD50 by nasal inoculation. The results in this study showed that recombinant rFPV co-expressing HA and HN genes or co-expression F and HN genes induced promising protective immunity in vaccinated chickens.