| A fowlpox-based recombinant virus rFPV-12LSFH9A co-expressing the fusion protein (F) gene from ZJ1 strain of Newcastle disease virus (NDV) and the HA gene from F strain of H9 subtype avian influenza virus (AIV) was developed, and its protective efficacy against either NDV or AIV challenge in chickens was evaluated.1. Construction of recombinant fowl pox virus (rFPV) co-expressing the Fgene of NDV and the HA gene of H9 subtype AIVThe transfer vector pP12LSFH9A was constructed by inserting the F gene of NDV, the HA gene of H9 subtype AIV and their promoters into the plasmid pP12LS which contains the genomic DNA fragments of fowlpox virus non-essential for replication. Chicken embryo fibroblast (CEF) monolayers pre-infected with Chinese vaccine strain 282E4 and large plaque strain of FPV were transfected with pP12LSFH9A and a rFPV was obtained and designated as rFPV-12LSFH9A. By selection of blue plaques on the CEF overlaid with agar containing X-Gal, rFPV-12LSFH9A was screened and subjected to further rounds of plaque purification until a pure population was achieved. PCR indicated that the F and HA gene had been inserted into the genomic DNA of FPV. Subsequently, F and HA expressed in CEF infected with rFPV-12LSFH9A were confirmed by indirect immunofluorecent assay (IFA).2. Vaccination trails for evaluating protective efficacy ofrFPV-12LSFH9A in chickens 2.1 Protective efficacy of rFPV-12LSF9HA against F48E8 strain of NDV challengein chickensNine groups of 7-day-old SPF chickens were immunized with rFPV-12LSFH9A (LP and 282E4 original), rFPV-FH9A, rFPV-12LSF(LP and 282E4 original) and FPV(LP and 282E4 strain), respectively, by subcutaneous (SQ) route in the nape of neck at the dosage of 104PFU in 0.2ml inoculum. One group vaccinated with killed vaccine for ND by intramuscular (IM) route. Another group was injected with PBS as control. Seven days after vaccination, ELISA antibody could be observed in groups except the PBS and FPV inoculated groups. All birds receiving either rFPVs or inactivated vaccine were completely protected against the virulent NDV challenge by oculonasal route 21 days post-vaccination while those receiving PBS or FPV experienced 100% mortality.Eight groups of 10-day-old commercial broiler chickens were subsequently vaccinated by SQ route at the dosage of 105PFU for rFPVLp-12LSFH9A, rFPV-FH9A, rFPV-12LSF(LP and 282E4 original) and FPV(LP and 282E4 strain), respectively. Other two groups were vaccinated with 0.2ml inactivated vaccine in oil emulsion and PBS for positive and negative control, respectively. During the observation period from day 7 to 28 post-vaccination, the ELISA antibody dropped all the time beside the killed vaccine group. All birds were challenged with the virulent NDV F48E8 strain (10~5ELD50/bird) by the oculonasal route 28 days post-vaccination. The protective efficacy of rFPVs against NDV challenge ranged from 33.3% to 70.8%.2.2 Protective efficacy of rFPV~12LSFH9A against H9 subtype AIV challenge inchickensNine groups of 7-day-old SPF chickens were inoculated with rFPVLP-12LSFH9A, rFPV282E4-12LSFH9A, rFPV-FH9A, rFPVLP-12LSH9A , rFPV282E4-12LSH9A, FPVlp, FPV282E4, H9 killed vaccine and PBS at the same approach and dose as descripted above. Seven days after vaccination, HI antibody could be detected in all groups except that inoculated with FPV and PBS. The HI antibody titers arised during the observation period from day 7 to 21 post-vaccination. And the titers were much higher in chickens vaccinated with killed vaccine than in chickens immunized with rFPVs . All birds were challenged with H9 subtype AIV (107ELD50/bird) by oculonasal route on day 21 post-vaccination in SPF birds respectively. Tracheal swabs were taken from all birds on day 5 after immunization for isolating challenged AIV in 10-day-oldThough the titers dropped all the time in experiments with commercial broiler birds that vaccinated with rFPVs, rFPVs could suppress commercial broiler chickens to shed virus from respiratory tract as well. The protective index ranged from 33.3% to 70.8%.In all the ex... |
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