| Both Newcastle disease (ND) and H9 subtype avian influenza (AI) have been serious disease problems and caused considerable economic losses in poultry industry in China. So far, vaccination with conventional vaccines including the attenuated live vaccines and inactivated vaccines in oil emulsion to protect birds against ND and H9 subtype AI has been a routine procedure in China. However, the use of these vaccines is restricted because of the disadvantages they inherit including their interference of routine serological surveillance, higher cost and side effect of greater streos in birds post-vaccination. Here, we described the construction of recombinant fowlpox virus co-expressing the fusion protein (F) gene of Newcastle disease virus (NDV) and the HA gene of H9 subtype avian influenza virus (AIV) and the evaluation of its protective efficacy against either NDV or AIV challenge in chickens for the development of better vaccine.1. Construction of recombinant fowlpox virus co-expressing the F gene of NDV and the HA gene of H9 subtype AIV and its biological propertiesThe genomic RNA of NDV strain F48E8 was prepared by phenol-chloroform extraction and used as template for RT-PCR to obtain the fusion protein (F) gene, which was cloned into pGEM-T easy vector to form pGEM-TF and its sequence was determined. The hemagglutinin (HA) gene of H9 subtype ATV in pUCHA was modified by using PCR. Its amplified product was then inserted into pGEM-T easy vector and the resulting plasmid pGEM-THA was determined by sequencing. In order to construct recombinant plasmid p7SHF, the F gene in pGEM-TF and the HA gene in pGEM-THA were excised, respectively, and inserted into the randomly selectednonessential region of the fowlpox virus (FPV) under the transcriptional control of fowlpox virus promoter Pe/L. and the synthetic promoter Ps, respectively, which were ligated in opposite direction. The P11-LacZ reporter gene from pppG18 was cloned into p7SHF resulting in recombinant transfer vector pFPVHF. Pre-infected with Chinese vaccine strain 282E4 of FPV (wt-FPV), chicken embryo fibroblast (CEF) monolayers was transfected with pFPVHF to generate recombinant fowlpox virus co-expressing F and HA, designated rFPV-F-HA. By selection of blue plaques on the CEF overlaid with agar containing X-Gal, rFPV-F-HA was screened and subjected to further rounds of plaque purification until a pure population was achieved. PCR and Southern-blot assay 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-F-HA were confirmed by indirect immunofluorescence assay (IFA).Comparison of the viral titer between rFPV-F-HA and wt-FPV grown in CEF demonstrated that the ability of rFPV-F-HA to replicate in CEF was not impaired for insertion of the target DNA fragments. The rFPV-F-HA was further detected by blue plaque assay and PCR after it was passaged for 20 times on CEF monolayers. These results showed that the inserted F gene and HA gene were stably integrated. Expression of F and HA in rFPV- F-HA confirmed by IFA suggested that it be genetically stable. 2. Detection of antibody in chickens to Newcastle disease virus fusion protein by indirect ELISAAn indirect ELISA for detecting antibody in chickens induced by the fusion protein of NDV expressed by recombinant fowlpox viruses (rFPVs) was developed with great sensitivity, high specificity and strong reproducibility, and subsequently evaluated with sera from SPF chickens vaccinated with or without rFPVs. The optimum coating concentration of NDV antigen was determined by checkerboard titration using pre-immune chicken antiserum to recombinant fowlpox virus expressing the F gene of NDV and amounted to 355ng of NDV antigen per well. The serum sample for detection was diluted to 1:40. A near-linear relationship existed between positive /negative ratio(P/N) of anti-sera at a single working dilution (1:40) and the In of the corresponding observed serum ELISA titers (ET) as determined by a standard serial-dilution method. Regres...
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