| Marek's disease (MD) is a highly contagious malignant MDV virus-induced lymphomatous disease of chickens. Marek's disease has been controlled efficiently since the early 1970s by vaccination with live, non-oncogenic strains of MDV and/or herpesvirus of turkeys (HVT). Today, this strategy has been unusually effective. The foregoing succession of MD vaccines paralleled, and indeed was prompted by, the continued evolution of field strains of serotype 1 MDV to greater virulence, and decreased efficiency of vaccine. The v pathotype emerged in the late 1950s and was responsible for the catastrophic looses from MD in the 1960s, after classical strains mMDV was isolated. The vv pathotype emerged in the late 1970s, HVT had been in use about 8-10 years. The vv+ pathotype emerged in the early 1990s after bivalent vaccine had been in use about 10 years. These viruses are characterized by higher cytolytic activity, unusual tissue tropism, increased atrophy of lymphoid organs, immunosuppression, enhanced capacity to transform T cells, and earlier host death. The vv+ pathotype was responsible for large losses, which could be controlled until new efficient vaccine was found. With development of the molecular biology, many researchers have made major commitments to rDNA-based vaccination for MD. The MDV vaccine viruses are considered one of the most potent vectors for polyvalent live vaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases. In this report, we constructed several recombinant viruses based on attenuated MDV1 strains that expressed GFP gene and major antigenic epitopes of differential MDVserotypes, and evaluated the abilities of these recombinants to protect against mortality and development of lymphoid tumors caused by challenge with virulent MDV in SPF chickens. This study includes:1. Construction and properties of CVI988 virus recombinant expressing the GFPThe US2 gene nonessential for viral replication was amplified by polymerase chain reaction (PCR) with extracted total DNA from MDV vaccine strain infected CEF cells as template, and was cloned into T-easy vector; The expression cassette including GFP gene controlled by CMV promoter and enhancer was cloned into US2 gene to give rise to the transferring vector pGUS2GFP, then the complex of pGUS2GFP and DOTAP was transfected into CEF cells infected CVI988 strain virus. Recombinant rCVIGFP expressing the green fluorescence protein was selected and purified many times in CEF cell. The characterization of recombinant virus was evaluated in vivo and in vitro, the growth curves of recombinant virus having EGFP gene are similar to those of parent virus in CEF cells. From the numbers of PFU in cells infected with recombinant virus, the PFU was most at 5 days post-inoculated with recombinant virus. The recombinant virus expressing GFP could be re-isolated from chickens following intra-abdominal inoculation of rCVIGFP. The recombinant virus reported here represents the first MDV1 mutant expressing GFP in China and demonstrated the possibility of CV1988 used as virus vector to construct recombinant virus.2. Construction of a CVI988 virus recombinant expressing the major antigenic epitopes of gB of HVTIn recent years and reported, combination vaccine containing MDV CVI988 strain and HVT against vvMDV provided better protection than the monovalent vaccines. Among several nonessential sites within US repeat for viral growth, the US 10 gene appears to be the most stable and not to be connected with vaccinal immunogenicity. Based on the information of obtained above, we constructed recombinant virus expressing the major antigenic epitopes of HVT gB inserted within the US 10 gene of CVI988 virus. Partial sorfZ and sorfi, the whole US1 and US 10 of CVI988/Rispens strain were amplified using primers synthesized according to unique short region nucleotide sequence of MDV I strain published in GenBank and obtained the vector pBUS10. The hemagglutinin (HA) gene and termination codon (TAA) were added to the forepart and the back... |
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