Comparative Studies Of The Biological Activities Of MDV SC9-2 And Construction Of Recombinant MDV Expressing The NDV-F Gene

Marek's disease(MD) is a highly contagious disease virus of chickens, which has the common characteristics of lymphoproliferative and immunosuppressive. It contains three serotypes. MDV-1 belongs to the MDV that can cause tumors; MDV-2 belongs to the natural MDV that can not cause tumors; HVT belongs to MDV-3. A serotype 1 of field recombination MDV strain GX0101 which integrated with the long terminal repeat (LTR) of reticuloendotheliosis virus (REV) was isolated from chickens that suffered from tumour in Guangxi province of south China by Zhangzhi of our lab. We constructed BAC clone of GX0101 BAC-GX0101 by bacterial artificial chromosome (BAC). On the basement of BAC-GX0101, we knocked out the two meq genes and induced the Kan+expression casstte that inserted when we recombinant getting meq-null strain MDV GXO101?meq?Kan, that is so called SC9-1 strain MDV. Researches showed that SC9-1 not only loses the pathogenicity of MDV-1, but also can provide superior immune protection effect than commercial CVI988/Rispens. On the basement of SC9-1, we knocked out the BAC sequence in SC9-1 by cre-loxp recombinant system. This study aims to compare the biological activities of different generation of SC9-2 and the construction of a recombinant MDV expressing the NDV-F gene with SC9-1 vector.1. The self-excision of molecular identification of bacterial artificial chromosome (BAC) sequence in SC9-1In order to study the self-excision of molecular identification of BAC in meq-null MDV SC9-1 clone genome. SC9-1 recombinant plasmid containing cre recombinase expression cassette transfected chickens embryo fibroblast (CEF) to save SC9-1 recombinant virus. Passage SC9-1 recombinant virus on CEF cells continuously, using cre recombinase knockout BAC sequence in process of virus passaged. Extract 1 to 10 generations DNA of SC9-1 recombinant virus as template, PCR detecting BAC sequence of viral genome by specific primers. At the same time, using specific primers for the BAC sequence of gpt gene, the MDV conserved gene pp38 as an internal standard, detect the content of BAC sequences in the viral genome of different passages by fluorescence quantitative PCR. PCR amplify the residual sequence of both sides of loxp site, future verifying the knockout of BAC sequence. The results showed that using the BAC sequence specific primers to detect BAC sequence of different generations in the viral genome showed, the first five generation of virus DNA can amplify 600 bp specific band meaning the viral genome contains the BAC sequence. The 6 to 10 generation of virus DNA couldn't amplify 600 bp specific band meaning the viral genome don't contain the BAC sequence. The result of fluorescence quantitative PCR showed that the content of BAC sequence in viral genome decrease gradually with the passage of the virus and couldn't detect BAC sequence completely until the sixth generation. In the process of the knockout of BAC sequence, PCR identifying the residual sequence of both sides of loxp site, the results showed only one loxp site left after the knockout of BAC sequence and the sequence homology is above 99.7%. The experiment confirmed that the cre/loxp system can delete the BAC sequence in SC9-1 completely by culturing virus on CEF cells for 6 generation of batches and the knockout of BAC sequence has high degree of consistency.2. Attenuation of MDV SC9-2 strain lacking the meq oncogene by serial cell culture passage and evaluation of attenuated strains for immunosuppression and protectionMD is a lymphoproliferative disease of chickens caused by a oncogenic alpha-herpesvirus known as MDV. Previous study showed that the meq-null very virulent MDV(w MDV) provided superior protection to commercial vaccine by culturing to 40 passage in primary duck embryonic fibroblasts to eliminates lymphoid organ (bursa and thymus) atrophy and body weight loss in maternal antibody negative (MAb-) chickens. In the study, we culture the meq-null vv MDV SC9-2 by serial passage in CEF cells. The weight of chickens and relative thymus and bursa to body weight of chickens inoculated with the 40th passages of SC9-2(SC9-2/40) were significantly higher than those inoculated with the 10th passages(SC9-2/10th), but lower than negative control group and the differences is significant. There were no obvious influence on antibody levels induced by avian influenza virus (AIV)/ newcastle disease virus (NDV) inactivated vaccines compared the SC9-2/40th and the SC9-2/10th, but significantly lower than the control group. The virus load in the feather capsule of chickens inoculated the SC9-2/40th is significantly lower than SC9-2/10th, but the immune protection effect has no difference, at the same time all showed significant higher protection index than CVI988/Rispens. The result showed that the immunosuppression of the SC9-2/40 decreased but still need cell culture passage to eliminate the immunosuppression. The result provides a reference for the cell culture attenuation of meq-null VV MDV.3. Construction of recombinant MDV expressing the NDV-F gene and its replication in chickensWe use a meq-deleted attenuated MDV-I strain SC9-1 (GXOlOlAmeqAKan)as a vector to construct a recombinant virus expressing the exogenous gene NDV-F. The open reading frame (ORF) of exogenous gene NDV-F was inserted into the eukaryotic expression vector pcDNA3.1(-). Then, the expression cassette of NDV-F (which contains the CMV promoter) was amplified. Simultaneously, we amplified the selected gene Kan+expression cassette and insert it into the pMD18-T vector. Tandem expression cassettes were amplified using primers containing the 50-bp homologous arm of MDV-US2. The polymerase chain reaction (PCR) product was electroporated into EL250 host bacteria containing SC9-1. Then, the Kan+ expression cassette was deleted from the recombinant virus genome using 1%arabinose. The plasmid of the positive clone was extracted, the Kan+ expression cassette was deleted, and both were transfected into CEFs to rescue the recombinant virus. The recombinant virus was injected into chickens to observe its growth and replication. The recombinant virus rMDV-F containing the exogenous gene NDV-F was rescued. The recombinant virus could duplicate and express well in CEFs and grow and replicate well in chickens. Using SC9-1 as a vector, combined with a recombinant system of Red E/T and FLP/FRT, we construct a recombinant virus that can express the exogenous gene NDV-F. This study can lay the foundation for further study of recombinant viruses.