Sequencing Of Whole Genome Of NDV V4 Strain And The Construction Of CDNA Clone

Reverse genetics technology, explored widely in virus genetic function research and chimeric vaccine , has got dramatic development in the last 10 years. The first recovery system for Newcastle disease virus(NDV) was constructed successfully in 1999, and nowadays a few rescue systems including La Sota,Clone30,BC,ZJ1 have been reported, but not V4 strain. V4 strain was firstly isolated by Australian scientist Simmons in 1966, which was natural avirulent strain and mediated cell immunity primely, the superiority of its unique thermostable characteristic makes it possible to be vaccined in developing countries.Firstly , the V4 stock was purified three times by plaque formation. It's difficult to see clear plaques in CEF cells due to the avirulent characteristic , so we treated the V4 stock with trypsin and then allantoic Fluid and Mg2+ were added in the layer gel. In this case, we could see small plaques occasionally, but not stably. Then we treated the single clone strains with 56℃water bath and -20℃freezing and thawing, and heattolerant and coldtolerant strain was selected for further experiments. The results showed that there were some differences among these clones ,which indicated that our V4 stock was a mixture virus, and we selected the number 11 clone . Meanwhile, the 47-435bp fragment of different clone strains were amplified and sequenced ,the results indicated that the V4 stock was relatively purified.The whole genome sequences of V4 strain were obtained using 13 pairs primers.The results showed that the genome length was 15186nt,including 55nt 3'leader region ,114nt trailer region and NP,P,M,F,HN,L gene, each gene was composed of non-coding sequences and open reading frame(ORF) individually. The analysis of homologous genes and phylogenetic tree suggested that NP,P,F and HN gene was most homogenous with QV4 strain , 99.9%,96.7%,100%,99.8% individually, and M and L gene were most homogenous with HB92 strain both 99.4%.10 pairs primers were designed for the construction of complete cDNA clone pFT7-final, in which the cDNA fragment was under control by T7 promoter. Meanwhile we constructed additional plasmid in which cDNA was controlled by polII promoter. Besides, fusion PCR was explored to introduce a PmeI restriction enzyme site into the whole cDNA fragment in 3166bp, and the VP2 ORF of IBDV was inserted into pFT7-final by PmeI site, named pBRT7-VP2.The 3'leader and 5'trailer region was amplified and fused together ,then was linked with pPolII-BDV by NgoMIV and SfiI site to construct pBR-LT; the ORF of EGFP was amplified and inserted into pBR-LT to construct minigenome plasmid pBR-EGFP. After that, BHK21 cells were transfected by pBR-EGFP,pCIneo-NP,pCIneo-P,pCIneo-NP and pCAGGS-T7, the expression of EGFP indicated that our rescue system and plasmids worked well.In order to compare the transfection efficiency of co-transfection with single plasmid transfection, we selected pEGFP-N1 as a positive control.The results showed that the transfection efficiency of each group was about 10% and 40-50% respectively. The packaging experiments with helper virus La Sota,V4 and F48E9 strain showed that the minigenome could be packaged by these virus to generate defficienct virus particles, indicating the leader and trailer region played an important role in packaging process.Although we have tried several times to rescue V4 strain in both BHK21 and BSR T7-5 cells, we still couldn't get rescued viruses. What we have finished nowadays should have based some reliable prime work for the successful recovery in future days.Our experiments purified V4 stock by plaque formation firstly three times ,and then compared the thermostable characteristic of different clone for future experiments. We sequenced the whole genome sequences of V4 clone and analysed the gene composition including NP,P,M,F,HN and L,then we compared them with other NDV strains for homogenous and phylogenetic tree, the results indicated that our V4 strain was mostly closed to QV4 strain genetically. Three complete cDNA clones named pFT7-final,pFpolII-V4-final and pBRT7-VP2 were constructed respectively; We also constructed the minigenome system for V4 strain named pBR-EGFP, further co-transfection suggested that our rescue system work well at the same time; Packaging experiments mediated by helper virus indicated that 3'leader and 5'trailer region were important in packaging progress.