| Peste des petits ruminants virus mainly occurs in developing countries,a serious threat to the development of the sheep industry,its ability to spread across the border and the mortality rate of up to 100%.The PPRV epidemic strains reported in China are mainly concentrated in areas such as Tibet and Xinjiang.Other areas are rarely reported.In this study,FY strain isolated and identified in 2014 was sequenced and analyzed in order to provide a new reference for the study of molecular epidemiology of PPRV in China,and to enrich the gene pool of PPRV.It will be helpful to study the genetic background of PPRV in China,to analyze the variation of virus,and then to help the selection of vaccine and the development of new vaccine.This paper includes the following three aspects.1.Isolation and identification of PPRV FY strain:The PPRV disease materials collected from Fuyang city of Zhejiang province were ground,centrifuged and filtered.Then it was inoculated with Vero cells,BHK cells and CV cells(CV).96 hours later,the cell culture was collected and the virus was inoculated 3 or 5 times in these cells.The results showed that there was no obvious pathological changes in Vero cells and BHK cells,but there were typical pathological changes in CV cells,which mainly showed as follows:the cells became round,fell off,and appeared in the syncytium.When the cells were occurred with 80%pathological changes,the cell cultures were harvested,and the virus were identified by RT-PCR,Western Blot,indirect immunofluorescence and electron microscopy.The results showed that PPRV could be detected specifically in CV cell culture.Western Blot,indirect immunofluorescence assay showed that the virus isolated from CV cells reacted with PPRV F Monoclonal antibody.The obtained CV cell culture was centrifuged and the virus particles with a diameter of 150-300nm were observed by electron microscope,which was consistent with the reported PPRV virus particle morphology.The above results showed that we successfully isolated the PPRV strain by using CV cells and named it FY strain.2.Prokaryotic expression and polyclonal antibody preparation of N gene:N gene sequence of PPRV FY strain was amplified,connected to pET-32a vector and sequenced.After sequencing,the recombinant N protein was expressed in E.coli BL21(DE3),The recombinant N protein was induced by IPTG and purified by inclusion body purification.Polyclonal antibody against N protein was obtained by rabbit immunized with purified protein and was identified by Western Blot and indirect immunofluorescence method.The results showed that the prepared antibody can be used for detection of PPRV.3.Complete genome sequencing and sequence analysis of PPRV FY strain:According to the published sequence of PPRV reference strains,11 pairs of specific primers were designed,and then the complete genome sequence of PPRV FY strain was amplified by RT-PCR method.DNAstar software and Mega software were used to compare and analyze the gene sequences of PPRV FY strain with reference strains.In addition,the phylogenetic tree was drawn according to the nucleotide sequence of N gene.The results showed that the full-length genome of PPRV FY strain was 15948nt.It is postulated that 6 structural proteins(N,P,L,M,F and H)are encoded and 2 non-structural proteins(C and V)are encoded.The transcription initiation sequence and transcription termination sequence of PPRV were highly conserved.Homology analysis showed that FY strain had the lowest homology with Ivory Coast strain,but had the highest homology with Tibet strain.From the length of the noncoding region,N-P had the highest similarity and M-F had the lowest similarity.Through the comparison of the PPRV FY strain with reference strains can also be seen that GP area and AGP area were high conserved in the measlesvirus,and were complementary in different degrees.In conservative regions,21 animo acid mutations were found by comparing PPRV FY strain and reference strains.These mutations on the structure and function of its encoding protein still needs further research.According to the PPRV sequence of N gene,we performed phylogenetic analysis of PPRV FY strains with reference strains.Analysis showed that PPRV FY strain had the same topological group as the PPRV strain from Asian,which belonged to the IV lineage.In summary,this study successfully isolated the PPRV FY strain,which was prevalent in Zhejiang province in 2014.The strain is not suitable for cell proliferation,and can only proliferate in the cells that express its receptor gene.On this basis,we have completed the complete genome sequencing,prokaryotic expression of N gene and bioinformatics analysis.The results showed that PPRV FY strain belonged to the IV lineage.It had low homology with the vaccine strain but it had a high homology with other wild strains reported in China... |
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