Construction Of PPRV Reverse Genetic System And Rescue Of Recombinant Virus

Peste des petits ruminants virus(PPRV)is a member of the genus morbillivirus of Paramyxoviridae.The genome of PPRV is a single stranded,non segmented negative strand RNA.The full-length sequencing of PPRV is 15948 nt or 15954 nt,which can translate six structural proteins and two non structural proteins.PPRV is distributed all over the world.At present,PPR epidemic is often reported in China.PPRV infection can cause high incidence rate and high mortality rate in small ruminants such as sheep.Currently,all countries in the world,including China,use the attenuated vaccine to prevent and control the disease.The immune effect is good and the protection period is long.However,the stability is poor,the safety needs to be improved,and there may be potential biological safety hazards.In addition,with the increasing number of PPRV infected hosts,the current vaccine can not be used to immunize wild animals,leading to the outbreak of one after another and expanding.Using reverse genetics technology to construct recombinant PPRV is not only a convenient way to develop a more safe,stable and novel vaccine that can distinguish infection from immunity,but also a tool to carry out the research on the pathogenic mechanism of PPRV.In this study,the full-length cDNA plasmid of PPRV Nigeria 75/1vaccine strain constructed and preserved in our laboratory was used as the material.On this basis,we planned to establish the reverse genetic system of PPRV attenuated vaccine strain.We tried to rescue PPRV from the microgenome and the full-length cDNA plasmid:(1)The microgenomic plasmid of PPRV was designed and constructed,which was cotransfected with three helper plasmids(plasmids expressing N protein,P protein and L protein of PPRV respectively)to rescue PPRV microgenome,which proved that the virus rescue system was effective and laid the experimental foundation for rescuing recombinant PPRV containing full-length genome;(2)Two kinds of PPRV full-length cDNA plasmids based on T7 RNA polymerase system were designed and constructed:one containing only the full-length genome of PPRV;The other introduced the gene sequence expressing enhanced green fluorescent protein(EGFP)into the P and M genes of PPRV cDNA sequence.The results provide basic materials for the rescue of recombinant PPRV;(3)The full-length cDNA plasmid was cotransfected into BSR-T7/5 cells with helper plasmids.The results of IFA and Western blotting showed that the N,P,V and H proteins of r PPRV and r PPRV-EGFP were expressed;after blind passages for several generations,RT-PCR proved that the virus rescue was successful;The replication of r PPRV and r PPRV EGFP was further analyzed by RT q PCR.In this study,we used the microgenome rescue system to verify the function of the helper plasmids;On this basis,two full-length cDNA plasmids of PPRV based on T7 RNA polymerase system were constructed and cotransfected with helper plasmids to rescue two recombinant viruses.These results have some practical value and theoretical reference in the construction of PPRV reverse genetic system and the development of new vaccines,and are of great significance for the prevention and elimination of PPR.