Isolation And Identification Of Sheeppox Virus Guyuan Strain And Screening Of Si Rna Targeting Its RNAolymerase Gene

Capripoxvirus is a genus of viruses in the subfamily Chordopoxvirinae and the family Poxviridae, consists of three species: sheeppox virus(SPPV), goatpox virus(GTPV), and lumpy skin disease virus(LSDV). Capripoxviruses are among the most serious animal poxviruses. They cause negative economic consequences by damaging hides and wool and forcing the establishment of trade restrictions in response to an outbreak.Capsid provided relatively stable protection for the genome before viral invade host cells. Capsid will be dissociated at the appropriate time and location by internal environment, then the viral genome will be rapid released into the cytoplasm for replication. Viral’s uncoating is the primary determinant of SPPV’s proliferation in cell cytoplasm. The uncoating process relies on the synthesis of RNA and protein. Therefore, this study worked on the relationship of RNA polymerase with the uncoating of sheep pox virus based on a sheep pox virus’ s isolation.The uninfected sheep were immunized with suspension made from the incidence skin rash, and infected to sheep primary lamb testicles(LT) cells. After 5 blind passages, infected products was harvested and infected to Vero cells. Then identify the virus by IFA and PCR. The classical symptom and featured CPE was occurred in unimmunized sheep and Vero cells respectively. IFA and PCR results suggested that virus existed in the samples. Sequencing results showed the homology of the nucleotide fragment is more than 99% between isolated virus strain and reported strains. The results suggested the sheep poxvirus strain designated as GY strain was isolated from sheep suspected sheep poxvirus infection.ORF051 and ORF065 were amplified by GY strain, and were analyized by bioinformatics software. si Direct version 2.0 was used to design and preliminary evaluate si RNA to avoid off-target effects at full stretch.The two genes were clone into p EGFP-N1 vector, and the result suggests that the expression quantity of fluorescent at 48 h was highest while they were transfered to BHK cell. Then the recombinant vectors were co-transfected in BHK cells with the corresponding si RNAs. All of the si RNA can inhibit the expression of the two genes, and si RNA2 target to ORF051 and si RNA3 target to ORF065 has the obvious effects.Infected Vero cells by GY strain of SPPV, virus step growth curve was established by harvesting culture medium and cells interval eight hours. The curve shows that the amount of virus reached stabilized after 88 h with the proliferation of the virus. Both of ORF051 and ORF065 were clone into plv-puro vector, and then the Vero cell lines expressing the two genes were established by screening with puromycin.The stability and conservatism of the two genes are confirmed for designing their targeted si RNA in this research. The establishment of Vero cell lines expressing the two genes made preparetions for the study on the effects of the over expression of the two RNA polymerases on uncoating and help to understand the kinetics of virus infection in a host cell. Their respective effective si RNA were obtained with GPF as a reporter gene, which will lay the foundation for the further studies about the relationship of the RNA polymerase with uncoating.