Isolation And Identification Of Sheeppox Virus Of Gansu Strain And Establishment Of Duplex-PCR Assay For Detecttion Of Sheeppox

Sheeppox and Goatpox are highly acurate, febrile and contagious disease casused by sheeppox virus (SPPV), goatpox virus (GTPV). All Capripoxvirus DNA are double-stranded with lengths of around150kbp. Strains from Goatpox and Sheeppox share at least147genes. P32gene in Capripoxviruses is an important immunogenic gene; G protein-coupled receptors contributes to immune evasion and signal transduction through cell membranes; the Capripoxvirus homologue of Vaccinia virus30kDa RNA polymerase subunit (RPO30) gene might be associated with toxicity and host of Capripoxviruses. P32, GPCR and RPO30genes are highly conserved among Capripoxviruses, and they can be used in diagnosis and molecular epidemiology of Sheeppox and Goatpox. In recent years, frequent outbreaks of Sheeppox and Goatpox in our country has caused great economic losses in animal husbandry and severely restricted their development. It is of great significance to establish a quick and high efficient detection method and research on the molecular epidemiology of epidemic strain in our country. Thus, this research included several aspects as follow:1. Isolation and identification of Sheeppox virus Gansu strainSamples were collected from sick sheep in Zhangye, Gansu, and one virus strain was isolated with testis cells of lambs. We identified the virus strain, named GansuZY-isolate, was a Sheeppox virus from the molecular level by amplifying highly specific P32gene and analyzing its gene sequence.2.Coloning and gene sequence analysis on P32, GPCR and RPO30of G ansuZY-isolateP32, GPCR and RPO30of GansuZY-isolate were cloned successfully. According to the gene sequence analysis, strains of these tree genes and other SPPV gene generally have genome identities similar by at least99.0%-100%, which demonstrated the three genes in different SPPV strains are highly conserved. We can learn from the evolution analysis that, GansuZY-isolate is belong to SPPV because the result of molecular biology was consistent. GansuZY-isolate is similar to other SPPV strains reported in China, which indicated that the epidemic strain in our country is relatively stable, and there is no dramatic gene variation.3. Establishment of duplex-PCR assay for detecttion of sheeppox The duplex-PCR reaction was developed based on P32gene and InS-1gene, GPCR gene and InS-1gene, RPO30gene and InS-1gene. The experimental results demonstrated, all of the three methods have high specificity. Sensitivity test showed that the sensitivities of the above three methods were652ng,652ng and65.2ng respectively.In general, this research enriched molecular epidemic and research data of SPPV in our country. The initial establishment of duplex-PCR detection has laid a solid foundation for the development of commercialized quick SPPV detection.