Genetic Evolution Analysis Of Three Main Constructive Protein Genes Of Orf Viruses Isolated From Different Areas

In this research, some pathologic materials were collected from suspected Orf sick sheep in Chifeng, Wuchuan and Shandong areas, after the treatment of cut up, grinding,3 times of freezing and thawing and sterilizing process, the supernatant of the pathologic material is inoculated into the Calf testicle cell and MDBK cell lines; meanwhile, one pair of primers were synthesized according to the Orf virus-B2L gene sequence published on the Genbank and B2L gene of Orf were amplified from suspected Orf pathologic materials and its cell cultures by PCR; the supernatant of the pathologic materials were subjected to differential centrifugation, negative staining and observed by electron microscopy. Based on the above results,3 pair of primers were synthesized according to the B2L、F1L and VIR gene sequence of Orf virus which published on the Genbank and the B2L、F1L、VIR genes of 3 Orf strains collected from the different areas were sequenced after a series operation of virus DNA extraction, PCR amplification, pMD 19-T vector ligation, DH5α competent cell transfer, selection and identification of positive clones. PCR amplifications of 3 different strains were compared and analyzed by DNAstar, then sequence homology of those genes were compared with the genes sequences of B2L、F1L、VIR which published on the Genbank by BLAST on line. Finely, phylogenetic tree was constructed by Clustal-V Method after Meg Align analysis.Results showed:significant cytopathic effects were appeared on the first and second generation inoculation; on the third inoculation, the cytopathic effect was not obvious, but PCR amplification of cell culture has significant DNA strand. Oval shaped typical virus were observed by the microscopy; above results indicating that we obtained 3 different Orf viruses, named as Orfv-W、Orfv-C�'�Orfv-Q. The homology of B2L、 F1L and VIR3 genes of the 3 strains were 94.8%～97.4%、93.2%～98% and 95.1%～ 100% respectively; shared a homology of 82.1%～98.0%、93.4%～98.0% and 93.8%～ 99.1% with the reference strains published from the different countries. Phylogenetic tree showed that strain Orfv-W is closely related to the goat isolation strain; Orfv-Q is related to the sheep isolation strain; Orfv-C is related to the both goat and sheep isolation strains, and genetic branch of the 3 genes is closely related to the Orf virus isolated in the northwest China.