China Animal Husbandry and Veterinary Medicine ›› 2019, Vol. 46 ›› Issue (12): 3725-3732.doi: 10.16431/j.cnki.1671-7236.2019.12.032

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Isolation and Identification of Orf Virus DZ Strain

LI Pengfei1, ZHANG You1, XIAN Simei1,2, LI Ting1, BAO Ximing1, ZHANG Yi1, RAO Tiyu1, WU Bomei1   

  1. 1. College of Animal Science, Guizhou University, Guiyang 550025, China;
    2. Institute of Animal Disease Research of Guizhou, Guiyang 550025, China
  • Received:2019-07-04 Online:2019-12-20 Published:2019-12-21

Abstract: This study was aimed to understand the prevalence of orf virus (ORFV) in Chongqing.18 samples of clinical suspected orf disease materials were collected from sheep farms in Dazu,Chongqing,etc,and DNA of the samples was extracted,which was identified as positive by PCR.Positive disease materials were routinely treated,inoculated with lamb testicular cells (LT),and blind transmitted to the F5 generation.Toxic LT cell lesion CPE was observed,and F5 generation cell cultures were systematically identified by indirect immunofluorescence test (IFA),PCR amplification,sequencing,homology alignment,genetic evolution analysis and TCID50 determination.The results showed that the nucleic acid positive rate of ORFV detected by PCR was 61.1% (11/18).After LT cells were inoculated with Dazu strain for 36 h,the long-spindle cells became round,fused,reticulated and shriveled,and most of the cells fell off after 72 h.IFA observed the specific green fluorescence in cytoplasm,and the fluorescence was distributed around the nucleus.Specific target bands of F1 to F5 cell cultures were amplified by PCR with a size of 1 137 bp.The sequencing results were compared with 19 strains of ORFV B2L genes in GenBank,and the nucleotide homologies were 97.9% to 100%,which was up to 100% of the homology with the SA00 isolates from the United States,and the genetic evolution analysis showed that they were in the same evolutionary branch,with close relationship.The TCID50 value of F5 generation cell culture was 10-5.68/0.1 mL,which could be stable proliferation in cells.In conclusion,this study successfully isolated and obtained an ORFV strain,provided biological materials for the follow-up ORFV research,and laid a foundation for the prevention and control of stomatitis in Chongqing.

Key words: orf virus (ORFV); isolation and identification; genetic evolution analysis

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