Isolation And Identification Of Orf Virus From Shihezi, Xinjiang And Construction Of It’s119Gene Deletion Strain

In this study, detection, isolation and identification of Orf virus（ORFV）were conductedin goats suspected ORFV infection from Shihezi area of Xinjiang. Two protective antigengenes B2L and F1L, and three virulence genes VIR, GIF and VGEF of ORFV isolates werecloned and sequencd, respectively. Then sequencing and genetic evolution were carried out tounderstand whether genetic variation has taken place in ORFV Xinjiang epidemic strains.Then, ORFV119gene deletion strain was constructed through the method of homologousrecombination. And growth characteristics and genetic stability of ORFV119gene deletionstrain were studied. This study laid foundation for prevention and control of ORFV infectionin Xinjiang region and development of new vaccine. Research methods and main results areas follows:1. Isolation and identification of ORFV Shihezi isolatesPositive samples were screened, suspected lip scabs to aphthous ulcer disease in goatswere collected from shihezi area of Xinjiang, with Orf virus B2L gene specific primeramplification. After milling and aseptic processing of positive samples, Vero cells wereinoculated for virus isolation, typical cytopathic effects were visible untill the5th passage.Electron microscopy showed the virus particles were ovally-shaped, enveloped and wrappedwith rope-like structures. In ultrathin sections of samples，mature virions were visible in thecells. The lips of2month-old lambs were inoculated with the viral culture medium forinfection experiments. Typical ORFV clinic symptoms such as papules and pustules appearedin mouths, lips and other parts after the inoculation, which confirmed three isolates wereORFV, named ORFV SHZ1, ORFV SHZ2and ORFV SHZ3, respectively.2. Cloning and genetic evolution analysis of important antigen and virulence gene ofORFV isolatesIn order to investigate the Orf virus (ORFV) epidemic strains of genetic variation inXinjiang, the protective antigen genes B2L, F1L and virulence genes VIR, GIF and VEGF ofthe three ORFV isolates were cloned, sequenced and analyzed for genetic evolution. Theresults showed that B2L and F1L in the three isolates were relatively conservative with97.28-98.37%and94.56-97.17%nucleotide sequence identities to those of other previouslydescribed isolates, respectively. At amino acid level, the identities were88.6-97.9%and86.7-97.4%, respectively. For the virulence genes VIR, GIF and VEGF the identities were91.67-98.01%,88.67-96.12%or75.85-97.96%at nucleotide sequence level, and91.8.-97.3%,85.2-97.0%or71.5-98.5%at amino acid sequence level, respectively. The VEGF geneshowed relatively larger variations, particularly at amino acid sequence level when comparedwith other reference strains. Phylogenetic trees based on B2L and VIR showed that ORFVisolates SHZ1and SHZ2were closer to isolates from Taiwan. Phylogenetic tree of VEGFindicated that the three isolates were all closely related to NZ2strain from New Zealand.Phylogenetic trees based on GIF gene revealed that the isolated were most closely related to MUK59/05of India. In the F1L phylogenetic tree, these isolates were divided into twobranches, each representing isolates OV/C2and OV/Torino (Italy) and OV-SAOO (USA),respectively. The results inferred that recombination had occurred among Shihezi isolates,Taiwan strains and other strains, causing greater variation in virulence gene.3. Construction and identification of119gene deletion mutant of ORFVFragment containing119gene and it’s flanking sequences of ORFV-SHZ1strain wasamplified by PCR,123bp sequences in119gene were deleted using enzyme digestion. Thenthe expression box with vaccinia late promoter P11and LacZ reporter gene was inserted into119gene mutant to generate recombinant shuttle vector. Vero cells infected by ORFV werethen transfected by shuttle vector for homologous recombination. The recombinant ORFVwas screened and purified, virus titer, growth characteristics and genetic stability ofrecombinant virus with119gene deletion were assayed. The results showed that TCID50ofrecombinant virus was1×10-4/mL. Compared with parental strains, there is no significantdifference in TCID50. The growth curve of the recombinant virus is similar to parental strainon Vero cells. The results revealed that119gene maybe irrelevant to ORFV virulence andreplication.