Isolation And Identification Of ORF Virus And Proliferation Characteristics In Different Cells

OrfV is a member of the Poxviridae and Parapoxviruses.It is a linear,double-stranded DNA virus with envelopes.It causes sheep and goats to develop papules in the skin and mucous membranes of the lips,tongue,nose,and breast.Pustules,ulcers,and the formation of verrucous scars as the main features of contact,epithelial,animal-animal communicable diseases.Studies at home and abroad have demonstrated that the virus is a highly mutated virus.Viruses isolated in different countries and regions have great differences in pathogenicity.The restriction enzyme mapping analysis of the genome shows that there are also differences.One of the most important tools for studying viruses is cells that can continuously proliferate viruses.Lamb or yak testicular cells are susceptible to OrfV proliferation,but the acquisition is limited by the source,and the acquisition process of primary cells is complex and large-scale use of experimental animals;OrfV cultured on passaged cells,there is weakened virulence after passage,cells Due to the instability of the disease（CPE）,the in vitro proliferation of the SF virus is an important technical bottleneck that directly affects the development of OrfV-related biological products in the basic research and practice of the biological characteristics of OrfV.In order to obtain OrfV endemic strains and provide data for OrfV basic research and vaccine production,the following studies were conducted:1.Isolation and identification of OrfV-DZ strainIn this study,18 samples of suspected sheep aphthous ulcers were collected from multiple sheep farms in Chongqing.After the DNA was extracted,a PCR assay established in the laboratory was used.After identification of positive diseased materials,the sheep’s testicular cells were inoculated to isolate OrfV.The virus was identified by indirect immunofluorescence,gene sequencing,homology analysis,and animal infection tests.The results showed that:11 of the 18 clinical samples were positive by PCR.After being inoculated with sheep testicular cells,after successive passage to the fifth passage,one case still showed typical cytopathological changes.Indirect immunofluorescence can be detected in cells.After the B2L gene was amplified and sequenced,the homology of the nucleotides and the encoded proteins of the 19 other OrfV B2L genes was between 97.9%and 100%.Inoculation of the oral lip of the cell culture inoculated goat’s mouth,appears to be consistent with the symptoms of thrush clinical symptoms.2.The establishment of a method for detecting live OrfV based on PMA-PCRAzidopropylammonium bromide（PMA）is a novel dye that can penetrate the damaged cell membrane and enter the cells to bind with nucleic acids.Under exposure to visible light（460 nm）,PMA can cross-link DNA and inhibit PCR amplification of DNA.Live mutton sore virus has a complete cell membrane structure that prevents PMA from binding to DNA.In this study,a combination of propidium azide bromide（PMA）and PCR was established to detect live acne outbreaks.Poisonous method.The results showed that:the water bath can inactivate OrfV at a temperature above 60°C.The optimal concentration range of PMA is 20-40μmol/L.Irradiation with inhaled OrfV(TCID50 10^-5.68/mL)can be performed at a distance of 20cm from a 500W halogen lamp for 15min.The DNA is fully bound and the PCR amplification of the inactivated virus from the sow virus is inhibited without affecting the PCR amplification of live avian sore virus.The method detects the lowest concentration of live oral sore virus is 10^-2.68 TCID₅₀/0.1 mL,and the specificity is good.3.Adaptive culture of DZ strain of the solitary herpes virus on different cellsIn this study,the primary cells of OrfV-DZ strain F5 generation isolated on the testis cells（LT）of Lamb were inoculated with two primary cells:yak testis cells（BT）and chicken embryo fibroblasts（CEF）;7 passages were inoculated.Cells:Vero,MDBK,BHK-21,MDCK,Hela,GSFs,NIH3T3,detected OrfV-DZ strains by observing the time of appearance of cytopathic effect（CPE）,TCID₅₀ assay,IFA,PMA-PCR and SYBR Green I real-time fluorescence quantitative PCR Proliferation characteristics of different cell lines.The results showed that F5 passages of OrfV-DZ strains were inoculated with BT cells.CPE appeared at 24h of F3 generation.No CPE was observed even after CEF cells were passed to F5.CPE was observed at 36 days of F5 generation inoculated with MDBK cells.HeLa cells were inoculated.CPE occurred in F5 for 48 h,and no CPE was seen in the other 5 passages passed blindly to the 5th passage;F5 passage cytotoxicity in BT,MDBK and Hela cells was collected and the TCID50 was determined as:10^-5.27/0.1 mL,10-4.86/0.1 mL,10-4.57/0.1 mL;specific fluorescence can be detected in BT,MDBK and HeLa cell pastes;PMA-PCR method can detect specific bands in BT and MDBK 1-8 cell cytotoxicity The specific band was detected in Hela cell line 1⁵ generations;the copy number of OrfV-DZ amplified DNA in LT from 1st to 8th generation was 3.02×10⁷、1.32×10⁷、3.02×10⁷、4.17×10⁷、5.01×10⁷、3.09×10⁷、2.30×10⁷、2.57×10⁷copies/μL;the number of copies of virus amplified from 1-8 generations in MDBK cells was 7.76×10⁶、5.89×10⁷、4.07×10⁷、2.88×10⁷、2.11×10⁶、1.29×10⁶、9.33×10⁵、7.59×10⁵copies/μL,respectively;The amplified copies of Hela cells at 1-5 generations were 6.46×10⁷、9.33×10⁶、6.17×10⁶、2.0×10⁶、1.38×10⁵copies/μL respectively.The results showed that OrfV-DZ strain was in MD.In the MDBK and Hela cells,the number of virus copies was gradually decreased as the passage increased,so the cell lines suitable for OrfV-DZ proliferation were LT and BT.Conclusion:This experiment successfully isolated a strain of Oral Aphthous Oral Virus which was isolated from the affected sheep and was named OrfV-DZ strain.A PMA-PCR method was established to detect live OrfV virus,and the optimal selection for OrfV-DZ strain proliferation was established.The cell lines were lamb testicular cells（LT）and bovine testicular cells（BT）.