Isolation And Identification Of Orf Virus And Establishment Of An Immortalized Ovis Aries Testis Cell Line

Orf virus?ORFV?,also known as contagious secthyma virus which belong to Parapoxvirus genus of the poxvirus family.It can infect sheep and goats that can cause blisters,pustules and ecdysis in the mucous membrane and anal area of the lamb.The disease has a high mortality rate and lethality.The disease has become widespread in China and has seriously affected the development of the aquaculture industry.In December 2016,an outbreak of suspicious orf occurred in a goat farm in Feixi,Anhui Province.After collecting suspected epidemic materials,the original Ovis aries testis cell was used to isolate the ORFV and the virus proliferation characteristics were investigated.In addition,we also performed genetic evolution analysis of ORFV that was popular in Anhui Province from 2013 to 2017.Considering the lack of a stable cell lines for the propagation of the ORFV,we utilized the Lentiviral packaging system to integrate the human telomerase reverse transcriptase?hTERT?into the chromosome of the original Ovis aries testis cells.The immortalized cells was successfully screened under the pressure of puromycin.This work laid the foundation for follow-up studies.The research content of this paper is detailed as follows.1.Isolation and identification of the AH-FX16 ORFV strain:The clinical manifestations of the diseased lambs were erythema,blisters,pustules,and scarring around the oral mucosa,tongue,mouth and lips.Collecting the lamb skin,The total viral DNA was extracted from the lamb skin and the VIR gene of ORFV was specifically amplified by PCR.The filter-sterilized material was inoculated into the sheep's primary testicular cells.Obvious cytopathic changes were observed after 48 hours,such as the appearance of cell shedding and a large intercellular distance.After filtrating and removing the bacteria of skin material,the filtered fluid inoculated with the original Ovis aries testis cells.There was a obvious cytopathic changes which can be observed after 48 hours virus affection,such as the appearance of cell shedding and a large intercellular distance.The TCID₅₀0 of the 5th cell ORFV was 10^-4.625/0.1 mL.The result of indirect immunofluorescence showed that ORFV was mainly distributed in the nucleus and a small amount of virus appeared in the cytoplasm.The presence of ORFV protein?B2L protein?in cell culture was confirmed by Western-Blot assays.The purified virus was observed under an electron microscope.Typical virus particles of the ORFV could be seen as ellipse-shaped,ball-like,capsular,with a diameter of 200³00nm.Finally,we inoculated the one-month-old lamb with the isolated ORFV through skin scratches.The results showed that the lamb present typical clinical symptoms of thrush.The above results prove that we have successfully isolated the infectivity of the ORFV and named it as AH-FX16 strain.2.Phylogenetic analysis of the ORFV in Anhui Province:11 genes?ORF007,ORF008,ORF011,ORF020,ORF059,ORF112,ORF117,ORF125,ORF129,ORF132?of the ORFV were amplified that is prevalent in Anhui Province from 2013 to 2017.After multiple sequence alignment analysis,there was a"universal"base insertion or deletion between the genes of these epidemic strains,which suggesting that ORFV is undergoing continuous evolution.Phylogenetic tree results showed that the prevalent ORFV strains in Anhui formed a large evolutionary group,and had close genetic relationship with the strains that were endemic to Fujian.2.Construct the recombinant plasmid pLOV-CMV-hTERT by the lentiviral expression system,use the liposome transfection method,and after screening by puromycin,obtain the Ovis aries testis cells with good cell state.DNA and RNA of 10 generations and 20generations of sheep testicular cell lines were extracted and detected by PCR and RT-PCR.The hTERT assay was positive.The corresponding carrier flag protein was detected by Western-Blot.Indirect immunofluorescence experiments using hTERT polyclonal antibody demonstrated that hTERT was expressed in cells.This cell line was named Ov-ST.This cell line can also be used for the propagation of ORFV.3.Establishment of immortalized Ovis aries testis cell line:First,the recombinant plasmid pLOV-CMV-hTERT was constructed,then the recombinant lentiviral vector three-plasmid system was co-transfected into 293T cells to package the recombinant particles,and the lentivirus particles were infected to the primary Ovis aries testis cells,which were positive by puromycin resistance screening.Positive clones were obtained by puromycin resistance screening.RT-PCR and Western blot were used to detect the transcription and expression levels of hTERT gene in the passaged cell lines.The positive clone was screened and named as Ov-ST.After 20 passages of positive cell clones were serially passaged,the cells were stable and had good splitting and growth capabilities.Finally,the expression of telomerase of10 th and 20th generations of immortalized cells was detected by RT-PCR,IFA and Western blot.The results showed that hTERT was stably expressed in all cells,demonstrating that we successfully established the immortalized cell line.OV-ST cells were inoculated with ORFV AH-FX16 strain and the cells were found to stably propagate ORFV,which laid the foundation for the development of the ORFV vaccine and the study of the mechanism of ORFV infection.