Isolation, Determination And Virulence Attenuation Of Yangling And Luxi Orf Virus

Orf, as known as contagious pustular dermatitis, is an acute and contagious virusdisease,which is caused by orf virus. Orf is aworldwide distribution disease, which has beenreported in Xinjiang, Qinghai, Yunnan, Shandong, Sichuan and Shaanxi provinces, and oftenoccurs in late summer and autumn/winter. In endemic areas, the morbidity of orf is as high as80%~90%. Huge economic losses were caused by orf. In this study, two orf virus strainsfrom yangling in Shaanxi province andluxi in Yunnan province were isolated and determined.The antigenic differences of these two orf viruses were identified by trace cells crossneutralization test and these two orf viruses were passaged to P86via continuous inoculationon calf testis primary cells. In order to detect the attenuated effect,3lambs of30-45d wereinoculated separately at the lip withthe P86orf virus. The main results were as follows:1. Isolation and determination of Yangling and Luxi ORFV strainThe5th generation of ORFV can cause apparent CPE of calf testis primary cells and thevirus titer of the5th generationisolated from Yangling and Luxi was107.66TCID50/mland108.2TCID50/ml, respectively. The similarities ofYangling orfv compared with its referencesequenceswere96.57%and96.43%, whichwere97.75%and96.33%for Luxiorfv.Accordingto the sequences alignment of Yangling and Luxi, the similarity of B2L gene is96.93%andF1L gene similarity is96.90%. In conclusion, two wild strain of ORFV in Yangling and LuxiCountywere isolated in this study.2. Trace cell cross neutralization testbetween Yangling andLuxi ORFV strainTo produce relevant immune serum, the two strains were separately used to immunerabbits. The corresponding serum antibody titer were1:2and1:4by double immunodiffusiontest, and the ELISA serum antibody titer were1:100000and1:1000000. The fixedvirus-dilution serum method was used to conduct trace cell neutralization test. Theneutralization antibody titer between Yangling ORFV immune serum and Yangling ORFVwas1:214, and which was1:186for Luxi ORFV. Theneutralization antibody titer of LuxiORFV immune serum versus Yangling and Luxi ORFV were1:224and1:316, respectively.The antigen ratio of these two ORFV was1.27by using R=r1.r2, which indicated that theneutralization antigenicdifferences between Yangling and Luxi ORFV is not obvious.3. Virulence attenuated ofYangling and Luxi ORFV The isolated Yangling and Luxi ORFV were passaged to P86on calf testis primary cells,and the P86Yangling ORFV virus titer was106.92TCID50/ml，and this was107.31TCID50/mlfor Luxi ORFV. After inoculation to lambs, the P86Yangling ORFV inoculation sites werenormal, not erythema, blisters, pus cell and ulcer lesions and other lesion appearance. Postinfection3days, the inoculation sites werecompletely healing. Although there was grain sizelupus in inoculation sites when the P86Luxi ORFV was given, it is still safefor the P86LuxiORFV. Thus, the virulence of P86Yangling and Luxi ORFV strains had been attenuated,whichwas no pathogenicity for lambs.In conclusion, the ORFV Yangling strain and Luxi strain are successfully isolated anddetermined in this study, and the neutralization antigenicdifferences between Yangling andLuxi ORFV are not obvious. Finally the relevant attenuated strains have obtained.