Cultivation Of Attenuated Strains Of Marek's Disease Virus And Their Characteristic Analysis

The attenuation of isolated virus is an effective method for cultivating attenuated vaccine. In this study, four Marek's disease virus (MDV) virulent strains (L-ZY, L-CZ, L-MS and L-SY) were passaged continually by 110 generations in vitro, and cultivated four high-passaged strains (L-ZYp110C, L-CZp110C, L-MSp110C and L-SYp110C).The pathogenicity in SPF chicken and growth characteristics of high-passaged strains in vivo and vitro were noticed. Meanwhile, genetic variation of the pathogenic related gene (Meq,132bpr,RLORF4 and RLORF12) was analyed. The results showed that the SPF chickens inoculated with strain L-SYp110 and strain L-MSp110C in variant doses were MD negative in experimental period (about 12 weeks).There was no significant difference in body weight between experimental and control groups. The replication capacity of high-passaged strains in vitro was higher than wild-type parental virus, conversely in vivo. After sequence alignment between high-passaged strains and parental virus, the results were as followed. (1) In strain L-ZYp110C, four Cytidines (cccc) were inserted in the position between 528 and 531 in the gene encoding Meq, resulting in ORF frame shift mutation. 69bp deletion from position 171 to 239 in the gene encoding RLORF4 was found. Unique"TGTTGGG"deletion in the position between 168 and 174 in the gene encoding RLORF12 resulted in ORF shift mutation. (2) strain L-MSp110C contained position 637 extra T (thymidine) insertion in the gene encoding Meq , resulting in frameshift mutation. The T (thymidine) deletion in position 67 in RLORF12 was found. The end result was a frameshift and translational termination after amino acid 33.If a downstream ATG was utilized then a protein of 76 aa would be produced.(3) 69bp deletion from position 165 to 233 in the gene encoding RLORF4 of strain L-CZp110C was found.(4) 51bp deletion from position 215 to 265in the gene encoding RLORF4 of strain L-SYp110C was found. (5) The number of copy of the 132bp repeats was increased apparently for four high-passaged strains.The results show that the ability of replication and characteristics of oncogenes of four high-passaged strains were changed in passage 110, resulting in attenuation of MDV virulent strains. This study provides experiment basis for screening vaccine strain.