Attenuation Of Epidemic Marek's Disease Virus Strains In Vitro And Mutational Analysis Of Main Tumor-associated Genes

Marek's disease(MD) is a highly contagious lymphoproliferative and demyelinating discorder of chickens which is caused by Marek's disease virus(MDV) serotype 1,a cell-associated, acuted-transforming alphaherpesvirus.MD had a worldwide distribution and caused enormous loss to the poultry industry,and was primarily controlled with vaccine.Recently,the continuous evolution of field strains towards greater virulence will likely constitute a threat to future poultry production,and further post vaccine failures.Therefore,research of new and efficious MD vaccine is a focus.Passage of virulent strains serially in cell culture is still an important access to achieve MD vaccine.And changes of some MDV's genetic fragments were regarded as be associated with virulence.This research would conduce to analysis of virulence and selecting of new vaccine strain.Four epidemic MDV strains isolated from Nouth Eastern and Sichuan area of China were named with L-SY,L-MS,L-CZ and L-ZY separately.These strains were cloned and passaged highly on Chick Embryo Fibroblast(CEF) to 75 to 85 passages.Therfore,four high-passaged MDV strains were obtained,they were L-SYp85C,L-MSp75C,L-CZp75C and L-ZYp75C.The biological characteristics in vitro,pathogenicity and virus load in feather tip of strain L-SYp85C and L-MSp75C were analysis dynamically.Meanwhile,mutational analysis of Meq,RLORF4,RLORF12 and 132bpr genes of these four high-passaged MDV strains were undertaked.The main purpose was to study changes during passaging both in biological and genetic characteristics,and provide theoretical basis for vaccine research.The results were as follow:1.Passaging on CEF of strain L-SY and L-MS resulted in increased replicaion rate and plaque sizes.1 day SPF chickens were inoculated with strain L-SYp85C and L-MSp75C in ten times doses of parental strains.After 12 weeks in isolated environment,no tumor was found and body weights not affected,and negative of MDV antigen in feather tip of AGP assay.Also, the virus load of feather tips was lower than 10~6 copies/10~6 cell which indicate that replicative captability of strain L-SYp85C and L-MSp75C has significantly decreased.All of above showed that both strain L-SYp85C and L-MSp75C had been attenuated 2.Mutational analysis of Meq,RLORF4, RLORF12 and 132bpr gene were studied which were thought to be related with virulence.The comparison of sequences and analysis of Meq genes indicated that two strains(L-ZYp75C and L-CZp75C) contained single nucleotide insertion and deletion.Extra Cytidine(C ) insertion in position 625 of strain L-CZp75C was predicted to cause Open Reading Frame(ORF) shift in three overlapping genes(Meq,RLORF6 and 23KD).The Meq gene from L-ZYp75C strain contained an extra cytidine(C) insertion at nucleotide position 529 and a single thymidine(T) deletion at nucleotide position 602, resulting in frameshift mutation.And this frameshift mutation could lead to changes in deduced amino acids from position 177 to 200 of Meq gene.The comparison of nucleotide sequences of RLORF4 genes in the study revealed that the RLORF4 gene of strain L-SYp85C contained a fragment deletion in ORF from nucleotide position 215 to 265,resulting in 17 amino acids deletions,which were not found in other sequenced strains.Comparison of nucleotide sequences of RLORF12 genes in the study revealed several mutations.The RLORF12 gene of strain L-MSp75C contained a single T deletion at nucleotide position 67 and of 814 vaccine strain a large fragment deletion from nucleotide position 18 to 86,and both of the deletions located in Origin of replication site(Ori) of MDV genome.But strain L-ZYp75C possessed an unique "TGTTGGG" deletion in its RLORF12 gene.When the four Chinese epidemic MDV strains were serially passaged on CEF,the number of copies of the 132bp repeats increased from 2 to more than 10 copies.All of above results indicated that deletion and/or insertion mutation occurred in Meq,RLORF4,RLORF12 and 132bpr after serial passage of these four Chinese epidemic MDV strains on CEF.In this experiment,we cultivated four high-passaged MDV strains,and analysed systematically the attenuation and attenuation-related genetic characteristics,which provided signigicant datas for the research of MDV1 pathogenic mechanism,variation,diagnose and new vaccines.