Mutational analysis of Meq, RLORF4, RLORF12 and 132bpr genes of epidemic Marek's disease virus strains highly passaged on chicken embryo fibroblast / 病毒学报

ABSTRACT

Recently, much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among many genes in the MDV genome, several genes such as Meq, RLORF4, RLORF12, and 132bpr have been considered recently associated with virulence of MDV. In this paper, primers of Meq, RLORF4, RLORF12 and 132bpr genes were designed and synthesized, based on the published whole genome sequence of MDV strain GA. The genes of Meq, RLORF4 and RLORF12 from four Chinese epidemic MDV strains highly passaged on chicken embryo fibroblast (CEF), i. e. L-SYp85C, L-MSp75C, L-CZp75C, and L-ZYp75C, as well as their corresponding parent strains, i. e. L-SY, L-MS, L-CZ, and L-ZY, the reference virulent strain J-1 and the vaccine strain 814 were amplified by PCR respectively. Then the PCR products of interest were cloned and sequenced respectively. The results of sequence comparison and analysis of Meq genes in the study indicated that Meq genes from the two strains L-ZYp75C and L-CZp75C contained single nucleotide insertion and deletion. The Meq gene from strain L-ZYp75C contained an extra cytidine (C) insertion at nucleotide position 529 and a single thymidine (T) deletion at nucleotide position 602, resulting in a frameshift mutation. And this frameshift mutation could lead to changes in deduced amino acid sequence from position 177 to 200 of Meq gene. The extra C insertion at nucleotide position 625 in Meq gene of strain L-CZp75C was also predicted to cause frameshift mutation in three overlapping genes (Meq, RLORF6 and 23KD genes). 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 Open Reading Frame (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, 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.