Molecular Evolution Study Of Chinese Isolates Of Marek’s Disease Virus

Marek’s disease(MD) is a lymphoproliferative disease affecting chickens. It is caused by Gallid herpesvirus type 2(GaHV-2), commonly known as Marek’s disease virus serotype 1(MDV-1), which is in the Mardivirus genus and the family Herpesviridae. The main clinical manifestations were T-cell lymphoma and paralysis.Since the early 1970’s, the disease has been controlled with vaccines. However, the current epidemiological data in China revealed that the MD occurring ratio was increasing in immunity chicken. Several virulence tests of isolates have indicated that the pathogenic characteristics changed significantly, and the virulence enhanced considerably in new Chinese MD virus(MDV) isolates. Under the circumstances, the precondition of prevention and control of MD were that study the molecular evolution of MDV field isolates, and search the key factors relate to virulence of MDV field isolates. Several molecular epidemiologic studies have indicated that Chinese MDV isolates constitute a separate clade from MDV isolates obtained from other regions at the gene level. Among 18 total MDV strains which have complete genome sequences available in GenBank, there are three Chinese MDV strains(814, GX0101, and LMS). However, the available genetic data are still insufficient information to reveal the molecular evolution and virulence factor of Chinese MDV isolates. In this study, we sequenced the genomes of 6 new Chinese MDV strains(LCC, LTS, WC/1203, JL/1404, CC/1409, and HS/1412) isolated from chickens with failed immunisation as well as one previously isolated Chinese MDV strain, J-1. The complete nucleotide consensus sequences of the 7 field strains were similar in size and organized into 6 regions that are characteristic of class E herpesviruses. Phylogenetic analysis of genomes demonstrated for the first time that MDV diverged over the last two decades in China and around the world at the genome level. A transition in the evolution of MDV occurred in the 1990’s. Strains isolated in China after 2000 were closely related at both the gene and genome levels. The results of multiple sequence alignments demonstrated that the Chinese isolates contained specific sequences, especially in meq, ICP4, UL36 and US/TRS junction regions. The specific sequences may work together to promote the virulence evolution of Chinese MDV isolates. The point mutations of meq, ICP4 and UL29, and a 151-bp insertion in US/TRS junction regions were more likely to leading to increased virulence. Moreover, mutations RLORF4 T11 M, RLORF7 P176R/T, UL29 R506 L, RSORF1 K44 R and ICP4 K1948 M are unique mutations of new Chinese MDV isolates.To future study the variations related to virulence in Chinese MDV isolates, we sequenced the genomes of passaging attenuated strains in vitro, L-MSp75 and L-MSp110, and parent strain L-MSp5. The results of multiple sequence alignments demonstrated that the attenuated strains contained several insertions, deletions and nonsynonymous single nucleotide mutations. The mutations occurring in RLORF1, RLORF2, RLORF12, ICP4, US7, US8, the upstream of IRS(corresponding to the downstream of TRS), and IRS/US junctions(corresponding to the US/TRS junctions) were more likely to leading to attenuated virulence. Taking these two part of the study,we found that the focus regions which contained the mutations related to increased virulence and attenuated virulence were ICP4 gene and US/TRS junction regions. They are the key regions in future study of Chinese MDV virulence factors.In this study, we first analysed the molecular evolution of Chinese isolates at the genome level, and explored the variations related to the virulence of new Chinese isolates. Provided research direction and theoretical basis for future virulence factors study, prevention MD, and development of new MD vaccines.