Comparative analysis of oncogenic genes revealed unique evolutionary features of field Marek's disease virus prevalent in recent years in China

Abstract

Background: Marek's disease (MD) is an economically important viral disease of chickens caused by Marek's disease virus (MDV), an oncogenic herpesvirus. This disease was well controlled since the widespread use of commercial vaccines, but field MDVs have shown continuous increasing in virulence and acquired the ability to overcome the immune response induced by vaccines. Nowadays, MD continues to be a serious threat to poultry industry, isolation and characterization of MDVs are essential for monitoring changes of viruses and evaluating the effectiveness of existing vaccines.

Results: Between 2008 and 2010, 18 field MDV strains were isolated from vaccinated chicken flocks in Sichuan province, China. Three oncogenic genes including Meq, pp38 and vIL-8 genes of the 18 isolates were amplified and sequenced. Homology analysis showed that the deduced amino acid sequences of these three genes exhibit 95.0-98.8%, 99.3-100% and 97.0-98.5% homology respectively with these of other reference strains published in GenBank. Alignment analysis of the nucleotide and deduced amino acid sequences showed that four amino acid mutations in Meq gene and two amino acid mutations in vIL-8 gene displayed perfect regularity in MDVs circulating in China, which could be considered as features of field MDVs prevalent in recent years in China. In addition, one amino acid mutation in pp38 gene can be considered as a feature of virulent MDVs from USA, and three amino acid mutations in Meq gene were identified and unique in very virulent plus (vv+) MDVs. Phylogenetic analysis based on Meq and vIL-8 protein sequences revealed that field MDVs in China evolved independently. Virulence studies showed that CVI988 could provide efficient protection against the field MDVs epidemic recently in China.

Conclusions: This study and other published data in the GenBank have demonstrated the features of Meq, pp38 and vIL-8 genes of MDVs circulating in recent years in Sichuan, China. Mutations, deletions or insertions were observed in these three genes, and some mutations could be considered as the unique marks of the MDVs circulating presently in China. The paper supplies some valuable information concerning the evolution of MDV which is useful for the vaccine development and control of MD in China.

Figure 1

PCR products of 132 bp repeats, Meq, pp38 and vIL-8 gene of 18 MDV isolates from China. Lane M: DL2000 Marker; Lane 1-20: ddH₂0, CVI988, LS, BY, YY, MS01, MS53, MS54, MS67, NC01, NC02, TQ20, DY01, DY04, XJ01, XJ4, WS03, WS04, YA, and 5079, respectively; A. analysis of copy numbers of 132 bp repeats in 18 MDV isolates; B. Amplification of Meq gene of 18 MDV isolates; C. Amplification of pp38 gene of 18 MDV isolates; D. Amplification of vIL-8 gene of 18 MDV isolates.

Figure 2

Comparison of deduced amino acid sequences of three oncogenic genes. A. Alignment analysis of amino acid sequences of Meq gene; B-1. Alignment analysis of nucleotide sequences of pp38 gene; B-2. Alignment analysis of amino acid sequences of pp38 gene; C-1. Alignment analysis of nucleotide sequences of of vIL-8 gene; C-2. Alignment analysis of amino acid sequences of vIL-8 gene. Vertical arrow refers to mutation of nucleotide or amino acid (aa) and two-way arrow refers to deletion of 59aa.

Figure 3

Phylogenetic analysis on Meq, pp38 and vIL-8 gene sequences of 18 Sichuan isolates and other reference MDVs. The phylogenetic tree was constructed using the MEGA version 4.0 by the neighbor-joining method with 1000 bootstrap replicates. Asterisks indicate the 18 isolates from Sichuan.

Figure 4

Clinical and histopathologic observation of diseased chickens in control group infected by MDV LS strain. A. Clinical anatomical change, "a" to "e" were heart, lung, liver, stomachus glandularis, intestinal tract, respectively, and tumors were observed in these tissues, even in the intestinal tract and mesenterium; B. Diagnosis by histopathologic slices (HE 400×). All tissues were fixed in 10% formalin paraffin and embedded, and 5 μm sections were stained with haematoxylin and eosin (HE), for light microscopy, "f" to "n" refer to bursa of fabricius, lung, liver, skeletal muscle, ovary, spleen, kidney, glandular stomach mucosa, cardiac muscle. Arrows show that multimorphology lymphocytes infiltrated patically and formed tumors (h, i, m, n), and other organs were infiltrated by lymphocytes extensively and their organizations were completely destroyed (f, g, j, k, i).