Marek's disease (MD) is currently one of the important contagious diseases in poultry industry. It not only causes the death of birds directly, but also causes immunosuppression of infected chickens which are more sensitive to other pathogens such as E. coli. Although CVI988 vaccine  is widely used now in China, but immune failure still occurred in some flocks. It had been reported that the virulence of MDV isolates have increased in the last four decades , and some of the recent isolates are more pathogenic for chickens than MDVs isolated before . The emergence of MDVs of increasing virulence is a significant problem for the poultry industry.
MDV is a member of the genus Mardivirus that consists of serotypes 1 and 2 (MDV-1 and MDV-2) as well as serotype 3 or herpesvirus of turkeys (HVT) . Among them, the only serotype 1 is oncogenic, and some of the unique genes such as Meq (MDV EcoRI-Q) , pp38 , vIL-8 (viral interleukin-8)  were reported to be associated with viral oncogenicity and pathogencity.
In this study, homology comparison of the nucleotide and deduced amino acid sequences of Meq, pp38 and vIL-8 of 18 isolates and other reference strains were conducted, and high nucleotides and amino acids identities of these three genes between isolated MDVs and other reference MDVs were observed, indicating that the genetic stability of these three genes was high in recent years. Currently, Meq gene is most likely considered to be the factor to induce tumors. It encodes 339 amino acids and contains a basic leucine zipper (bZIP) domain at the N terminal closely resembling the jun/fos oncogene family , and its transactivation can lead to cell transformation [27, 28]. The length of Meq gene of JM, CU-2 and CVI988 was 177bp longer than these of virulent MDVs [29, 30], which result in a 59 aa insertion. It is reported to have a suppressive effect on the Meq expression . After sequence alignment, this insertion was also found in Chinese vaccine 814 and Russian vaccine strains 3004, but not found in all 18 field isolates. In addition, it had been reported previously that amino acid change at position 71 (serine → alanine) and 77 (glutamate → lysine) was the feature of high virulent MDVs . In this study, the same mutation at position 71 was also observed in all wild strains from China, but the mutation at position 77 was not observed in field isolates from China, and this mutation seemed to be a feature of virulent MDVs from USA. Besides, it was also found that some amino acid mutations at position 80, 115, 139 and 176 in most isolates from China displayed perfect regularity. Mutation at position 80 (aspartate → tyrosine), 115 (valine → alanine), 139 (threonine → alanine), and 176 (proline → arginine) accounted for 96.3% (26/27), 100% (27/27), 90.0% (24/27) and 96.3% (26/27) of all field MDVs (not including vaccine 814 strain) from China, respectively. The mutation in position 80, 115, 139, 176 of Meq protein could be used as virulent genetic characteristics of the circulating MDVs MDV in China. Moreover, it was observed that the three amino acids at position 119 (arginine), 153 (glutamine) and 176 (alanine) were unique in very virulence plus (vv+) strains 684A and 584A, but we can not determine that whether this is the character of all vv+ MDVs.
pp38 codes a 38Ku phosphoprotein and plays a role in viral reactivation in latent period [33, 34] and cell transformation [35, 36]. Minor differences in amino acid sequences of different MDVs have been observed for pp38. Originally, it was believed that pp38 was not expressed in CVI988 ; however, it was showed to be present in CVl988 later, and glutamine in position 107 was replaced by arginine as defined by monoclonal antibody (MAb) H19 [38, 39]. In this study, after alignment of the amino acid sequence of pp38 gene of 25 MDVs, it was found that no mutation at position 107 was observed in all the field isolates, but glutamate at position 109 in virulent MDVs (GA, RB1B, 584A and 648A) from USA were replaced by Glycine in field isolates from China. So the mutation (Glutamate → glycine) in position 109 can be considered as a feature of virulent MDVs isolated from USA.
The vIL-8 gene locates in the long repeat region and was originally identified as a spliced Meq variant . This gene consists of 3 exons and is expressed during cytolytic infection. vIL-8 attracts T cells, especially after vIL-8 receptors are up-regulated by interferon-γ (lFN-γ). vIL-8 may be important for the switch of infection from B to T lymphocytes. This gene was very conservative in 648A, RB1B, MD11 and GA strains , but after comparison of the amino acid sequence, two special point mutations at position 4 (leucine→ serine) and 31 (aspartate → glycine) were found only in the field MDVs from China. We speculate that the point mutations at position 4 and 31 could be considered as the features of field MDVs in China.
Phylogenetic tree, based on the Meq and vIL-8 amino acid sequences, revealed that the field MDVs from China formed an independent cluster, while vaccine strains, mild virulent MDVs and virulent MDVs from USA formed another cluster. In addition, in the phylogenetic tree of Meq, the MDVs in the latter cluster could be further divided into two different branches. This result implied that field MDVs from China may evolve independently, and genetic difference of MDVs displayed in phylogenetic tree of the Meq was more obvious than these of vIL-8 and pp38 gene. Meq gene could be a gene of priority for Phylogenetic analysis.
Virulence study in this study revealed that virulent MDVs prevalent in China in recent years could break through the protection provided by HVT vaccine. Histopathologic diagnosis of the diseased chicken showed multimorphous and immature lymphocytes infiltrated and proliferated in all kinds of organs, leading to complete destruction of the structure of organs and tissues. CVI988/Rispens vaccine is a better choice for immunization. Currently, MD occasionally occurred in some chicken farms immunized with CVI988 vaccines in China; however, result of virus isolation and virulence test did not show that the very virulence plus (vv+) strain, such as 684A in USA, was epidemic in China, and the immune failure may be due to the improper selection of vaccines and the incorrect way of vaccination.
In conclusion, high nucleotides and amino acids identities of Meq, pp38 and vIL-8 genes between 18 MDV isolates and other reference MDVs was observed. Four mutations at position 80, 115, 139 and 176 of the amino acid sequence of Meq gene, two mutations at position 4 and 31 of the amino acid sequence of vIL-8 gene can be considered as main features of field MDVs prevalent in recent years in China; one mutation at position 109 in amino acid sequence of pp38 gene can be considered as a feature of virulent MDVs isolated from USA. Phylogenetic analysis of Meq genes could provide the evolution difference of different MDVs. CVI988/Rispens vaccine could provide enough protection against the challenge of prevalent MDVs, and should be widely used commercially. Nevertheless, as new variant strains may emerge in the future, constant surveillance of new filed MDVs is necessary to reveal the character of epidemic MDVs and to develop better vaccines and control program of MD.