3.3 Sequencing of oncogenes and Sequence analysis
Atleast one oncogene was amplified and sequenced for each of the 17 MDV positive samples, a total of 13 Meq, 11 pp38 and 13 vIL-8 gene sequences were sequenced. The obtained sequences were deposited in GenBank with accession numbers. The details are given in Table 1.
The Meq sequences analysis of the current field MDV strains showed 99.8-100% and 99.5-100% similarity at nucleotide and amino acid levels, respectively. All strains showed a meq gene length of 1020 bp encoding for meq protein comprising of 339 aa. In the present study, five proline rich repeats (PPPPs) were observed at positions 152-155, 175-178, 191-194, 216-219 and 232-235 in all these strains. The amino acid mutations in the Meq displayed similarity at several positions including 71, 77, 80, 88, 93, 119, 139, 153, 176, 180, 217, 277, 283, 320 and 326. Two mutations associated with MDV virulence were observed in all 13 Meq sequences at positions 71 (Alanine) and 80 (Tyrosine). Four distinctive substitutions including one in basic region and three in leucine zipper domain were observed in all sequences except one (MK388077) as follows: 80 Y (Tyrosine; D80Y), 88 T (threonine; A88T), 93 R (arginine; Q93R) and 139 A (alanine; T139A) (Table 2).
As for pp38 gene, the identity ranged from 98.6 - 99.9 % and 98.6 -100% at nucleotide and amino acid levels, respectively among the field MDV sequences. 11 sequences of this study were compared with five reference MDVs at amino acid levels. The analysis revealed mutation at aminoacid position109 (glutamate→glycine). All the 11 sequences had glutamine at position 107 (Table 3).
vIL-8 comprises of 132 amino acids encoded by 682 nucleotides from three exons (1..64,240..375,474..678). The homology of the nucleotide and deduced amino acid sequences of 13 sequences were 99.5-100% and 100% respectively. The complete nucleotide and amino acid sequences of five reference MDVs were compared with the sequences of the present study. All the 13 sequences had two mutations at nucleotide position 11 (T →C) and 92 (A→G) resulting in amino acid substitutions at positions 4 (leucine to serine; L4S) and 31 (aspartate to glycine; D31G) (Table 4).