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Turkey hemorrhagic enteritis virus (THEV) is a member of the genus Siadenovirus and causes disease in turkey poults characterized by splenomegaly, bloody diarrhoea and death. The mechanism responsible for intestinal lesion formation and mortality is not known, although there is strong evidence that it is immune-mediated. All strains of THEV are serologically indistinguishable, although there are naturally occurring avirulent strains of THEV that replicate efficiently in turkeys without the intestinal haemorrhage or mortality associated with more virulent strains. The purpose of this study was to determine which viral genes are involved in virulence. The full-length genome of an avirulent vaccine strain was sequenced and compared with the genome of a virulent field isolate from Israel that was sequenced in 1998. Comparison of the two 26.3 kb genomes revealed 49 nucleotide differences resulting in 14 putative amino acid changes within viral proteins. Sequencing of the regions surrounding the 14 missense mutations revealed variations in ORF1, E3 and the fiber (fib) knob domain in five additional strains with varying degrees of virulence. Complete sequences of these genes were determined in a total of 11 different strains of THEV. All strains had at least one missense mutation in ORF1, and all but two of the strains had one missense mutation in E3. At least one missense mutation was found in the fiber knob domain in six out of seven virulent strains. Sequence variation of ORF1, E3 and fib in strains of THEV with different phenotypes strongly indicates that these genes are the key factors affecting virulence.