This report documents the first sequence analysis of the entire M1, M2, and M3 genome segments of the muscovy duck reovirus (DRV) S14. The complete sequence of each of the three M gene segments was determined. The M1 genome segment was 2283 nucleotides in length and was predicted to encode muA protein of 732 residues. The Escherichia coli expressed M1 transcripts generated a 108kDa protein, as expected for muA. A cleavage product of muA, muA1, could be detected by Western blotting with duck anti-reovirus and mouse anti-muA polyclonal serum. muA was distributed diffusely in the cytoplasma and nucleus of transfected Vero cells, which provides evidence that muA might be functional related to the mammalian reovirus (MRV) mu2. The M2 gene was 2155 nucleotides in length and was predicted to encode muB major outer capsid protein of 676 amino acids. The M3 genome segment was 1996 nucleotides in length and was predicted to encode a muNS protein of 635 amino acids. It was unexpectedly found that 5'-termini of the M1 and M2 genes ended with 5'-ACUUUU and 5'-UCUUUU, respectively, instead of 5'-GCUUUU, which is present on most mRNAs of other avian reoviruses (ARV). The UCAUC 3'-terminal sequences of the S14 M1, M2, and M3 genome segments are shared by DRV, ARV, and MRV. Alignment of the DRV muA-, muB-, and muNS-encoding genes with ARV revealed 72.9-73.9%, 67.1-69.6%, and 69.4-70.8% nucleotide identity, respectively. The amino acid sequence homology between DRV and ARV ranged from 85.3 to 86.2% (muA), 75.0 to 76.5% (muB), and 78.4 to 79.8% (muNS). Phylogenetic analyses of the M1, M2, M3, and S-class [Kuntz-Simon, G., Le Gall-Recule, G., de Boisseson, C., Jestin, V., 2002. Muscovy duck reovirus sigmaC protein is a typically encoded by the smallest genome segment. J. Gen. Virol. 83, 1189-1200; Zhang, Y., Liu, M., Hu, Q.L., Ouyang, S.D., Tong, G.Z., 2006a. Characterization of the sigmaC-encoding gene from muscovy duck reovirus. Virus Genes 36, 169-174; Zhang, Y., Liu, M., Ouyan, S.D., Hu, Q.L., Guo, D.C., Han, Z., 2006b. Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses aggregated the same specified genogroup in Orthoreovirus Genus II. Arch. Virol. 151, 1525-1538] genome segments suggests that DRV and ARV share a recent common ancestor and that the two lineages have subsequently undergone host dependent evolution.