Characterization of the sigma B-encoding genes of muscovy duck reovirus: sigma C- sigma B-ELISA for antibodies against duck reovirus in ducks.

摘要

The sigma B/ sigma C-encoding genes of muscovy duck reovirus (DRV) S12 strain were cloned, sequenced, and expressed in Escherichia coli. The sigma C-encoding gene of DRV showed only 21-22% identity to that of avian reovirus (ARV) at both nucleotide and amino acid level. The sigma B-encoding gene of DRV comprised 1163 bp with one open reading frame (ORF). The ORF comprised 1104 bp and encoded 367 amino acids with a predicted molecular mass of 40.44 kDa. A zinc-binding motif and a basic amino acid motif were found within the predicted amino acid sequence of sigma B. The identities between the S12 and ARV were 59.3-64.0% and 60.9-62.5%, respectively, at the nucleotide and deduced amino acid levels. Phylogenetic analysis of the sigma B-encoding gene sequence indicated that S12 separated as a distinct virus relative to other avian strains. The expressed sigma B/ sigma C fusion proteins in E. coli could be detected, approximately 45 and 50 kDa, respectively, by duck anti-reovirus polyclonal serum. In addition, an ELISA ( sigma B- sigma C-ELISA) using the expressed sigma B- sigma C proteins as coating antigen for detection of antibodies to DRV in ducks was developed. In comparison with the virus neutralization test and agar gel immuno-diffusion test (AGID), the sigma B- sigma C-ELISA showed perfect specificity and sensitivity. The sigma B- sigma C-ELISA did not react with the antisera to other duck pathogens, implying that these two proteins were specific in recognition of DRV antibodies. Taken together, the results demonstrated that sigma B- sigma C-ELISA was a sensitive and accurate method for detecting antibodies to DRV..