Characterization of the sigmaB-encoding genes of muscovy duck reovirus: sigmaC-sigmaB-ELISA for antibodies against duck reovirus in ducks.
The sigmaB/sigmaC-encoding genes of muscovy duck reovirus (DRV) S12 strain were cloned, sequenced, and expressed in Escherichia coli. The sigmaC-encoding gene of DRV showed only 21-22% identity to that of avian reovirus (ARV) at both nucleotide and amino acid level. The sigmaB-encoding gene of DRV comprised 1163bp with one open reading frame (ORF). The ORF comprised 1104bp 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 sigmaB. 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 sigmaB-encoding gene sequence indicated that S12 separated as a distinct virus relative to other avian strains. The expressed sigmaB/sigmaC fusion proteins in E. coli could be detected, approximately 45 and 50kDa, respectively, by duck anti-reovirus polyclonal serum. In addition, an ELISA (sigmaB-sigmaC-ELISA) using the expressed sigmaB-sigmaC 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 sigmaB-sigmaC-ELISA showed perfect specificity and sensitivity. The sigmaB-sigmaC-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 sigmaB-sigmaC-ELISA was a sensitive and accurate method for detecting antibodies to DRV.
Zhang
Yun
Y
Avian Infectious Disease Division, National Key Laboratory of Veterinary Biotechnology, Harbin, Veterinary Research Institute of Chinese Academy of Agricultural Sciences, No. 427 Maduan Street, Harbin 150001, PR China. yunzhang03@yahoo.com
Guo
Dongchun
D
Liu
Ming
M
Geng
Hongwei
H
Hu
Qilin
Q
Liu
Yin
Y
Liu
Nihong
N
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005779:Immunodiffusion / Q000662:veterinary; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000662:veterinary; D030101:Orthoreovirus, Avian / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D010802:Phylogeny; D011201:Poultry Diseases / Q000235:genetics / Q000276:immunology / Q000821:virology*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012088:Reoviridae Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment
D000914:Antibodies, Viral; D036022:Capsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C031728:sigma 1 protein, reovirus; C053179:sigma protein 3, Reovirus