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Clinical and Diagnostic Laboratory Immunology, May 2002, p. 698-703, Vol. 9, No. 3
1071-412X/02/$04.00+0     DOI: 10.1128/CDLI.9.3.698-703.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Importance of M-Protein C Terminus as Substrate Antigen for Serodetection of Equine Arteritis Virus Infection

Laboratory of Molecular Virology and Immunology, Department of Biological Sciences, University of Québec at Montréal, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3P8

Received 30 October 2001/ Returned for modification 4 January 2002/ Accepted 2 March 2002

Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombinant glutathione S-transferase-M fusion protein. Various deletion mutant clones, which covered the entire sequence of the M protein, were then generated by inverse PCR and expressed in Escherichia coli to examine, by a Western blot assay, the antigenic reactivity of the clone-derived truncated M proteins with sera from horses either experimentally or naturally infected with EAV. Deletion of the hydrophobic N-terminal 87 aa did not abolish immune reactivity of the protein with serum antibodies to EAV, thereby demonstrating the antigenicity of the C-terminal region (aa 88 to 162) of the M protein. Further truncations of the M-protein C-terminal domain defined particular linear epitope-containing amino acid sequence regions. However, only the M-protein C-terminal region was readily recognized by all EAV-specific horse antisera tested in this study. Based on these findings, only the M-protein C-terminal polypeptide composed of aa 88 to 162 is necessary to identify horse serum antibodies specific to the EAV M protein. Thus, this polypeptide might be useful for serodetection of EAV-infected animals.