Antibody Responses of Pigs to Defined E super(rns) Fragments after Infection with Classical Swine Fever Virus

Antibody responses of pigs to defined E super(rns) fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA). Selection of various E super(rns) fragments was based on an immunodominant E super(rns) region encompassing three overlapping antigenic regions, amino acids 65 to 145 (E super(rns) sub(aa) sub(65-145)) (AR1), 84 to 160 (E super(rns) sub(aa) sub(84-160)) (AR2), and 109 to 220 (E super(rns) sub(aa) sub(10) sub(9- 220)) (AR3), identified earlier by our group (M. Lin, E. Trottier, J. Pasick, and M. Sabara, J. Biochem., in press). Defined E super(rns) fragments, including AR1, AR2, AR3, E super(rns) sub(aa) sub(65-160) (AR12), E super(rns) sub(aa) sub(84-220) (AR23), E super(rns) sub(aa) sub(65-220) (AR123), E super(rns) sub(aa) sub(109-145) (the consensus region defined by the three overlapping regions), and E super(rns) sub(aa) sub(109-160) (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV. Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the E super(rns) fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100%. Detection of antibody responses to refolded E super(rns) sub(aa) sub(109-145) and E super(rns) sub(aa) sub(109-160) by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational. When cutoff values were raised to give a specificity of 100%, four E super(rns) fragments (AR2, AR23, E super(rns) sub(aa) sub(109-145), and E super(rns) sub(aa) sub(109-160)) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%). E super(rns) sub(aa) sub(109-145) and E super(rns) sub(aa) sub(109-160) were capable of detecting antibody responses in infected pigs as early as 7 days postinfection. Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection. An advantage of such a test would be its utilization for serologica