Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications

Abstract

The development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n = 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

Keywords: ELISA; PEDV; antibody response; cross-reactivity; multiplex FMIA; recombinant structural proteins; whole virus.

FIG 1

PEDV multiplex (6-plex) fluorescent microbead-based immunoassay (FMIA) sample/positive-result (S/P) ratios of serum antibody (IgG) responses (means and standard errors [SE]) over time to the 5 recombinant polypeptides (S1 non-S-INDEL, S1 S-INDEL, N, M, and E) and to the whole-virus (WV) antigen in pigs (n = 12 per group) inoculated with PEDV (USA/IN/2013/19338E), TGEV Miller (ATCC VR-1740), TGEV Purdue (ATCC VR-763), PRCV (ATCC VR-2384), or PDCoV (USA/IL/2014) or with a negative control (sham inoculation). Different letters denoted statistical differences (P = ≤0.05). Samples above the estimated S/P cutoff (dashed line) were considered positive.

FIG 2

Performance of PEDV whole-virus (WV) indirect ELISA on experimental samples of precisely known porcine coronavirus infectious status, i.e., pigs inoculated with PEDV (USA/IN/2013/19338E; n = 12), TGEV Miller (ATCC VR-1740; n = 12), TGEV Purdue (ATCC VR-763; n = 12), PRCV (ATCC VR-2384; n = 12), or PDCoV (USA/IL/2014; n = 12) or with a negative control (sham inoculation; n = 12). (A) Sample/positive result (S/P) ratios of serum antibody (IgG) responses (mean, SE) over time in each inoculation group. (B) Distribution of cumulative ELISA WV IgG sample/positive result (S/P) ratios in serum samples (n = 792 total; n = 132 per group) collected at DPI −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42. Different letters denoted statistical differences (P = ≤0.05). Samples above the estimated S/P cutoff (dashed line) were considered positive.

FIG 3

Distribution of cumulative FMIA IgG sample/positive result (S/P) ratios obtained for each recombinant polypeptide (S1 non-S-INDEL, S1 S-INDEL, N, M, or E) on serum samples (n = 792 total; n = 132 per group) collected at DPI −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 from pigs (n = 72 total; n = 12 per group) inoculated with PEDV (USA/IN/2013/19338E), TGEV Miller (ATCC VR-1740), TGEV Purdue (ATCC VR-763), PRCV (ATCC VR-2384), or PDCoV (USA/IL/2014) or with a negative control (sham inoculation). The FMIA S/P cutoff values estimated for each individual antigen are presented in the graph (dashed line).

FIG 4

Amino acid sequences of 5 recombinant polypeptides derived from the following PEDV structural proteins: (i) envelope (E) full-length protein (CV777 PEDV strain); (ii) full-length nucleocapsid (N) protein from a consensus among PEDV strains (CV777, non-S-INDEL, and S-INDEL); (iii) truncated (C-terminal intravirion topological domain) membrane (M) protein from a consensus among PEDV strains (CV777, S-INDEL, and non-S-INDEL); (iv) truncated spike protein (globular head; S1) from a consensus among PEDV non-S-INDEL strains; (v) S1 from a consensus among PEDV S-INDEL strains.