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

Luis Gabriel Gimenez-Lirola; Jianqiang Zhang; Jose Antonio Carrillo-Avila; Qi Chen; Ronaldo Magtoto; Korakrit Poonsuk; David H. Baum; Pablo Piñeyro; Jeffrey Zimmerman

doi:10.1128/JCM.02507-16

DOI: 10.1128/JCM.02507-16

Article Figures & Data

Figures

Tables
Additional Files

Open in new tab
Download powerpoint
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.
Open in new tab
Download powerpoint
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.
Open in new tab
Download powerpoint
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).
Open in new tab
Download powerpoint
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.

TABLE 1

Optimal S/P cutoff points and diagnostic sensitivity of serum IgG antibody response to each PEDV antigen by 6-plex FMIA or WV ELISA

Assay	PEDV marker(s)	Cutoff (S/P)^a	No. of samples that tested positive/total no. (%) of positive samples; 95% CI^b
6-plex FMIA	S1 (non-S-INDEL)	0.10	69/72 (95.8); 88.4–98.6
	S1 (S-INDEL)	0.11	68/72 (94.4); 86.5–97.8
	N	0.59	59/72 (81.9); 71.5–89.1
	M	0.21	58/72 (80.6); 69.9–88.0
	E	0.90	1/72 (1.4); 0.25–7.5
	WV particles	0.67	48/72 (66.7); 55.2–76.5
ELISA	WV particles	0.70	63/72 (87.5); 77.9–93.3

↵a The optimal S/P cutoff points were selected to ensure 100% diagnostic specificity.
↵b CI, confidence interval.

TABLE 2

Detection of serum IgG antibody responses among inoculation groups by day postinoculation^a

Inoculation group	Total no. of pigs	PEDV marker(s)	Assay	No. of pigs positive by day postinoculation:
Inoculation group	Total no. of pigs	PEDV marker(s)	Assay	−7	3	7	10	14	17	21	28	35	42
PEDV	12	S1 (non-S-INDEL)	6-plex FMIA	0	0	6	11	11	11	12	12	12	11
		S1 (S-INDEL)		0	0	5	10	11	11	12	12	11	11
		N		0	0	2	6	10	10	10	10	10	9
		M		0	0	1	8	10	10	10	10	9	9
		E		0	0	0	0	0	0	0	0	1	0
		WV particles		0	0	0	3	7	9	9	8	8	7
		WV particles	Single ELISA	0	0	2	7	11	10	11	11	10	10
TGEV Purdue	12	S1 (non-S-INDEL)	6-plex FMIA	0	0	0	0	0	0	0	0	0	0
		S1 (S-INDEL)		0	0	0	0	0	0	0	0	0	0
		N		0	0	0	0	0	0	0	0	0	0
		M		0	0	3	9	9	9	9	8	8	8
		E		0	0	0	0	0	0	0	0	0	0
		WV particles		0	0	1	1	1	1	1	1	1	1
		WV particles	Single ELISA	0	0	1	1	1	1	1	1	0	1
TGEV Miller	12	S1 (non-S-INDEL)	6-plex FMIA	0	0	0	0	0	0	0	0	0	0
		S1 (S-INDEL)		0	0	0	0	0	0	0	0	0	0
		N		0	0	2	2	1	0	0	0	0	0
		M		0	0	2	10	11	11	12	11	11	11
		E		0	0	0	0	0	0	0	0	0	0
		WV particles		0	0	1	1	1	1	1	1	1	1
		WV particles	Single ELISA	0	0	1	1	1	1	1	1	1	0
PRCV	12	S1 (non-S-INDEL)	6-plex FMIA	0	0	0	0	0	0	0	0	0	0
		S1 (S-INDEL)		0	0	0	0	0	0	0	0	0	0
		N		0	0	0	0	0	0	0	0	0	0
		M		0	0	3	5	6	5	7	7	3	7
		E		0	0	0	0	0	0	0	0	0	0
		WV particles		0	0	0	1	0	0	0	0	0	0
		WV particles	Single ELISA	0	0	0	0	0	0	0	0	0	0
PDCoV	12	S1 (non-S-INDEL)	6-plex FMIA	0	0	0	0	0	0	0	0	0	0
		S1 (S-INDEL)		0	0	0	0	0	0	0	0	0	0
		N		0	0	0	0	0	0	0	0	0	0
		M		0	0	0	0	1	0	0	0	0	0
		E		0	0	0	0	0	0	0	0	0	0
		WV particles		0	0	0	0	0	0	0	0	0	0
		WV particles	Single ELISA	0	0	0	0	0	0	0	0	0	0

↵a Data are presented as numbers of samples (with values above selected S/P cutoff values) that tested positive with respect to each PEDV antigen by 6-plex FMIA or WV ELISA. Data in bold indicate true-positive results, and data in italics indicate false-positive results (cross-reactivity).

TABLE 3

Amino acid sequence homology of PEDV recombinant polypeptides S1, N, M, and E compared to homologous regions of PEDV-related porcine coronaviruses TGEV Miller, TGEV Purdue, PRCV, and PDCoV used during experimental inoculation^a

Strain used for exptl inoculation	% homology for amino acid sequence of PEDV recombinant polypeptide
Strain used for exptl inoculation	S1 PEDV non-S-INDEL (consensus)	S1 PEDV S-INDEL (consensus)	N PEDV (consensus)	M PEDV (consensus)	E PEDV (CV777 strain)
PEDV non-S-INDEL (USA/IN/2013/19338E)	100	92	100	100	98
TGEV Purdue (ATCC VR-763)	28	28	31	55	28
TGEV Miller (ATCC VR-1740)	28	28	31	55	28
PRCV (ATCC VR-2384)	29	29	31	55	30
PDCoV (USA/IL/2014)	23	23	20	20	19

↵a Sequence analysis was performed using UGene multiplatform software (version 1.25; Unipro, Novosibirsk, Russia). S1, spike (globular head); N, nucleocapsid (full length); M, membrane (C-terminal intravirion topological domain); E, envelope (full length).

Additional Files

Figures
Tables

Supplemental material

Supplemental file 1 -
Tables S1 (Porcine coronaviruses strains, inoculum doses, and routes used during experimental inoculations) and S2 (PEDV strains used to determine consensus amino acid sequences corresponding to PEDV recombinant polypeptides)

PDF, 210K