Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types I and II

Abstract

To further characterize the humoral immune response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV), direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses directed against PRRSV nonstructural proteins in pigs experimentally exposed to the virus. The highest immunoreactivities were against nsp1, nsp2, and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual ELISA for the simultaneous detection and differentiation of serum antibodies against type I and type II PRRSV. Receiver operating characteristic analysis based on 1,334 known-positive and 1,357 known-negative samples showed good specificity (98.3% to type I and 99.3% to type II) and sensitivity (97.4% for type I and 99.8% for type II). To differentiate type I and type II PRRSV, 470 sera originating from experimentally inoculated pigs were tested, and positive sera were correctly differentiated in 469 of 470 samples. The capability of the nsp7 dual ELISA to detect serum antibody responses from pigs infected with various genetically different field strains was determined. The nsp7 dual ELISA possessed 97.6% agreement with the Idexx HerdChek PRRS 2XR ELISA. In further testing of Idexx ELISA suspected false-positive samples, the nsp7 dual ELISA resolved 98% of the samples as negative. Taken together, these results indicate that the nsp7 dual ELISA can be used as a differential test for PRRSV serology with high levels of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of recombinant PRRSV nsp preparations, followed by Coomassie blue staining. The left lane shows the protein molecular mass standard; the remaining lanes represent nsp1, nsp2, nsp4, nsp7, and nsp8 preparations, as indicated. NA, North American genotype (type II); EU, European genotype (type I). Note that nsp1 is further cleaved into nsp1α and nsp1β subunits (6, 13). Intact nsp1 and 26-kDa nsp1β eluted from the immobilized metal affinity column are shown in the second lane from the left.

FIG. 2.

Kinetics of antibody response to PRRSV nsps. Pigs were experimentally infected with type II PRRSV, VR2332. The serum samples were from 0 to 202 days postinoculation (DPI) as indicated. For nsp4 and nsp8, serum samples from 10 pigs were tested; for nsp1, nsp2, and nsp7, serum samples from 30 pigs were tested.

FIG. 3.

Two-graph ROC plot of the PRRSV nsp7-based ELISA. The graphs were calculated using 965 (type I nsp7) and 1,726 (type II nsp7) individual animal serum samples and GraphROC software. The downward-pointing histogram on the left side of the figure represents the uninfected animals, and the upward-pointing histogram on the right side of the figure represents the PRRSV-infected animals. The green line represents the diagnostic sensitivity (D sens) of the assay as the cutoff S/P ratio is moving from 0 to 2.7. The red line represents the diagnostic specificity (D spec) of the assay as the cutoff S/P ratio is moving from 0 to 2.7. The black dotted vertical line represents the optimized cutoff value of 0.51 (type I) (A) and 0.52 (type II) (B), which corresponds to the maximum diagnostic sensitivity and specificity.

FIG. 4.

Differentiation of type I and type II PRRSV using the nsp7 dual ELISA. The distributions of individual samples with S/P values above the cutoff in the type I and type II nsp7 ELISAs are shown according to the calculated r values. The percentage of serum samples compared to the total number of positive sera in each test is shown on the vertical axis. For each positive sample, an r value, representing the log₁₀ of the ratio obtained by dividing the S/P ratio observed in the type I nsp7 ELISA by the S/P ratio observed in the type II nsp7 ELISA, was calculated. Thus, r values of >0 represent positives in the type I nsp7 ELISA, and r values of <0 represent positives in the type II nsp7 ELISA.