Seroepidemiology for Orthorubulavirus suis in Mexican Pigs by Development of an Indirect ELISA Based on a Recombinant NP Protein

Abstract

Orthorubulavirus suis (LPMV) is the etiologic agent of blue eye disease (BED), which affects pigs of all ages, and it has been endemic in central Mexico since the 1980s. To date, no disease control program has been established. Therefore, there is a need for a serological diagnostic method with high sensitivity and specificity. In this study, the recombinant protein NP of LPMV was produced in the E. coli BL21 system and then purified using affinity chromatography. The purified protein was used to coat plates for an indirect ELISA assay (iELISA). To determine the sensitivity and specificity of the test, a 2 × 2 contingency table was constructed using positive and negative control sera. The specificity and sensitivity levels were 98.1% and 98.7%, respectively. According to our findings, 45% of serum samples (378/839) were positive, with seropositivity percentages in the analyzed states ranging from 72.5% to 6%. To confirm the presence of antibodies, the indirect immunofluorescence technique was applied to iELISA-positive serum samples. In this study, antibodies against the LPMV nucleoprotein were detected, indicating that the virus or defective particles may be circulating in Mexican pigs and highlighting the risk of LPMV spreading to disease-free areas.

Keywords: ELISA; Orthorubulavirus suis; recombinant nucleoprotein.

Figure 1

Map and features of pET SUMO-NP-LPMV vector construction. The red arrow indicates the cloned NP gene.

Figure 2

Overproduction of rNP-LPMV. (a) Coomassie staining and (b) Western blot with specific anti-his antibodies. (1) and (2) inclusion bodies from induced BL21-NP-LPMV; (3) rNP-LPMV purified by affinity chromatography.

Figure 3

Distribution of OD450 mn of positive and negative reference sera in the rNP-LPMV iELISA test represented by box-and-whisker plot.

Figure 4

Distribution of the positivity rate of the serum samples from the eight states of Mexico used in the present study. The dotted line indicates the cut-off point of the test.

Figure 5

Map of Mexico showing the central states of study indicating the positivity rate of the serum samples.

Figure 6

Indirect immunofluorescence in SCP cell culture observed at 20×. (A) Positive hyperimmune control serum. (B) Negative reference serum. (C) Positive sample from the State of Mexico. (D) Positive serum from Michoacán. (E) Positive serum from Jalisco. (F) Positive serum from Aguascalientes. (G) Positive serum from Guanajuato. (H) Positive serum from Querétaro. (I) Positive serum from Morelos. (J) Positive serum from Veracruz. Red arrows indicate some specific fluorescent foci.