Development and evaluation of antigen-specific dual matrix Pestivirus K ELISAs using longitudinal known infectious status samples

Authors: Arruda, Bailey; Falkenberg, Shollie; MORA-DIAZ, JUAN-CARLOS - Iowa State University; MATIAS FERREYRA, FRANCO - Iowa State University; MAGTOTO, RONALDO - Iowa State University; GIMENEZ-LIROLA, LUIS - Iowa State University

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2022
Publication Date: 10/12/2022
Citation: Arruda, B.L., Falkenberg, S.M., Mora-Diaz, J., Matias Ferreyra, F.S., Magtoto, R., Gimenez-Lirola, L. 2022. Development and evaluation of antigen-specific dual matrix Pestivirus K ELISAs using longitudinal known infectious status samples. Journal of Clinical Microbiology. 60(11). Article e00697-22. https://doi.org/10.1128/jcm.00697-22.
DOI: https://doi.org/10.1128/jcm.00697-22

Interpretive Summary: Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is no robust assay to detect antibodies targeting APPV to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n=6; negative control) or APPV (n=16). Serum and pen-based oral fluid samples were collected through 70 days post inoculation (DPI). The immune response to three recombinant APPV antigens (Erns, E2, or NS3) was evaluated via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by a real-time reverse transcription PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 DPI, respectively. All samples remained genome positive until 70 DPI. Antibodies were first detected in oral fluids at 14 DPI, ten days prior to serum detection and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.

Technical Abstract: Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is limited information on the infection dynamics of and immune response against APPV and no robust serologic assay to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n=6; negative control) or APPV (n=16). Serum, pen-based oral fluid samples, and nasal swabs were collected through 70 days post inoculation (DPI). The immune response to recombinant APPV Erns, E2, or NS3 antigens was evaluated using both serum and oral fluids via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by a real-time reverse transcription PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 DPI, respectively. All samples remained genome positive until 70 DPI. Detection of nasal shedding was less consistent, with APPV being detected by RT-qPCR in all inoculated animals at 42, 49, and 56 DPI. Antibodies were first detected in oral fluids at 14 DPI, ten days prior to serum detection and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.