Development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays.

For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals.

Interleukin-1ß expression by a recombinant porcine reproductive and respiratory syndrome virus.

The cytokine interleukin-1 beta (IL-1ß) is a potent inflammatory mediator in response to infection, and can be used as an immunological adjuvant. In this study, we constructed a recombinant porcine reproductive and respiratory syndrome virus (vP129/swIL1ß) expressing swine IL-1ß from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. MARC-145 cells infected with vP129/swIL1ß secreted 1947 pg of IL-1ß per 2 × 10(5)cells at 36 h post-infection. In vitro growth kinetics analysis in MARC-145 cells showed that the vP129/swIL1ß virus had a similar replication rate as that of parental virus. We further performed in vivo characterization of the vP129/swIL1ß virus in a nursery pig disease model. The vP129/swIL1ß infected pigs did not show visible clinical signs, while respiratory distress and lethargy were evident in pigs infected with the parental virus. The expression of various cytokines from peripheral blood mononuclear cells measured by fluorescent microsphere immunoassay showed that IL-1ß, IL-4 and IFN-γ expression levels were up-regulated in pigs infected with vP129/swIL1ß at 7 and 14 days post-infection. However, no detectable level of IL-1ß was found in serum samples from pigs infected with either vP129/swIL1ß or parental virus. In summary, this study demonstrated a recombinant PRRSV as a useful tool to study the role of different cytokines in disease progression and immune responses, which represents a new strategy for future therapeutic application and vaccine development.