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.

Background and ObjectivesInfluenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes.Methods and ResultsEach chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates.ConclusionThis study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.

The cytokine interleukin-1 beta (IL-1β) 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/swIL1β) expressing swine IL-1β from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. MARC-145 cells infected with vP129/swIL1β secreted 1947 pg of IL-1β per 2 × 10(5)cells at 36 h post-infection. In vitro growth kinetics analysis in MARC-145 cells showed that the vP129/swIL1β virus had a similar replication rate as that of parental virus. We further performed in vivo characterization of the vP129/swIL1β virus in a nursery pig disease model. The vP129/swIL1β 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-1β, IL-4 and IFN-γ expression levels were up-regulated in pigs infected with vP129/swIL1β at 7 and 14 days post-infection. However, no detectable level of IL-1β was found in serum samples from pigs infected with either vP129/swIL1β 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.

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