Master of Science / Department of Diagnostic Medicine/Pathobiology / Dana Vanlandingham / Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus maintained among swine and avian species. In infected pigs, replication of JEV leads to the onset of viremia and the development of neurological and reproductive disease in young and naïve pregnant animals. The high-titer viremia levels associated with JEV infection in pigs, whilst important to the enzootic transmission cycle responsible for viral maintenance, also have human health implications within the zoonotic cycle. Sensitive and specific veterinary diagnostic methods capable of readily detecting JEV infection are critical components of JEV surveillance programs in the Asian Pacific region. In this study, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assays were evaluated for use in veterinary diagnosis of JEV. Our hypotheses for this research project were that RT-qPCR assays with fewer oligonucleotide mismatches between the primers and probes of the assays and JEV genomes will be more sensitive for the diagnosis of JEV infection and that oral shedding of JEV in swine would allow for detection of viral RNA using oral fluids. The sensitivity and specificity of three RT-qPCR assays for the detection of JEV were determined using tissue culture fluids of five representative JEV strains belonging to four endemic genotypes. The first assay (assay #1), targeting the highly conserved NS5 gene and 3UTR regions, provided optimum detection for the current predominant genotype, GI-b. All three assays were highly specific for JEV when tested against other selected flaviviruses in the JEV serocomplex. A rope-based collection method allowed for the simplified collection of oral fluids from three-week-old piglets challenged with endemic JEV strain JE-91. These fluids were then evaluated using RT-qPCR assays for the presence of viral RNA. The results suggest that the shedding of JEV in oral fluids can be readily detected and that non-invasive oral fluid collection can serve as a novel sampling method for the diagnosis and surveillance of JEV in swine.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Classical swine fever virus (CSFV) is an enveloped, positive strand RNA virus, and member of the genus Pestivirus. It is a highly infectious and transmissible swine pathogen that threatens the global swine industry. The United States has been free of CSFV since 1977, however, monitoring the millions of domestic and feral pigs present in the US puts a significant strain on national surveillance efforts. There are no validated diagnostic techniques that can simultaneously sample multiple pigs (i.e. all pigs in a pen or barn). Similarly, there are no validated serological assays that can quickly test for CSFV without cross-reacting with other pestiviruses. The purpose of the first study was to establish a moderate CSFV-infectious model and determine how a single oral fluid sample from a pen of pigs can function as a diagnostic sample for detecting CSFV. Oral fluid (OF) and serum samples were collected from 10 pigs experimentally infected with CSFV Paderborn strain. Using RT-PCR, CSFV was detected in OF on 8 days post infection (dpi), and in the serum of one pig on 6 dpi. A single OF sample can, therefore, take the place of 10 serum samples to detect CSFV in a population. In a second study, monoclonal antibodies reactive to CSFV glycoproteins were generated in mice immunized with recombinant E2 and Erns antigens. Five E2-specific clones and two Erns-specific clones showed reactivity to CSFV-infected. Epitope mapping of the E2 clones showed that all reacted with the N-terminal portion of E2; a region highly variable among pestiviruses. Together with OF sampling, monoclonal antibodies can be used to develop new tools for improving CSF surveillance in large swine populations.