Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is an enveloped, segmented, negative-sense RNA virus that infects avian species and mammals. Its segmented feature enables antigenic shift which can generate novel IAVs that pose a threat to animal and public health due to lack of immunity to these viruses. Pigs have been considered the “mixing vessels” of influenza A viruses to generate novel reassortant viruses that may threaten animal and public health. Therefore, it is necessary to understand the pathogenicity and transmissibility of newly emerged reassortant viruses in swine. Adding to this complexity is the newly identified bat influenza A-like viruses which have roused interest in understanding the evolutionary history and pandemic potential of bat influenza. At least 10 different genotypes of novel reassortant H3N2 IAVs with gene(s) from 2009 pandemic H1N1 [A(H1N1)pdm09] have been identified in pigs in the United States. To date, only three genotypes of these viruses have been evaluated in animal models leaving the pathogenicity and transmissibility of the other seven genotype viruses unknown. We showed that reassortant viruses with genes from A(H1N1)pdm09 are pathogenic and transmissible in pigs. Further studies showed that avian-like glycine at position 228 of the HA receptor binding site is responsible for inefficient transmission of the reassortant H3N2 IAV with five A(H1N1)pdm09 genes. Studying the recently discovered IAV-like sequences from bats has been hindered by the lack of live virus isolation or culturing. Using synthetic genomics, we successfully rescued modified bat influenza viruses that had the HA and NA coding regions replaced with two classical IAVs. Additional studies were performed with truncations on NS1 protein and substitution of a putative virulence mutation in bat influenza PB2. Virus reassortment experiments demonstrated that bat influenza has limited genetic and protein compatibility with other influenza viruses; however, it readily reassorts with another divergent bat influenza virus. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 IAVs in pigs. It also indicates that the bat influenza viruses recently identified are viable viruses that pose little pandemic threat to humans. Moreover, they provide new insights into the evolution and basic biology of influenza viruses.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Richard Hesse / Bovine Coronavirus (BCoV) is known to cause enteric and respiratory diseases, such as calf diarrhea, winter dysentery, calf respiratory disease, and bovine respiratory disease complex (BRD). All of these diseases are believed to be caused by the same genotype of BCoV. BCoV exhibits tissue tropism for both the gastrointestinal and respiratory tracts. This tropism is due to 9-O-acetylated sialic acid receptor on both epithelial cells in the respiratory and enteric tract. Currently, the only vaccine available for BCoV targets the enteric form of the disease. This study addresses the hypothesis that antibodies from the enteric form of the disease can cross neutralize the respiratory form of the virus. Data from surveillance studies suggest that BCoV is one of the major contributors to BRD, for which there is no currently approved vaccine for the respiratory form of the disease. Our approach to answering this question is to sequence and analyze the complete genome of 11 respiratory and enteric coronavirus isolates using next generation sequencing (NGS). Following the NGS, viruses were selected based on phylogenetic analysis and ability to grow and be maintained in cell culture. These viruses were then be used as serum neutralization indicator viruses in SN assays. 147 bovine serums submitted to KSVDL were used to determine if there are any serological differences between the immune response to respiratory versus enteric viruses based on the antibodies produced by the animal. The overall results show that there are few differences between the enteric and respiratory isolates at the genomic level and the serological response from the animal to these viruses. The differences between enteric and respiratory virus will need to be further addressed and analyzed to conclude if there is a noteworthy difference between the viruses with different tropisms. Other factors, such as host immune response and environment, are believed to be involved in the virus tropism to certain areas of the body.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most costly virus to the swine industry, worldwide. This study explored the application of deep sequencing techniques to understand better the virus-host interaction. On the virus side, PRRSV exists as a quasispecies. The first application of deep sequencing was to investigate amino acid substitutions in hypervariable regions during acute infection and after virus rebound. The appearance and disappearance of mutations, especially the generation of a new N-glycosylation site in GP5, indicated they are likely the result of immune selection. The second application of deep sequencing was to investigate the quasispecies makeup in pigs with severe combined immunodeficiency (SCID) that lack B and T cells. The results showed the same pattern of amino acid substitutions in SCID and normal littermates and no different mutations were identified between SCID and normal littermates. This suggests the mutations that appear during the early stages of infection are the product of the virus becoming adapted to replication in pigs. The third application of deep sequencing was to investigate the locations of recombination events between GFP-expressing PRRSV infectious clones. The results identified different cross-over occurred within three conserved regions between EGFP and GFPm genes. And finally, the fourth goal was applied to develop a set of sequencing tools for analyzing the host antibody repertoire. A simple method was developed to amplify swine VDJ repertoires. Shared and abundant VDJ sequences that are likely expressed by PRRSV-activated B cells were determined in pigs that had different neutralization activities. These sequences are potentially correlated with different antibody responses.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Mechanisms for mutations in RNA viruses include random point mutations, insertions, deletions, recombination and re-assortment. Most viruses have more than one of these mechanisms operating during their life cycle. Impact of sequence divergence is seen in the areas of evolution, epidemiology and ecology of these viruses. Immediate negative consequences of genetic diversity include failure of vaccination, resistance to anti-virals, emergence and re-emergence of novel virus isolates with increased virulence or altered tropism. To identify specific sequence features that influence recombination, a new in-vitro system was developed using an infectious cDNA clone of PRRS virus that expressed fluorescent proteins. The in-vitro experimental system involved the co-transfection of a pair of closely related PRRSV infectious clones: a fully functional non-fluorescent PRRS virus infectious clone that possessed a single mutation in a green fluorescent protein (GFP) and a second infectious clone that contained a defective fluorescent virus. The readout for successful recombination was appearance of a fully functional fluorescent virus. The model system creates the opportunity to study several aspects of recombination, including the requirement for sequence homology between viruses undergoing recombination.

Doctor of Philosophy / Department of Plant Pathology / Anna E. Whitfield / Tomato spotted wilt virus (TSWV) is an economically important plant virus dependent on insects (thrips) for transmission to plant hosts. Like many animal-infecting viruses, TSWV replicates in the cells of its insect vector. The virus is an emergent disease threatening food and fiber crops worldwide. The aim of this work was to develop novel control strategies against TSWV through a better understanding of the virus-vector interaction. Previously, the TSWV GN protein was shown to be the viral attachment protein, a molecule mediating attachment of virus particles to the midgut epithelial cells of vector thrips. The specific goals of my research were to further examine the utility of disrupting the virus-vector interaction for effective virus control by exploiting GN properties, and to track the route of TSWV in thrips using confocal microscopy. To achieve these goals, I expressed soluble and insoluble forms of GN fused to green fluorescent protein (GFP) transiently and transgenically and examined their cellular localization in planta. GN::GFP recombinant protein localized to Golgi stacks throughout the cells as indicated by a punctate pattern or co-localization to a Golgi marker. In contrast, the soluble form of GN, GN-S::GFP, localized to the ER and apparently also to the cytoplasm. Virus acquisition and transmission assays with GN-S::GFP transgenic tomato plants demonstrated that transmission of TSWV by F. occidentalis was reduced by 35 to 100%. These results indicated that transgenic expression of GN-S in tomato plants may have the potential to prevent secondary spread of the virus. Novel features of the morphology of principal (PSGs) and tubular salivary glands (TSGs) of the insect vector F. occidentalis and of their infection with TSWV were described. The virus colonized different cell types and regions within the PSGs with variable intensity and distribution; and accumulated at the lumen of individual cells. The TSGs of F. occidentalis are proposed as a route for TSWV infection into the PSGs. The transgenic plants and the new knowledge of the virus vector interaction are promising tools to control TSWV and a model approach for the control of other vector-borne viruses.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / With introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds, reassortment between the pH1N1 and endemic swine influenza viruses (SIVs) has been reported worldwide. Recently, reassortant H3N2 and H1N2 variant SIVs that contain the M gene from pH1N1 virus and the remaining seven genes from North American triple-reassortant (TR) SIVs have emerged. These variant viruses have caused more than 300 cases of human infections and one death in the USA, creating a major public health concern. To date, the pathogenicity and transmissibility of H1N2 variant viruses in pigs has not been investigated. Through passive surveillance, we have isolated two genotypes of reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. Full genome sequence and phylogenetic analysis showed that one is a swine H1N2 variant virus (swH1N2v) with the M gene from pH1N1; the other is a reassortant H1N2 virus (2+6 rH1N2) with six internal genes from pH1N1 and the two surface genes from endemic North American TR H1N2 SIVs. Furthermore, we determined the pathogenicity and transmissibility of the swH1N2v, a human H1N2 variant (huH1N2v), and the 2+6 rH1N2 in pigs using an endemic TR H1N2 SIV (eH1N2) isolated in 2011 as a control. All four viruses were able to infect pigs and replicate in the lungs. Both H1N2 variant viruses caused more severe lung lesions in infected pigs when compared to the eH1N2 and 2+6 rH1N2 viruses. Although all four viruses are transmissible in pigs and were detected in the lungs of contact animals, the swH1N2v shed more efficiently than the other three viruses in the respective sentinel animals. The huH1N2v displayed delayed and inefficient nasal shedding in sentinel animals. Taken together, the human and swine H1N2 variant viruses are more pathogenic and the swH1N2v more transmissible in pigs and could pose a threat to public and animal health.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) employs multiple functions to modulate host’s innate immune response, and several viral nonstructural proteins (nsps) are major players. In this dissertation, the research was mainly focused on identification and functional dissection of ORF1a-encoded nsps. PRRSV replicase polyproteins encoded by ORF1a region are predicted to be processed into at least ten nonstructural proteins. In chapter 2, these predictions were verified by using a panel of newly established antibodies specific to ORF1a-encoded nsps. Most predicted nsps (nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8) were identified, and observed to be co-localized with de novo-synthesized viral RNA in the perinuclear region of the cell. Among all PRRSV proteins screened, nsp1β is the strongest type I interferon antagonist. In chapter 3, mutagenesis analysis of nsp1β was performed to knock down nsp1β’s IFN antagonist function. A highly conserved motif, GKYLQRRLQ, was determined to be critical for nsp1β’s ability to suppress IFN-β and reporter gene expression. Double mutations introduced in this motif, K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV), improved PRRSV’s ability to stimulate the expression of IFN-α, IFN-β and ISG15. In addition to its critical roles involving in modulating host innate immune response, in the studies of Chapter 4, we demonstrated that PRRSV nsp1β functions as a transactivator to induce the -2/-1 ribosomal frameshifting in nsp2, which results in expression of two novel PRRSV proteins, nsp2TF and nsp2N. The conserved motif GKYLQRRLQ is also determined to be critical for the transactivation function of nsp1β. In chapter 5, the interferon antagonist, de-Ub and de-ISGylation activity of newly identified nsp2TF and nsp2N were evaluated. In vitro and in vivo characterization of three nsp2TF-deficient recombinant viruses indicated that all mutant viruses have improved ability to stimulate the innate immune response and provide improved protection in mutant virus-vaccinated animals. In summary, this study verified the previously predicted PRRSV pp1a processing products, further evaluated the function of nsp1β and nsp2-related proteins. These data obtained here will provide basic knowledge for future development of vaccines and control measurements.