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Obesity is a major global health concern due to its steadily increasing rates and significant contribution to numerous diseases, including cancer. Ovarian cancer specifically, is associated with a 30% increased risk with obesity, although the mechanisms for this are unknown. Waist-to-hip ratio has been especially associated with ovarian cancer, suggesting that visceral fat may be the greatest contributor. Here, we investigated individual visceral fat depots as independent contributors to cancer progression, specifically focusing on adipose tissue-derived stem and progenitor cells, which have previously been shown to be recruited by cancer cells and participate in cancer progression. We confirmed that ovarian cancer tumor burden was indeed significantly increased in mice on a high fat as compared to low fat diet. To further investigate mechanisms, we examined changes in progenitor populations that occurred in intra-abdominal parametrial (pmWAT), retroperitoneal (rpWAT), and omental (omWAT) white adipose tissue (WAT) depots with cancer presence. The greatest tumor burden was evident in omWAT, which also displayed an increase in CD45- cells but a decrease in adipose progenitor cells (APC) and endothelial progenitor cells, suggesting that there was an increase in stromal cells, but that the stem cells were pushed towards differentiation. PmWAT and rpWAT showed remarkably stable progenitor populations. However, a tumor from pmWAT had a significant presence of CD45- cells, actually matching that of its surrounding tissue and differing from the omWAT tumors, indicating that microenvironment has a major influence on tumor stromal cells. We also found that with high fat diet, many cancer-associated changes were exacerbated, such as an increased inflammatory response in all tissues and further decreases in APCs in omWAT. In vitro studies further confirmed that ovarian cancer cells and SVF cells were able to directly interact. Additionally, SVF cells were able to increase the proliferation, mobility, and invasiveness of cancer cells. Conversely, co-culturing also enhanced the proliferation and mobility of SVF cells, providing further evidence that SVF cells may be recruited by cancer cells and that their relationship may be bilateral. Thus, this study provides a good foundation for examining the cellular contributions of adipose tissue to cancer. By further characterizing the mechanism for the association between obesity and cancer development, we could find novel targets to decrease the progress of cancer development in at-risk obese individuals. Ph. D.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important swine pathogen. As the emergences of novel virulent strains of PRRSV continue to occur worldwide, rapid vaccine development is the key for effective control of ongoing PRRSV outbreaks. With the availability of the PRRSV reverse genetics systems, rapid vaccine development against PRRSV through the manipulation of the reverse genetics becomes feasible. To facilitate the vaccine development effort and study of PRRSV genes, we first established a DNA-launched infectious clone of the passage 14 PRRSV strain VR2385, pIR-VR2385-CA, and identified a spontaneous 435-bp deletion in the nsp2 gene. To characterize the biological and pathological significance of this nsp2 deletion, we restored deleted nsp2 sequence back to pIR-VR2385-CA and constructed another clone pIR-VR2385-R. VR2385-CA and VR2385-R were successfully rescued in vitro. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no significant effect on the pathogenicity of the virus. With the availability of the DNA-launched infectious clone of PRRSV, we successfully applied the molecular breeding approach to rapidly attenuate PRRSV. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred. DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were successfully rescued in vitro and shown to be attenuated both in vitro and in vivo. Furthermore, DS722, but not DS5M3, still elicit similar protection against PRRSV challenge as its parental virus. This study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positive-strand RNA virus. We subsequently utilized the novel synthetic attenuated virus engineering (SAVE) approach to attenuate PRRSV. The GP5 and nsp9 genes of PRRSV were codon-pair deoptimized with the aid of a computer algorithm. SAVE5 and SAVE9 with deoptimized GP5 gene and SAVE9 with deoptimized nsp9 gene, were successfully rescued and shown to be attenuated in vitro. An in vivo pathogenicity study indicated the attenuation of SAVE5 virus in vivo. The results have important implications for rapid vaccine development against PRRSV and other important viruses. Ph. D.

Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease. Master of Science

Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications. Master of Science

Cancer research is evolving. Historically concerned with the mechanisms by which malignant cells circumvent cell death signaling and maintain unchecked proliferation, focus has shifted to the complex interactions between the tumor cell and the surrounding microenvironment. Ovarian cancer has one of the highest incidence-to-death ratios of all cancers, and is typically asymptomatic until the later stages, often resulting in metastasis prior to discovery. Naturally occurring phenotypes like lactation and child-bearing (parity) reduce ovarian cancer incidence, but the mechanisms are not understood. As the primary site for ovarian cancer metastasis, and a secondary lymphoid organ capable of mounting potent innate and adaptive immune responses, we believe the omental fat band (OFB) provides a unique opportunity to study complex interactions within the tumor microenvironment. Additionally, we hypothesize that once understood, leukocyte populations within the OFB could be modulated to disrupt the pro-tumorigenic cascade. Using fluorescence-activated cell sorting (FACS) and quantitative realtime PCR (qRT-PCR), we comparatively evaluated the changes in the compositional immune profile of the OFB as a result of parity and cancer. Parous mice were associated with a reduction in macrophages and neutrophils in the OFB, resulting in an inherent "protective state" that was refractory to metastatic cancer cell growth after intraperitoneal implantation. This indicates that the leukocyte populations within the OFB play an important role in tumor development. Therefore we utilized the potent TH1-type immunomodulatory cytokine IL-12 in a membrane-bound form to circumvent reported side effects, such as hepatic and renal damage, cardiotoxicity and death. Targeted IL-12 delivery to the OFB resulted in delayed disease development, although not protection from subsequent challenge. This was also associated with a reduction tumor-associated macrophages (TAMs) and neutrophils (TANs) within the OFB. Kinetic studies demonstrated that these changes were induced by a significant reduction in neutrophil and macrophage chemoattractants early on in the pro-tumorigenic cascade (7 days post-implantation). This work demonstrates that the OFB is a functionally plastic tissue that can be harnessed and re-mobilized to display an anti-tumorigenic microenvironment. Ph. D.

Ovarian cancer is known as the "silent killer," due to its late diagnosis and frequent recurrence after initial treatment. Finding a new way to diagnose and treat ovarian cancer in conjunction with current therapies is paramount. By capitalizing on metabolic changes that occur during cancer progression, interventions can be developed. The Nobel laureate Otto Warburg is credited with discovering an altered metabolic state within cancer cells known as the Warburg effect. In the Warburg effect, cancer cells participate in an increased rate of aerobic glycolysis with an excess secretion of lactate, allowing for carbon flux into biosynthetic pathways. Exactly which metabolic pathways are altered in ovarian cancer and at which stage in the progression of ovarian cancer they are occurring was unknown. Therefore using the recently established mouse ovarian surface epithelial (MOSE) progression model, we were able to measure metabolic changes in varying states of disease and levels of aggressiveness. As cells progressed from a benign early stage (MOSE-E), through a transitional intermediate stage (MOSE-I), to an aggressive late stage (MOSE-L), the MOSE cells became more glycolytic and lipogenic, establishing the MOSE model as a valuable model for studying ovarian cancer metabolism. Treating the MOSE cells with the naturally occurring chemotherapeutic agent sphingosine decreased p-AKT protein levels in the cell, decreased the glycolytic rate and decreased de novo cholesterol synthesis. Cancer stem cells are known to be resistant to chemotherapy treatments and targeting their metabolism may be promising for combinatorial treatments. Therefore, the metabolism of highly aggressive tumor-initiating cells (TIC), harvested from ascites of C57Bl/6 mice injected with MOSE-L cells were characterized. Although the basal metabolism of the TICs was similar to the MOSE-L cells, TICs were more resistant to cell death as a consequence of external stresses and substrate depletion. The TICs could also up-regulate oxygen consumption rate (OCR) when uncoupled and increase glycolysis when ATP Synthase was inhibited, highlighting their resiliency. Taken together, we have identified targets for treatment strategies that could suppress the growth of primary tumors and may be effective against TICs, thereby suppressing tumor recurrence and possibly prolonging the life of women with ovarian cancer. Ph. D.

Influenza epidemics and pandemics remain a significant burden to world health and economy. Low efficacy of current inactivated influenza vaccines in the elderly and immunocompromized and the inability to protect against antigenically drifted or shifted strains of influenza virus are the two major problems in influenza vaccine research. To overcome these hurdles, we have utilized an in vitro cell culture vaccine platform, which results in whole inactivated influenza vaccine (WIV) bearing bioactive membrane-anchored immunomodulatory proteins such as cytokines on the virion surface, collectively known as CYT-IVACs (Cytokine bearing-Inactivated Vaccine). In addition, we tested whether a multimeric M2e peptide presented on WIV can serve to enhance immunogenicity and augment protective efficacy of whole virus vaccines. Our panel of cytokines includes IL-2, IL-4, IL-12, IL-23, and Flt3L as well as the multimeric M2e peptide, all fused to the membrane anchoring regions of influenza virus hemagglutinin protein and constitutively expressed in virus permissive MDCK cell line. Subsequent infection with influenza virus results in incorporation of fusion constructs directly into budding progeny virions that are harvested, purified and inactivated to generate distinct CYT-IVAC formulations. Following validation of immunomodulator incorporation, vaccines were tested for in vivo efficacy in either "young adult" or "aged" female Balb/c mice. Our results demonstrate that our CYT-IVAC~IL-12/HA and CYT-IVAC~IL-23/HA serve as potent mucosal adjuvants in young adult mice elicited significantly high levels of mucosal IgA antibodies and afford superior protection against lethal virus challenge. Our Flt3L/HA formulation was the most effective stimulator of systemic anti-viral antibody levels. In "aged" mice a single dose formulation of IL-12 bearing CYTIVAC was superior at affording protection against lethal homotypic virus challenge. Finally, administration of multimeric M2e molecule co-presented on WIV elicited prolonged antibody responses in "young adult mice" and provided cross-protection from challenge with the heterologous influenza A pandemic strain 2009 H1N1. In conclusion, the CYT-IVAC approach represents a novel tailored advancement to current WIV approaches that has the potential to elicit both potent mucosal and systemic immune responses in young and old. Ph. D.

HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains. Ph. D.

Mosquito-borne diseases continue to be a burden to global health. The viruses that cause these diseases are maintained in nature through a biological transmission cycle involving susceptible vertebrate and mosquito hosts. While knowledge of the interactions occurring between mosquito-borne viruses and vertebrates is considerable, much less is known about the interactions of these viruses with their disease vectors. Studies with Drosophila melanogaster have been important in understanding how insects respond to viral infections. However, mosquitoes and the viruses they vector have co-evolved during a long period of time. Unfortunately, many of the genetic advantages of a fly model are not available when working with mosquitoes. Nevertheless, a sequenced genome, and molecular tools such as high-throughput sequencing and RNAi knockdown are helping to bridge these gaps. Here we describe several additional tools for the study of virus-vector interactions in the mosquito. Ph. D.

Mosquito-borne diseases remain a significant burden on global public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although infection of the vertebrate host is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. It is well known that invertebrates rely on small RNA pathways as an adaptive antiviral defense. The canonical antiviral response in these organisms involves dicer enzymes that cleave viral double-stranded RNA replicative intermediates (RIs) into small interfering RNAs (siRNAs; ~21-24 nucleotides). One strand of the siRNA duplex guides the targeting and destruction of complementary viral RNAs when loaded and retained in a multi-protein complex called the RNA-induced silencing complex. Here, we show that mosquito vectors mount a redundant double defense against virus infection mediated by two different small RNA pathways. Specifically, we demonstrate that in addition to a canonical antiviral response mediated by siRNAs, virus infection of the mosquito soma also triggers an antiviral immune pathway directed by ping-pong-dependent PIWI-interacting RNAs (piRNAs; ~24-30 nucleotides). The complexity of mosquito antiviral immunity has important implications for understanding how viruses both induce and modulate RNA-silencing responses in mosquito vectors. In mammals, viral RIs induce a range of relatively nonspecific antiviral responses. However, it remains unclear if viral RIs also trigger RNA silencing in mammals. Mosquito-borne viruses represent an ideal model for addressing this question as their transmission cycles involve alternating replication in mammalian and invertebrate hosts. Although we report identifying a subset of virus-derived small RNAs that appear to be products of RNA silencing in two mammalian cell lines infected with the mosquito-borne chikungunya virus (CHIKV), our studies suggest these small RNAs have little biological relevance in combating virus infections. Thus, while the accumulation of virus-derived siRNAs is essential to the survival of mosquitoes infected with CHIKV, they appear to have little functional significance in mammalian antiviral immunity. Ph. D.

Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform. Ph. D.

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA viruses. A large and diverse host species are infected by paramyxoviruses, including avian, porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of them also made the species jump to humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous system disease from South and North Central United States. To understand their importance as swine pathogens, molecular characterization and pathogenicity studies were undertaken. The complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix (M, fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be conserved in most members of Paramyxoviridae were also found in swine viruses. These include: conserved and complementary 3â leader and 5â trailer regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal sequences. The length of each gene of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt "U" rich 3â untranslated region (UTR). The P gene of these viruses were predicted to express the P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins and the C protein was expected to be expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence specific features related to virus replication and host specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis based on individual genes as well as predicted amino acid sequences suggested that these viruses were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The screening ELISA developed by us could be used to detect seroprevalence of sPIV3 in animal and human populations. Master of Science

Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. Our group has developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Here we report methodologies for the construction of membrane-bound cytokine fusion constructs in which our cytokine of interest (mouse GM-CSF, mouse IL-2, mouse IL-4) was fused to the membrane anchoring regions of viral Hemagglutinin (HA). Progeny virions, produced from influenza infected MDCK cells expressing membrane-bound cytokines, readily incorporated membrane-bound cytokines during budding and these cytokines on the virus particles retained bioactivity following viral inactivation. In vivo vaccination studies in mice showed enhanced antibody titers and improved protection following lethal challenge in those mice vaccinated with IL-2 and IL-4-bearing CYT-IVAC's compared to the conventional wild-type vaccine without membrane-bound cytokines. In addition, the immune response induced by IL-2 and IL-4-bearing CYT-IVACs was skewed toward Th1 (cellular) mediated immunity compared to the Th2 (humoral) dominated response induced with wild-type vaccination. Cellular mediated immunity afforded by IL-2 and IL-4 CYT-IVACs was manifested as enhanced influenza specific T cell proliferation and activation. In conclusion, we have developed a novel methodology to introduce bioactive membrane-bound cytokines directly into virus particles in order to augment the immunogenicity of inactivated, whole virus influenza vaccines. Ph. D.