Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

The West Nile virus (WNV), part of the flavivirus group, is one of the most recognized arboviruses in the world. Following its 1937 discovery in Uganda, it subsequently expanded into the Middle East, then to Europe, and ultimately established a presence in North American in 1999. The virus is maintained in the environment as an enzootic cycle primarily between Culex species of mosquitoes and various avian hosts. WNV has gained much attention for both its effect on avian hosts and for its ability to cause serious infections in dead-end hosts such as humans. With the WNV circulating within the United States now for 20 years, significant mutational changes have occurred resulting in the selection of a new, more virulent WNV genotype. There are limited studies on potential factors involved in the selection of new WNV phenotypes. Further study of potential factors, geographical correlation, and variants found in WNV genome warrants investigation. WNV samples collected in Ohio to screen for incidence provide a resource for investigation of potential genetic variation of WNV relative to specific biomes. This study has piloted a strategy wherein cDNA from WNV positive RNA extracts and subsequent amplification by the polymerase chain reaction (PCR) affords WNV genetic material for evaluation. Because major changes in WNV pathogenicity have corresponded to changes in the envelope gene product, my study has targeted that region of the WNV genome. These efforts resulted in the amplification and sequence determination of a region of the envelope gene that showed variation relative to WNV isolates from other regions of North America.

Doctor of Philosophy, The Ohio State University, 2020, Plant Pathology

Production of soft red winter wheat (Triticum aestivum), the type of wheat used for baking cookies, crackers, and cakes, is known to be reduced by several biotic diseases; however, the impact of virus diseases on wheat health is widely underestimated in Ohio. This is partially due to a lack of knowledge of which specific viruses are in Ohio and their distribution, which results in fewer resources for growers to control virus diseases. Our overarching goal was to identify and assess the prevalence of wheat viruses in Ohio and begin to fill knowledge gaps from identification, to impact on wheat production, to effective management. Two distinct research pathways were undertaken to accomplish this goal. In the first pathway, we conducted multiple surveys of Ohio wheat fields to identify viruses that pose a risk to wheat production. In the second pathway, we examined the impact of brome mosaic virus, one of the viruses identified in the surveys, on wheat production across multiple growth stages of infection and cultivars. Surveys of Ohio wheat fields were conducted in 2012, 2016, and 2017 in which samples showing virus-like symptoms were collected and subjected to high throughput sequencing, reverse-transcription PCR, or ELISA to assess virus sequence diversity, prevalence, and incidence within fields. From our surveys we identified barley yellow dwarf virus, cereal yellow dwarf virus, wheat streak mosaic virus, and wheat spindle streak mosaic virus as viruses that are a high risk to wheat production in Ohio based on sequence diversity, prevalence, and incidence within fields. Other viruses, including High Plains wheat mosaic virus, soilborne wheat mosaic virus, and oat necrotic mottle virus, were detected in Ohio but were determined to currently be low risk to wheat. Agropyron mosaic virus and cocksfoot mottle virus were identified in Ohio for the first time. We also identified and characterized novel members of Rymovirus, Luteovirus, and Endornaviridae. Further inves (open full item for complete abstract)

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2016, College of Sciences and Health Professions

Pneumonia Virus of Mice (PVM) and Respiratory Syncytial Virus (RSV) are negative sense, single-stranded, enveloped RNA viruses from Pneumovirus genus, Paramyxoviridae family. RSV is the leading cause of respiratory diseases in infants. PVM causes similar respiratory illness in mice. PVM is used as an animal model to study RSV pathogenesis because of its similarity with RSV infection. Viral infection induces type I interferon (IFN) response as an antiviral strategy. PVM and RSV both have two non-structural (NS) proteins that are known to be IFN antagonists. While RSV can target different signaling components of IFN pathway, the mechanism of IFN suppression for PVM was unknown. We have identified that PVM can also target different signaling components of IFN pathway to circumvent the host immune system. Our observations showed that PVM NS proteins facilitate proteasome-mediated degradation of RIG-I, IRF3, STAT2 in IFN pathway by direct interactions with them. Production of several Interferon Stimulated Genes (ISGs) is the distal part of the IFN pathway. We have identified that NS proteins of PVM can also target a few of them such as TRAFD1, IFITM1, ISG20, and IDO for complete suppression of the host immune system. RSV NS proteins play a similar role to suppress IFN pathway by targeting TBK1, RIG-I, IRF3, IRF7, and STAT2. Our study has identified one ISG, OASL, that has antiviral properties against RSV and documented that to counteract this antiviral property of OASL, RSV NS proteins can degrade OASL in a proteasome-dependent way. These above observations help us to delineate the complete suppression mechanism for the whole Pneumovirus genus, both for PVM and RSV by providing the first experimental evidence of signaling components from the IFN pathway targeted by PVM to suppress the IFN response. PVM is a clinically relevant animal model that will help us to find new therapeutic strategies against Pneumovirus infection. RSV study with one of those important ISGs, OA (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2016, Biomedical Engineering

Each year, one million new cases of cancer are diagnosed in the United States and each case is unique, making it hard disease to prevent and treat. In the past few decades, nanoparticles have emerged as a promising platform for the development of cancer therapies. Unlike small molecule drugs, nanoparticles can be both passively and actively targeted towards tumor sites (both primary and metastatic sites). Additionally, they are able to carry large cargos for delivery of monotherapy or combination therapy, while also decreasing systemic side effects often associated with small molecule cancer therapies. Nanoparticles in the clinic, as well as in clinical and pre-clinical trials have been predominantly spherical in shape. However, recent data suggest that high aspect ratio nanoparticles may have advantages for cancer treatment, including decreased uptake by phagocytic cells, improved margination, and enhanced tumor homing and penetration. Despite this, synthesis of these materials remains challenging using chemical approaches. Therefore, I turned to towards the study of plant virus-based nanoparticles, and my dissertation focused on the study and development of high aspect ratio viral nanoparticles. Specifically, I focused on the plant viruses tobacco mosaic virus (TMV) and potato virus X (PVX). My initial studies evaluated TMV as a model high aspect ratio nanoparticle and determined that it had improved diffusion into a 3D spheroid compared to a spherical virus. Additionally, I determined that it could be stably loaded with via non-covalent interactions with a cationic photosensitizer for photodynamic therapy. In the central body of my thesis I developed the filamentous virus PVX for cancer therapy. Stealth coated PVX filaments exhibited substantially extended circulation time, while also decreasing recognition by PVX-specific antibodies, an important step towards translation of this platform. Addition of targeting ligands on the surface of the PVX filament led to se (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Understanding the viral infection and lifecycle of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is important in improving disease outcomes and reducing viral prevalence. In our SFTSV study, we discovered specific cytokine profiles associated with the severity of clinical symptoms. We used single cell RNA sequencing (scRNAseq) on patient blood samples to identify a unique expansion of the B cell population in SFTSV-induced fatal cases which indicated that plasma B cells are a primary reservoir of SFSTV replication. These findings present a potential method of reducing the severity of SFTSV infection, especially in aged patients who are more susceptible to adverse outcomes. In our KSHV study, we developed a novel oral 3D infection model and demonstrated that KSHV can only infect exposed basal epithelial cells in the oral epithelia. We used scRNAseq to show that keratinocyte differentiation and cell death pathways were affected by KSHV infection, suggesting that epithelial differentiation could contribute to KSHV reactivation through changes in epigenetic regulation. In addition, we found a unique population of infected cells with limited early lytic gene expression and a unique gene expression profile, which we termed latent-2 cells. These findings demonstrate that our in vitro 3D epithelial ALI culture model should be a valuable tool to further understand oral KSHV infection for the development of future anti-viral therapeutics to block KSHV transmission.

MS, University of Cincinnati, 2023, Medicine: Immunology

The emergence of pneumonia-like symptoms in late 2019 heralded the beginning of a global health crisis, resulting in the COVID-19 pandemic. Following the identification of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, scientists and pharmaceutical companies have worked to develop a safe and effective vaccines against the virus. Numerous vaccine candidates have been evaluated, including the mRNA-based vaccines, viral-vector vaccines, protein subunit vaccines, and inactivated vaccines. This study focuses on Virus-like Particles (VLPs) as an effective booster dose after initial priming with the Parainfluenza virus 5 (PIV5) vector, which are known to protect animal models against influenza, RSV and rabies. Here, we obtained PIV5 vaccines expressing the SARS-CoV-2 Spike (S) glycoprotein, and administered them intranasally in BALB/cJ mice, followed by boosting with virus-like particles containing the structural proteins of SARS-CoV-2. We analyzed spike-specific immune responses generated by the recombinant PIV5 prime/VLPs boost regimen (heterologous) vs a VLP prime/VLP boost or a single intranasal dose of the recombinant PIV-5 (homologous). We demonstrated that the heterologous regimen induced superior S-specific antibody titers, including neutralization antibody responses, as compared to either PIV5 alone or a VLP/VLP regimen. Preliminary results supported a trend toward higher neutralization titers when the interval between priming and boosting was extended from 3 weeks to 7 weeks. Overall, our data indicate that VLP-based vaccines are promising as an effective booster dose after initial priming with recombinant PIV5 vaccine. These studies support further development of the VLP boosting approach including extension of findings into rodent and non-human primate challenge models.

Doctor of Philosophy, The Ohio State University, 2023, Molecular, Cellular and Developmental Biology

Severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic which has led to nearly 400 million cases and over 5 million deaths. Unprecedented effort from the scientific community led to the development of two messenger ribonucleic acid (mRNA) vaccines that have helped to control the worst impacts of the pandemic. However, the durability of the protection of these vaccines and how they compared to immunity from natural infection, remained critical concerns. Additionally, the impact of immunocompromising disease and disease therapies threatened the efficacy of these vaccines in certain patient populations. Further, the emergence of SARS-CoV-2 variants of concern (VOCs) which exhibited resistance to vaccine-induced neutralizing antibodies (Nabs) continued to threaten the efficacy of the mRNA vaccines. To address these concerns our lab developed a highly sensitive pseudotyped lentivirus-base virus neutralization assay to examine Nab titers in COVID-19 patients and mRNA vaccine recipients. These Nabs are those antibodies which directly block viral entry, and are key determinants for protection from viral infection. With this I demonstrate that Nab titers induced by natural infection are dependent on disease severity with only hospitalized and intensive care unit (ICU) COVID-19 patient serum exhibiting strong Nab response, yet ~15% exhibited no detectable Nabs. However, mRNA vaccinated healthcare workers (HCWs) reliably exhibit strong Nab responses, with every HCW tested exhibiting detectable Nab titers. We further demonstrated that the Alpha, Beta, Delta, and Omicron variants exhibit varying degrees of Nab escape with Omicron exhibiting near complete escape from 2 dose mRNA vaccine-induced Nabs. Administration of an mRNA booster dose recovers neutralization of the Omicron variant. In addition, titers of Nabs wane over time more quickly for recipients of 2 mRNA vaccine doses compared to (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

Viral infections are an adverse occurrence that the body must work to control via the induction of a strong immune response. This generally includes the induction of a type I interferon response to halt the spread of virus early on and put the site of infection into an antiviral state. Programmed cell death pathways are also initiated as a means of clearing out infected cells and quelling further infection. However, over-activation of both of these pathways can lead to poor outcomes for the host and therefore require tight regulation of all players involved. There is perhaps no better example of the importance of immune regulation than in the context of pregnancy. Indeed, infections during pregnancy can cause a multitude of complications as the mother works to balance tolerance of the fetus with simultaneous fetal protection. A recent landmark study has indeed demonstrated that during Zika virus infection, activation of the maternal type I interferon (IFN) response is responsible for adverse fetal outcomes. We demonstrated that downstream of IFN, innate immune proteins, IFITM1, 2 and 3, block proper and necessary placental cell fusion. Specifically, cells expressing any of the IFITMs displayed decreased fusogenic ability. However, when the IFITMs were knocked down, cells fused spontaneously at high levels even in the presence of type I IFN. Our work demonstrated a negative function for typically beneficial antiviral proteins. Additionally, we have provided an evolutionary explanation as to why these proteins are tightly regulated and constantly ubiquitinated outside of the context of infection. On the other hand, the IFITMs, particularly IFITM3 continue to be critically important antiviral restriction factors in a wide array of viral infections. Indeed, individuals with single nucleotide polymorphisms (SNPs) in the IFITM3 gene are at increased risk of severe influenza virus infection. Therefore upon emergence of a new pandemic virus, SARS-CoV-2, it was critica (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2021, Microbiology

Biogeochemical fluxes across the planet are driven by microbial metabolisms and interactions. Viruses in the oceans play important top-down roles on microbial communities through lysis and mortality. Viral lysis keeps microbial abundances in check while also releasing large amounts of carbon and nutrients to the surrounding water column, which is either fed back into microbial respiration or sequestered in the deep ocean via aggregates of sinking particles. Viruses are also known to exert physiological controls on microbial communities by altering the transcriptional, translational, and metabolic functioning of infected host cells, termed “virocells”. Lastly, viruses are known to have a strong impact on host evolution through an arms race for infective or defensive strategies respectively and by acting as vectors for horizontal gene transfer. While these characteristics have been well described for bacteriophages (viruses of bacteria) and a few archaeal viruses from extreme environments, archaeal viruses in relatively non-extreme environments, particularly the oceans, are all but unknown. Here we make the first concerted efforts to establish a means to identify marine archaeal viruses, develop datasets from regions of the oceans where archaeal viruses are prevalent, and conduct the first global-scale effort to identify and investigate the ecology of marine archaeal viruses. Establishing a means to identify archaeal viruses comes in two parts. In the first part, described in chapter two, we establish the basic genomic principles needed to distinguish between marine archaeal viruses and bacteriophage in sequence data. We then test this approach in the Eastern Tropical North Pacific (ETNP) Oxygen Minimum Zone (OMZ) region, identifying 43 new marine archaeal viruses. In the second part, described in chapter 5, we use these principles and a growing database of marine archaeal viruses to develop a robust, high-throughput machine learning platform for archaeal virus disc (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Influenza virus is a leading cause of human mortality globally and presents a persistent pandemic threat. In addition to lung pathology, severe infections may be accompanied by extrapulmonary complications, including cardiac pathology. Influenza virus is recognized as a cardiotropic virus and an etiologic agent of viral myocarditis; thus, the virus can cause cardiac pathology in the absence of pre-existing cardiovascular disease. However, the underlying mechanisms of influenza-associated cardiac pathology are poorly understood. Influenza virus infections of extrapulmonary tissues have been reported for various mouse models of infection, but relevant models in which significant cardiac pathogenesis occurs and the animal's susceptibility is relevant to human infections are lacking. Single nucleotide polymorphisms in the IFITM3 gene are among the only genetic defects that have been reproducibly associated with severe influenza in humans. Given that numerous studies have linked IFITM3 defects to severe influenza virus infections, and that severe influenza virus infections may include cardiac complications, we sought to investigate whether IFITM3 plays a role in protecting the heart during infection. We showed that IFITM3 deficiency in a knockout mouse model exacerbated weight loss and mortality following influenza virus infections, and observed increased replication of virus in the lungs and hearts of IFITM3 KO mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, which was accompanied by fibrosis of heart tissue, prior to succumbing to infection. These findings revealed an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and established IFITM3 KO mice as a powerful model for studying influenza virus-induced cardiac dysfunction. We next sought to explore whether direct infection of heart tissue by influenza virus contributed to the observed cardiac pathologies following infection. We genera (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Sciences

HIV is a worldwide pandemic that remains incurable. Recent statistics show that in the United States alone, ~15 per 100,000 people were newly infected with HIV-1 in one year. The barrier to a cure is a reservoir of cells with viral DNA stably integrated into their genome, yet are not killed by the immune system. The integration step of the retroviral life cycle is crucial in reservoir formation. Viral DNA integration is catalyzed by protein integrase (IN). We study HIV-1 IN and prototype foamy virus (PFV) IN. PFV IN is used to model for HIV-1 integration, as HIV-1 IN inhibitors also block PFV IN activity. This implies that the two proteins have similar catalytic mechanisms. However, we have found differences between PFV IN and HIV-1 IN function. We determined that PFV IN could utilize calcium for strand transfer, unlike HIV-1 IN. Additionally, though HIV-1 IN has been reported to rapidly commit to its target DNA, PFV IN does not commit within an hour. There are likely differences in searching and target capture mechanisms between the two INs. A benefit to using PFV IN is that it can be readily assembled with oligomers that mimic viral cDNA ends to form an intasome complex. The PFV intasome contains a tetramer of PFV IN and two oligomer DNAs. We found in vitro that these intasomes aggregate at 37°C. Full-length intasomes aggregate more than those containing truncated PFV IN outer subunits, particularly deleting the carboxyl terminal domain (CTD). Aggregation can be prevented by using high non-physiological salt concentrations or with addition of small molecule protocatechuic acid (PCA). This finding is useful for future experiments that require longer lifetimes of PFV intasomes. Integration into chromatin is not well understood. Chromatin is comprised of basic units called nucleosomes. Our goal is to understand how IN chooses its site when integrating into nucleosomes. We altered either nucleosomes or PFV IN to understand how changes impact integration acti (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2018, Comparative Veterinary Medicine

Porcine reproductive and respiratory syndrome (PRRS) continues to be a huge threat to the swine industry globally. Development of effective vaccines is still a best measure for controlling PRRS and reducing economic losses. Due to advancements in global trade, co-infection of both PRRSV species occurs in the same herd. In this study, we employed new vaccine development strategies to induce an increased breadth of immunity against both PRRSV species. In PRRSV-1 and PRRSV-2 co-infected MARC-145 cells we observed a strong interference of PRRSV-2 on PRRSV-1 replication, especially in concurrent co-infected cells. We used PRRSV-1 and PRRSV-2 modified live virus vaccines (MLV) to analyze the efficacy of both consecutive and concurrent vaccination methods administered intramuscularly, either 3 days apart (PRRSV-1 MLV followed by PRRSV-2 MLV, (consecutive) or together on the same day (concurrent) against homologous challenge in pigs. We showed that the consecutive vaccination method appears to provide better clearance of both PRRSV species through induction of increased frequency of PRRSV-1 and PRRSV-2 specific IFN-¿ positive T-helper/memory and cytotoxic T cells responses, with increased virus neutralization antibody titers against both PRRSV-1 and PRRSV-2. Further, PRRSV MLVs have a safety concern, however killed vaccines are safe but have an efficacy concern due to their less immunogenicity. In that study, we employed killed vaccines against PRRSV-1 and PRRSV-2 species along with a potent mucosal adjuvant, like non-toxic heat labile (LT) enterotoxin for priming, followed by oral boosting with immunogenic conserved T-cell epitopes of PRRSV expressed by non-pathogenic E. coli against respective homologous challenge virus infections in pigs. We showed that our vaccination strategy induced a robust cell-mediated immune response, especially T cell responses against both PRRSV-1 and PRRSV-2, and improved the overall adaptive immune response in pigs. Further studies are nee (open full item for complete abstract)

Master of Environmental Science, Miami University, 2018, Environmental Sciences

Mosquito-borne diseases are one of the leading causes of illness and death in the world and pose significant risks to people in the US (US EPA, 2012). Both locally transmitted mosquito-borne diseases (e.g. West Nile virus) and travel-associated mosquito-borne diseases (e.g. Zika virus) threaten the health of Ohioans. A primary step in preventing the spread of these diseases is to carefully plan, implement, and evaluate mosquito surveillance plans. To assist local health departments (LHDs) with creating effective surveillance plans, the Ohio Environmental Protection Agency requested my assistance with creating guidance materials that will outline essential elements and necessary steps for mosquito surveillance plans. This report summarizes the research I conducted to develop a mosquito surveillance plan template and guide to aid the preparation of mosquito surveillance plans in Ohio. The guide outlines the important components of a surveillance plan including planning steps such as goals and objectives, and essential surveillance program elements- adult mosquito, larval mosquito, human (epidemiological), and vertebrate (non-human) surveillance. The template provides LHDs with an easy-to-use document to fill in. This project was completed in collaboration with the Ohio Environmental Protection Agency Division of Materials and Waste Management and the Ohio Department of Health Zoonotic Disease Program.

Master of Science (MS), Wright State University, 2016, Microbiology and Immunology

In this study an assessment (VirEMiA) on college students' knowledge and misconceptions of the Ebola virus was created and validated. VirEMiA was then used to determine what misconceptions college students have about Ebola, if there is a difference in misconceptions between students with and without a strong science background, and if Just-in-Time Teaching (JiTT) increases students' knowledge of Ebola and decreases their misconceptions. VirEMiA was shown to be a valid and reliable assessment whether confidence was integrated (seprel=0.97) or not (seprel=0.98), and for measuring misconceptions (seprel=0.97). If psychology and nursing majors were considered to have a strong background in science, the difference in misconceptions between students with and without a strong background in biology was not statistically or practically significant (tdf=392=1.86, p=0.06, d=0.19). However, if psychology and nursing majors were not considered to have a strong science background, there was a statistically and practically significant difference in misconceptions between students with and without a strong science background (tdf=392=4.18, p<<0.001, d=0.64). When VirEMiA was used as pre-homework for a class utilizing JiTT, student got about 4.4 more questions on the post-test correct compared to the pre-test, and the difference in their scores is statistically and practically significant (tdf=116=9.11; p<<0.001; d=0.84). Students also had about 7 fewer misconceptions after learning about Ebola, and this difference was practically and statistically significant (tdf=116=-9.80; p<<0.01; d=-0.91). These results show VirEMiA to be a valid and reliable instrument for measuring students' knowledge and misconceptions. It also showed that students' with a strong background in science do have fewer misconceptions than students without a strong background in science, as expected.

Doctor of Philosophy, The Ohio State University, 2015, Integrated Biomedical Science Graduate Program

Live attenuated respiratory syncytial virus (RSV) vaccine candidates are produced in Vero cells, a cell line that cleaves the attachment (G) glycoprotein. As a result, Vero-derived virus is 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cultures than virus grown in HeLa. HAE cultures are isolated directly from the human airways, so it is likely that Vero-grown vaccine virus would be similarly inefficient at initiating infection of the nasal epithelium following vaccination, requiring a larger inoculum, thereby raising the cost per dose. Using protease inhibitors with increasing specificity, we identified cathepsin L as the responsible protease and confirmed that virus grown in the presence of protease inhibitors was more infectious for HAE cultures. Our evidence suggests that the G protein interacts with cathepsin L in the late endosome or lysosome via endocytic recycling. While essential for Nipah virus F protein cleavage, endocytic recycling is detrimental to the production of infectious RSV from Vero cells. We found that cathepsin L is able to cleave the G protein in Vero-grown, but not in HeLa-grown virions suggesting a difference in G protein posttranslational modification. Using mutagenesis, we identified a cluster of amino acids that are important for G protein cleavage and they contain a likely cathepsin cleavage site. Virus grown in Vero cells and containing a G protein resistant to cleavage is 5-fold more infectious for HAE than the same virus grown in Vero. Live attenuated RSV vaccine virus containing this mutation would reduce the cost of vaccine production for infants.

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Human norovirus (HuNoV), hepatitis A virus (HAV), and rotavirus (RV), are responsible for the majority of foodborne illnesses. Seafood, particularly bivalve shellfish, is one of the major high risk foods for enteric viruses contamination. Therefore, there is an urgent need to develop effective thermal and non-thermal processing technologies to eliminate virus hazards in seafood. This study aims to determine the bioaccumulation patterns of human enteric viruses in shellfish tissues, to determine whether heat or high pressure processing (HPP) are capable of effectively inactivating enteric viruses in shellfish, and to determine whether viruses can develop resistance to processing technologies. Oysters (Grassostrea gigas) were cultivated in seawater artificially contaminated with HuNoV surrogates (Tulane virus, TV; murine norovirus, MNV-1), HAV, or RV at level of 1 × 10^4 PFU/ml, or 1 × 10^4 RNA copies/ml of a HuNoV GII.4 strain. Oysters were harvested after 24, 48, and 72 h post-inoculation, and the presence of viruses was determined in gills, digestive glands, and muscles by plaque assay or real time PCR (RT-qPCR). It was found that caliciviruses and HAV were localized in the stomach at a high level within the first 24 h, while RV was detected in the highest level in the gills. Next we determined the thermal stability of each of the viruses. It was found that Decimal reduction time (D-values) of TV, MNV-1, HAV, and RV ranged from 0.13 to 1.81 min and 1.26 to 7.29 s at 62 and 72°C, respectively. At 80°C the time to first log10 reduction (TFL-value) ranged between 0.46 and 32 s in cell culture medium, and ranged between 0.61 to 19.99 min in oysters. In terms of thermal resistance the four viruses can be ranked as the following: HAV>RV>TV>MNV-1. This study also compared the baro-sensitivity of seven RV strains (G1: Wa, Ku, and K8, G2: S2, G3: SA-11 and YO, and G4:ST3) following HPP. It was found that RV strains showed varying responses to HPP based on the initial temp (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

Purpose. Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses have shown efficacy against ovarian cancer. We studied the application of an oncolytic herpes simplex virus expressing two anti-tumor genes: ICP34.5 under the stem cell-specific nestin promoter, and anti-angiogenic molecule Brain-specific Angiogenesis Inhibitor-1 (BAI1) under the strong viral promoter IE4/5 for use against ovarian cancer. We also studied the use of this viral vector in combination with a second-line standard of care chemotherapeutic drug, doxorubicin. Experimental Design. The 34.5 Expressing Nestin-driven Vasculostatin-120 Expressing (34.5ENVE) virus was tested for treatment of ovarian cancer in vitro and in vivo. Efficacy of the virus, and its antiangiogenic effects on endothelial cells were assessed in vitro in cancer cell lines and in primary patient ascites samples. Scope of cytotoxic interactions between 34.5ENVE and chemotherapeutic agent doxorubicin were evaluated using Chou-Talalay synergy analysis. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of progressive human ovarian cancer. Results. 34.5ENVE showed robust efficacy against ovarian cancer cell lines, and even greater efficacy against ex vivo mouse and patient ascites. When combined with doxorubicin, 34.5ENVE killed synergistically with a robust increase in caspase-3/7 cleavage. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors. Conclusions. This study establishes the potential for use of oncolytic HSV in combination with doxorubicin for the treatment of late-stage ovarian cancer.

Master of Science (MS), Wright State University, 2009, Pharmacology and Toxicology

Influenza viruses are consistently responsible for an average of 20,000 deaths and 114,000 hospitalizations per year. To a great extent, these viruses always stay one step ahead of the available vaccines and people's immunity year after year because they have the ability to either mutate part of their genetic material, or to be transmitted from one species to another. That same genetic variability explains why highly pathogenic influenza viruses emerge that cause great mortality over several countries resulting in pandemics. Highly pathogenic strains of influenza A virus have emerged occasionally in recent history, producing pandemics such as the one in 1918. The Spanish influenza pandemic of 1918–1919 was uniquely severe, causing an estimated 50 million deaths worldwide. Also unique was the age distribution of its victims: the death rate for young, previously healthy adults, who rarely suffer fatal complications from influenza, was exceptionally high. More recently we have seen the emergence of influenza cases and fatalities involving the H5N1 avian influenza strains. Until an outbreak in Hong Kong claimed six human lives in 1997 (A/Hong Kong/156/97 [H5N1]), avian influenza viruses were thought to be incapable of infecting humans directly. However, the initial H5N1 outbreak has revealed that avian influenza viruses could infect humans without prior adaptation and even cause significant morbidity and mortality in the human population. It has been shown that the 1918 viral hemagglutinin sequence is more closely related to avian strains although it is a human HA. This indicates that the 1918 pandemic strain may have also jumped from avian to human with no prior adaptation or reassortment with a human virus. For these reasons, scientists have been interested in finding out what makes the 1918 virus different from all others, why avian influenza can be so pathogenic, and how to prevent and better treat infections with this virus. For this study the 1918 pandemic influe (open full item for complete abstract)

Master of Science, University of Toledo, 2012, Biology (Cell-Molecular Biology)

Viral hemorrhagic septicemia virus (VHSv), a member of the family Rhabdoviridae, is a highly contagious fish virus responsible for large-scale fish die-offs worldwide. A new strain of VHSv, designated IVb, has recently spread to the Great Lakes threatening the tourism, sports fishing and fishery industries of the region. Research on virus-host interactions in VHSv infected fish has been mostly limited to population and ecological based studies while the molecular basis of the disease remains widely uncharacterized. To study virus-host interactions on a molecular level, we cloned four of the six VHSv genes including the matrix (m), nucleocaspid (n), non-virion (nv), and phosphoprotein (p) genes. Of primary interest, the M protein encoded by VHSv seems to share similar characteristics with the matrix protein of a related rhabdovirus. Comparable to the well-studied rhabdovirus vesicular stomatitis virus (VSV) M protein, ectopically expressed VHSv M inhibits promoter activity of both an interferon stimulated response element containing promoter and a constitutively active simian vacuolating virus 40 (SV40) promoter in Epithelioma Papulosum Cyprini (EPC) cells. Interestingly, real-time PCR data suggest mRNA levels remain steady, while protein levels decrease. Together, these data may suggest a similar interaction found in VSV between the M protein and the RAE1-NUP98 complex preventing nuclear export of mRNA. Furthermore, as seen in related rhabdoviruses, we have cell rounding after ectopic expression of the M protein for greater than 48h. Annexin V staining suggests these morphological changes in the cells are due to the induction of apoptosis. Together, these observations suggest two novel functions for the VHSv M protein. Future work will focus on determining the mechanisms utilized by VHSvM to inhibit transcription and induce apoptosis. Additionally, we identified the VHSv phosphoprotein as an inhibitor of both IFN gene activation and IFN-mediated activation of ISGs. (open full item for complete abstract)

Master of Science, The Ohio State University, 2011, Food Science and Technology

Viruses are the leading cause of foodborne illness worldwide (67%). Specifically, human norovirus (HuNoV) is the major foodborne virus. Fresh produce is often at high risk for norovirus contamination because it can be easily contaminated at both pre-harvest and post-harvest stages and it undergoes minimal or no processing. There is an urgent need to develop novel interventions to eliminate foodborne enteric viruses in fresh produce. High pressure processing (HPP), a non-thermal processing technology may provide a new approach to reduce the virus load in fresh produce and related products. In the present study, we systematically investigated the effectiveness of HPP on inactivating norovirus in aqueous medium, lettuce, strawberry, and fruit puree using murine norovirus (MNV-1) as a surrogate for noncultivable HuNoV. Approximately 5 log virus reduction was observed in all food items upon treatment at 400 MPa for 2 min at 4°C, demonstrating that HPP is effective in reducing the MNV-1 load in fresh produce. Moreover, our results showed that pressure, pH, temperature, and the food matrix affected the virus survival. MNV-1 was more effectively inactivated at 4°C than at 20°C. MNV-1 was also found to be more sensitive to high pressure at neutral conditions (pH 7.0) than at acidic conditions (pH 4.0). Taken together, these findings support the notion that HPP is a promising intervention to eliminate the norovirus risk in fresh produce and related products while the organoleptic and nutritional properties of these foods are affected to the minimal extent. To further evaluate the potential of HPP in inactivating viruses, we continued to investigate the effectiveness of HPP on the inactivation of human rotavirus (HRV), vesicular stomatitis virus (VSV), and avian metapneumovirus (aMPV). HRV represents a major foodborne virus other than HuNoV and is non-enveloped; VSV and aMPV are enveloped and their virion structures are strikingly different from those of MNV-1, HuNoV, and HRV (open full item for complete abstract)