Master of Science, The Ohio State University, 2018, Comparative and Veterinary Medicine

Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the U.S. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been seriously hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model. The goal of this study is to develop lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. To do this, a LAB bacteria strain (Lactococcus lactis) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector. Subsequently, a novel microencapsulation technology was developed to enhance the stability of LABs in low and high pH environments. To test whether LAB-based HuNoV vaccine is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1 either with or without microencapsulation. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. Live LABs or LAB DNA were found in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces at day 13 post-inoculation, respectively, and further increased at late time points. After challenge with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less or no HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.

Committee: Jianrong Li (Advisor); Prosper Boyaka (Committee Member); Melvin Pascall (Committee Member) Subjects: Microbiology; Virology

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)

Committee: Jianrong Li (Advisor); Hua Wang (Committee Member); Melvin Pascall (Committee Member); Gireesh Rajashekara (Committee Member) Subjects: Food Science; Microbiology; Veterinary Services; Virology

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

Human norovirus (NoV) is responsible for more than 95% of nonbacterial gastroenteritis and over 60% of foodborne illness each year in the US. However, it has been a challenge to conduct research on human NoV because it cannot be grown in cell culture and lacks a small animal model. Despite major efforts, there is no antiviral therapeutic or vaccine for human NoV. In addition, the survival of human NoV is poorly understood. Fresh produce is major high risk food for human NoV-related outbreaks of gastroenteritis because it can easily become contaminated at any point during pre-harvest and post-harvest stages of production. However the ecology, persistence, and interaction of human NoV in produce systems is poorly understood. Determining the mechanisms underlying human NoV fresh produce contamination can facilitate the development of effective preventative and control measures to limit human NoV outbreaks. The objectives of this research are to determine whether human NoV and its surrogates become internalized via the root of growing produce and disseminated to the edible portion of the plants grown in soil; to determine whether biotic and abiotic stress can affect the rate of internalization and dissemination of human NoV in fresh produce; and to determine whether non-thermal food processing technologies can effectively inactivate internalized viruses while maintaining the fresh quality of produce. Romaine lettuce grown in soil was inoculated with 2 × 108 plaque forming unit (PFU) of human NoV surrogates [Murine norovirus-1 (MNV-1); Tulane virus (TV)] via the roots of plants. Plants were grown for 14 days and leaves, shoots, and roots of each plant were harvested and homogenized and viral titer was determined by plaque assay. It was found that both MNV-1 and TV can efficiently be internalized via plant roots and disseminated to shoots and leaves of lettuce. At day 14 post-inoculation, the titer of MNV-1 and TV in leaves of lettuce plants reached a level of (open full item for complete abstract)

Committee: Jianrong Li PhD (Advisor); Melvin Pascall PhD (Committee Member); Mark Peeples PhD (Committee Member); Wondwossen Gebreyes (Committee Member) Subjects: Food Science; Virology

Master of Science, The Ohio State University, 2014, Food Science and Technology

Human norovirus (NoV) is responsible for more than 95% of outbreaks of acute non-bacterial gastroenteritis worldwide and over 50% of foodborne illnesses in the US. Despite significant health, social, and economical burden it causes, no vaccine or antiviral drug available for this virus. This is due to the fact that human NoV cannot be grown in cell culture and lacks a small animal model for pathogenesis study. Recent epidemiological studies showed that severe clinical outcomes including death are often associated with high-risk populations such as infants, children, the elderly, and immunocompromised individuals. There is an urgent need to develop an effective therapeutic agent for human NoV. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases such as rotavirus. In the present study, we developed an efficient approach to generate a high titer of human NoV-specific IgY in chicken yolks using recombinant vesicular stomatitis virus (rVSV-VP1) expressing human NoV capsid protein (VP1) as an antigen. We first demonstrated that rVSV-VP1 replicated efficiently in a chicken cell line and VP1 protein can be highly expressed by VSV vector. Subsequently, White Leghorn chickens were immunized with recombinant rVSV-VP1 by intramuscular route or combination of intramuscular and nasal drop route. After immunization, eggs were collected daily and IgYs were purified from each egg. It was found that the purified IgY strongly reacted with human NoV virus-like particles (VLPs) by both enzyme linked immunosorbant assay (ELISA) and Western blot. In addition, hens vaccinated by intramuscular route triggered significantly higher human NoV-specific IgY than by the combination of intramuscular and nasal drop route. At week 4 post-vaccination, human NoV-specific IgY reached 4.8 mg per yolk in intramuscular vaccinated group, which was approximately 3 times more than that in the combined vaccin (open full item for complete abstract)

Committee: Jianrong Li (Advisor); Xin Li (Committee Member); Melvin Pascall (Committee Member) Subjects: Food Science

Master of Science, The Ohio State University, 2012, Food Science and Technology

Fresh produce is a high risk food for human norovirus (NoV) contamination, because it can easily become contaminated at both the pre- and post-harvest stages of cultivation. Disease surveillance has shown that human NoV is attributed to 40% of all fresh produce related outbreaks reported each year in the U.S. However, the ecology, persistence, and interaction of human NoV and fresh produce are all poorly understood. Increasing outbreaks of viruses in fresh and fresh-cut vegetables and fruits give high urgency to understanding the interaction of human NoV with fresh produce in order to develop effective preventive measures. In this research, the attachment, uptake, internalization, and dissemination of human NoV and its surrogates (murine norovirus, MNV-1; and Tulane virus, TV) were evaluated. First, the attachment of human NoV surrogates to fresh produce was visualized using confocal microscopy. Purified human NoV virus-like particles (VLPs), TV, and MNV-1 were conjugated with biotin, and subsequently applied to either Romaine lettuce or green onion. The biotinylated virus particles were visualized by incubation with streptavidin coated Quantum Dots (Q-Dots 655), which emit fluorescence that can be viewed using a confocal microscope. It was found that all three surrogates attached to the surface of Romaine lettuce leaves and were found aggregating in and around the stomata. Similarly, human NoV VLPs, TV, and MNV-1 were found to attach to the surface of Romaine lettuce roots. In the case of green onions, human NoV VLPs were found between the cells of the epidermis of both the shoots and roots. However, TV and MNV-1 were found to be covering the surface of the epidermal cells in both the shoots and roots of green onions. The results indicate that different viruses vary in their attachment patterns to different varieties of fresh produce. A quantitative assessment of the level of attachment of a human NoV GII.4 strain, TV, and MNV-1 was executed using Romaine lettuce a (open full item for complete abstract)

Committee: Jianrong Li PhD (Advisor); Ken Lee PhD (Committee Member); Yael Vodovotz PhD (Committee Member) Subjects: Food Science; Virology

Doctor of Philosophy, The Ohio State University, 2007, Veterinary Preventive Medicine

Human noroviruses (HuNoV) are the leading cause of food- and water-borne non-bacterial gastroenteritis worldwide. In this study we delineated the local and systemic immune responses to a GII. HuNoV (HS66 strain) in gnotobiotic pigs and calves and evaluated the immune responses to HuNoV-HS66 virus-like particles vaccines adjuvanted with either ISCOM or mLT and also protection against the homologous virus in Gn pigs. In our study, low antibody titers and antibody-secreting cell (ASC) numbers were elicited in the HuNoV-HS66-inculated pigs, but 65% of the pigs seroconverted. A higher Th1 (high IL-12 but low and transient IFN-gamma), but also low Th2 (IL-4 and IL-10), low transient pro-inflammatory (IL-6) cytokine and delayed innate (IFN-alpha) responses were induced by the HuNoV-HS66 in the serum of the Gn pigs. Intestinal IFN-alpha and IL-12 (late) were also significantly elevated after infection. Higher numbers of Th1 (IL-12 and IFN-gamma) cytokine-secreting cells (CSC) were elicited when compared to Th2 (IL-4) and pro-inflammatory (IL-6) cytokines. The HuNoV-HS66 caused diarrhea and intestinal lesions, mainly in the proximal part of the intestine of one of the calves euthanized at PID 3. Viral shedding was detected from post-inoculation day 1-6 and 67% of the animals seroconverted with HuNoV-HS66-specific IgA and IgG antibodies. Both Th1 (IL-12 and IFN-gamma) and Th2/T-reg (IL-10) cytokines, but also pro-inflammatory (TNF-alpha) were induced in the serum and fecal samples of the HuNoV-HS66-inoculated calves. One-hundred percent seroconversion rate was observed in the vaccinated pigs, regardless of the vaccine regimen, and 100% of coproconversion was detected in the VLP+ISCOM pigs compared to 75% of the VLP+mLT group. However, only 57% of the control pigs shed virus post-challenge. The VLP+mLT vaccine induced a balanced Th1/Th2 response, whereas the VLP+ISCOM induced a more Th2 biased response, but both vaccine regimens induced high levels of protection against virus (open full item for complete abstract)

Committee: Linda Saif (Advisor) Subjects: Biology, Veterinary Science

Doctor of Philosophy, The Ohio State University, 2013, Food Science and Technology

Human norovirus (NoV) is the leading cause of acute non-bacterial gastroenteritis worldwide. Despite the significant health, emotional, and economic burden caused by human NoV, there are no vaccines or therapeutic interventions for this virus. This is due in major part to the lack of a cell culture system and an animal model for human NoV infection. Thus, a vector-based vaccine may be ideal for controlling this disease. The major capsid gene (VP1) of a human NoV was inserted into the VSV genome at the glycoprotein (G) and large (L) polymerase gene junction. Recombinant VSV expressing VP1 protein (rVSV-VP1) was recovered from an infectious cDNA clone of VSV. Expression of the capsid protein by VSV resulted in the formation of human NoV virus-like particles (VLPs) that are morphologically and antigenically identical to the native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 stimulated a significantly stronger humoral and cellular immune response compared to baculovirus-expressed VLP vaccination. These results demonstrated that that the VSV-based human NoV vaccine induced strong humoral, cellular, and mucosal immunity in a mouse model. To further improve the safety and efficacy of the VSV-based human NoV vaccine, the gene for the 72kDa heat shock protein (HSP70) was inserted into rVSV and rVSV-VP1 vectors as an adjuvant, which resulted in construction of recombinant VSV expressing HSP70 (rVSV-HSP70) and VSV co-expressing human NoV VP1 protein and HSP70 (rVSV-HPS70-VP1), respectively. At the same inoculation dose, both rVSV-HSP70-VP1 and rVSV-VP1 triggered similar levels of specific immunity, even though VP1 expression by rVSV-HSP70-VP1 was approximately five-fold less than that of rVSV-VP1. To compensate for the reduced VP1 expression levels, the inoculation dose of rVSV-HSP70-VP1 was increased five-fold or same dosage of rVSV-VP1 and rVSV-HSP70 was combined vaccinated. Mice (open full item for complete abstract)

Committee: Jianrong Li (Advisor); Hua Wang (Committee Chair); Mark Peeples (Committee Member); Steven Krakowka (Committee Member) Subjects: Food Science; Virology