Identification of Effective and Practical Thermal and Non-thermal Processing Technologies to Inactivate Major Foodborne Viruses in Oysters DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Elbashir Mohamed Araud Graduate Program in Comparative Veterinary Medicine The Ohio State University 2015 Ph.D. Examination Committee: Dr. Jianrong Li, Advisor Dr. Hua Wang Dr. Melvin Pascall Dr. Gireesh Rajashekara Copyright by Elbashir Mohamed Araud 2015 ABSTRACT Human enteric viruses, such as 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 major high risk foods for enteric viruses contamination. Currently, the ecology, bioaccumulation, and persistence of enteric viruses in shellfish are poorly understood. There is no standard method to effectively inactivate these viruses in seafood. Therefore, there is an urgent need to develop effective thermal and non-thermal processing technologies to eliminate virus hazard in seafood. The goals of this study are to: i) determine the natural bioaccumulation patterns of human enteric viruses in shellfish tissues, ii) to determine whether heat or high pressure processing (HPP) are capable of effectively inactivating enteric viruses in shellfish, iii) and to determine whether viruses can develop resistance to processing technologies. Live oysters (Grassostrea gigas) were cultivated in a tank containing 4 liters of synthetic seawater and were artificially contaminated with HuNoV GII.4, HuNoV surrogates (Tulane virus, TV; murine norovirus, MNV-1), HAV, or RV at level of 1 × 104 PFU/ml of TV, MNV-1, HAV, or 1 × 104 RNA copies/ml of HuNoV. At 24, 48, and 72 h post-inoculation, oysters were harvested, different portions of oyster tissue including gills, digestive glands, and muscles were isolated, and the presence of the viruses in each ii tissue was determined by plaque assay or real time PCR (RT-qPCR). It was found that all viruses were bio-accumulated to a high titer within the oyster tissues within 72 h; however, the pattern of the bioaccumulation varied for each individual virus. Caliciviruses (HuNoV, TV, and MNV-1) and HAV were localized in the stomach at a high level within the first 24 h, while RV was bio-accumulated to the highest level in gills after 24 h. In order to determine the thermal stability of the four cultivable viruses (TV, MNV-1, HAV, and RV), each virus was diluted in cell culture medium, and the kinetics of viral inactivation was determined at temperatures of 62, 72, and 80 ˚C. It was found that the biphasic reduction model was the best fit to describe the virus inactivation curves at 62 and 72 ˚C. Decimal reduction time (D-values) of the low and high thermal resistant fractions of the four cultivatable viruses ranged from 0.13 to 1.81 min, and from 1.26 to 7.29 s at 62 and 72˚C, respectively. In contrast, the Weibull distribution was the best fit for the inactivation curves at 80 ˚C, and the time to first log10 reduction (TFL- value) ranged between 0.46 and 32 s. Within the oyster tissues, the TFL at 80 ˚C ranged between 0.61 to 19.99 min. The four viruses can be ranked from the most heat resistant to the least stable as the following: HAV>RV>TV>MNV-1. At 80 ˚C, time required for complete inactivation of HAV, RV, TV, and MNV-1 in cell culture medium is 12 s, 10 s, 10 s, and 6 s, respectively. However, it required 4, 3, and 3 min at 80 ˚C to completely inactivate RV, MNV-1, and TVin oysters, respectively, HAV survived the treatment even after 6 min. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted whereas viral genomic RNA remained intact. However, a iii lethal dose of heat treatment was not sufficient to disrupt the receptor binding activity of HuNoV, HuNoV virus-like particles (VLPs), and TV. These data demonstrated that enteric viruses were efficiently bioaccumulated in oyster tissues and different viruses exhibited different distribution patterns. Although foodborne viruses have variable thermal stability to heat, 80˚C for 6 min was not sufficient to completely inactivate all the tested viruses in oysters. High pressure processing (HPP) is a promising non-thermal technology that inactivates foodborne viruses while maintaining the organoleptic properties of processed foods. However, one interesting observation for HPP inactivation of viruses is that different viruses are variable in their susceptibility to high pressure. To date, the HPP sensitivity of different virus strains in the same genus, species or serotype is not known. RVs are genetically diverse which makes it a good model to study the role of strain diversity in HPP inactivation of viruses. This study compared the baro-sensitivity of seven RV strains derived from four serotypes (G1: Wa, Ku, and K8, G2: S2, G3: SA-11 and YO, and G4:ST3) following high pressure treatment. It was found that RV strains showed varying responses to HPP based on the initial temperature and had different inactivation profiles. Ku, K8, S2, SA-11, YO, and ST3 showed enhanced inactivation at 4°C compared to 20°C. In contrast, Wa strain was not significantly impacted by the initial treatment temperature. Within serotype G1, Wa stain was significantly (p<0.05) more resistant to HPP compared to Ku and K8. Overall, the resistance of the human RV strains to HPP at 4°C can be ranked as Wa>Ku=K8>S2>YO>ST3 and in terms of iv serotype G1>G2>G3>G4. In addition, pressure treatment of 400 MPa for 2 min was sufficient to eliminate the Wa strain, the most pressure resistant RV, from oyster tissues. HPP disrupted virion structure, but did not degrade viral protein or RNA, providing insight into the mechanism of viral inactivation by HPP. Therefore, HPP is capable of inactivating RV at commercially acceptable pressures and the efficacy of inactivation is strain dependent. In general, microorganisms are capable of acquiring resistance under stress. However, whether enteric caliciviruses can develop resistance during thermal and nonthermal processing is not known. This study utilizes TV, an enteric primate calicivirus, as a surrogate for HuNoV, to screen for heat and high pressure (HPP) resistant strains. Wild type (WT) TV was subjected to either heat treatment at 70˚C for 6 s in culture medium for 5 passages or HPP at 300 MPa for 2 min at 20˚C for 3 passages. Viral plaques were purified after each heat or HPP treatment and were subsequently re- treated or re-pressured and the change in log reduction after each treatment cycle was determined by plaque assay. At each passage, individual plaques were isolated, and tested for potential heat or pressure resistance. It was found that TV gradually developed resistance under heat and HPP stress. It appears that TV more rapidly developed resistance to HPP compared to development of resistance to heat. At passage 3, plaques isolated from HPP treated viruses had 2-3 logs more resistance compared to wild type TV. At passage 5, plaques isolated following heat treatment had 1-3 logs more resistance compared to wild type TV. Interestingly, these heat or pressure resistant TVs had delayed v growth replication kinetics, delayed cytopathic effects, and smaller plaque size, suggesting that they were attenuated in cell culture. Finally, the entire genome of two heat resistant and two pressure resistant TVs were amplified by RT-PCR and sequenced. Interestingly, the majority of mutations were found in the major capsid protein (VP1) and the minor capsid protein (VP2) although some of mutations were located in RNA dependent RNA polymerase and the N-terminal domain of nonstructural polyprotein. Collectively, we demonstrated that TV can easily develop resistance under both heat and HPP, and mutations in the genome may be responsible for the resistance. In summary, these data demonstrated that: (i) enteric viruses can be efficiently bio- accumulated in oyster tissues; (ii) different viruses have different bioaccumulation patterns in oyster tissues; (iii) enteric viruses have variable thermal stability and resistance to heat treatment can be ranked as HAV (most resistant)>RV>TV>MNV- 1(most sensitive); (iv) thermal treatment at 80˚C for 6, 4, 3, and 3 min was sufficient to completely inactivate HAV, RV, TV, and MNV-1 in oyster, respectively; (v) RV strains derived from different serotypes had different inactivation profiles and the resistance of the RV strains to HPP can be ranked as Wa>Ku=K8>S2>YO>ST3 and in terms of serotype G1>G2>G3>G4; (vi) TV can easily develop resistance to both heat and HPP treatments and majority of mutations responsible for resistance phenotype are located in major and minor capsid proteins; and (vii) heat and pressure resistant TVs are attenuated in cell culture. The findings in this study will facilitate the development of effective thermal and non-thermal processing technologies to eliminate enteric viruses in shellfish vi and will facilitate the establishment of new standards for industry and regulatory agencies to improve seafood safety. vii Acknowledgments It is my pleasure to thank all the people who made this work possible. I would like to thank my advisor Dr. Jianrong Li for his support and guidance in the execution of my research and pursuit of a career in science. I appreciate him giving me the opportunity to advance my education and for fostering my ability to think critically. Besides my advisor, I would like to thank the rest of my dissertation committee: I would like to thank Dr.