DECONTAMINATION OF FOOD AND FOOD-PROCESSING SURFACES FROM NOROVIRUS BY COLD ATMOSPHERICPRESSURE GASEOUS PLASMA A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Hamada Abdelsattar Ahmed Metwally Aboubakr IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor: Dr Sagar M Goyal Dec, 2017 © Hamada Abdelsattar Ahmed Metwally Aboubakr, 2017 ACKNOWLEDGEMENTS First, I thank and offer praise to the almighty ALLAH who granted me the capacity, understanding, and passion for learning, which enabled me to accomplish this work He also facilitated seeking knowledge for me, which is one of the greatest goals of human life, as the Prophet Muhammad (peace be upon him) has advised I would like to express my sincere thanks, deepest appreciation and heartful gratitude to Dr Sagar M Goyal, Professor of Virology, Department of Veterinary Population Medicine, University of Minnesota, for his continuous scientific and moral support, constant assistance, useful advice, valuable criticism, encouragement and motivation, supervision and guidance throughout the course of this I also thank him for his patience and immediate responses to my research needs and questions My deepest appreciation, thanks, and sincere gratitude are also due to Dr Peter Bruggeman, Professor of Mechanical Engineering and Director of High Temperature and Plasma Laboratory, Department of Mechanical Engineering, University of Minnesota, for fruitful collaboration, wise supervision, interest and care, guidance, useful advice, valuable scientific suggestions and technical recommendations throughout the course of this work as well as during the preparation of manuscripts I am grateful and indebted to Dr Jim Collins, Professor of Pathology and former Director of Veterinary Diagnostic Laboratory, Department of Veterinary Population Medicine, University of Minnesota, for his constant motivation and encouragement, unlimited support and help, and scientific consultations during his role as a committee member, and specially for granting me a Research Assistantship by which I could accomplish this work My acknowledgment is due to Dr Fernando Sampedro, Associate Professor, Center for Animal Health and Food Safety and Department of Veterinary Population Medicine, University of Minnesota, for his valuable consultation and guidance throughout the course of this work during his role as a committee member My deepest thanks are to Dr Mohammed Youssef and the spirit of Dr Amr El-Banna, Professors of Food Science and Technology, Faculty of Agriculture, Alexandria University, Egypt, for sincere advice, support, recommendations, and encouragement and motivation they provided me during their role as former Advisers in my graduate studies at Alexandria University I thank Gaurav Nayak, Paul Williams, and Urvashi Gangal from the Department of Mechanical Engineering, University of Minnesota, with whom I have done all the cold plasma treatments and plasma diagnostic studies I learnt a great deal of cold plasma techniques and plasma diagnosis from them Without their fruitful collaboration, I would not have been able to accomplish this work i My thanks to Dr Sunil Kumar Mor, Assistant Professor, Dr Anibal Armien, Professor, Veterinary Diagnostic Laboratory, University of Minnesota, for the scientific guidance and technical help during performing some experiments of the thesis work In addition, my thanks to Wendy Wiese and Lotus Solmonson, staff members of the virology laboratory, and to Nhungoc Ti Luong and Dr Yishan Yang who provided technical help in performing some experiments in this study I express my love to my sincere wife “Walaa Hamada” for her endless love and care, which empowered me to rise above all the hardships and overcome the challenges that we faced together I also, express my love to my father Abdelsattar Abuobakr and my mother, Soad Hamada, who planted in my heart the passion of learning and for facing all hardships and challenges regardless of how big they appear to be and to be stubborn to reach my goals Without their support and sacrifices, I would not have reached this success I appreciate the funding provided by the Agriculture and Food Research Initiative of the USDA’s National Institute of Food and Agriculture, grant number # 2017-67017-26172 and the funding from the Egyptian Ministry of Higher Education and Scientific Research, which was granted to me during the era of Prof Dr Mohamed Morsi, the first legitimate and democratically elect President in the history of Egypt I witness that he, unprecedently increased and dedicated a huge budget from Egyptian money for education, scientific research, and scientific mission for young scientists Finally, I would like to thank the Egyptian people whose money partially supported the expenses of my PhD The money that would have changed the lives of many poor people I owe them too much and I hope I will be able to pay them back in the future ii DEDICATION This work is dedicated to the spirit of my dear father, great mother, to my children Haneen, El-Baraa and Omar, and very specially to my beloved wife, Walaa, for her love and support that made this achievement possible iii Table of Contents List of Tables…… ………………………………………………………………… V List of Figures…………………………………… ……………………………… VII GENERAL INTRODUCTION…………………………………………………… CHAPTER 1: Literature review…………………………………………………… 1.1 NOROVIRUSES………………………………………………………………… 1.1.1 History, taxonomy and classification and structure…………………… 1.1.2 Infection and clinical symptoms……………………………………… 1.1.3 Burden of HuNoVs on public health and economy…………………… 10 10 16 19 1.1.4 Transmission routes, point of infection, and implicated food………… 19 1.1.5 Role of food and food-contact surfaces in foodborne HuNoV infection and outbreaks………………………………………………………… 23 1.1.6 Uncultivability and infectivity determination methods of HuNoVs… 25 1.2 COLD ATMOSPHERIC-PRESSURE GASEOUS PLASMA……………… 1.2.2 Atmospheric-pressure plasma sources………………………………… 1.2.3 Plasma chemistry……………………………………………………… 1.2.4 Antimicrobial efficacy of cold atmospheric plasma on foods………… 1.2.5 Mechanisms of germicidal efficacy of CAP………………………… CHAPTER 2: Virucidal effect of cold atmospheric gaseous plasma against feline calicivirus, a surrogate to human norovirus………………………… CHAPTER 3: Inactivation of virus in solution by cold atmospheric pressure plasma: identification of chemical inactivation pathways………… CHAPTER 4: Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus, a surrogate of human norovirus……… CHAPTER 5: Inactivation of human norovirus GII-4 and feline calicivirus on stainless-steel and Romaine lettuce using a novel 2D air-based plasma micro-discharge array……………………………………… CHAPTER 6: Factors affecting the virucidal efficacy of cold plasma against HuNoV as compared to its surrogate, feline calicivirus…………… CHAPTER 7: Comparison of cold atmospheric-pressure plasma and ultraviolet C irradiation on inactivation of feline calicivirus…………………… CHAPTER 8: General Discussion………………………………………………… BIBLIOGRAPHY………………………………………………………………… 31 36 42 44 59 71 108 159 200 241 263 281 288 APPENDIX 1.……………………………………………………………………… 325 iv List of Tables Table 1.1: Primary transmission routes for noroviruses by setting and by characteristics of the settings…………………………………………… 22 Table 1.2: Foodborne outbreaks of NoV transmitted by fresh produce from 2005 to 2016……………………………………………………………………… 24 Table 1.3: A Summary of research on inactivation of bacteria in foods by cold plasma…………………………………………………………………… Table 1.4: A Summary of research on inactivation of molds and yeasts in foods by cold plasma.……………………………………………………………… Table 1.5: A Summary of research on inactivation of viruses by cold plasma……… Table 2.1: Exposure levels and their corresponding distances between plasma jet nozzle and the surface of the virus suspension………………………… Table 2.2: Changes in the temperature of distilled water after exposure to various types of atmospheric gaseous plasma jet for various exposure times…… Table 2.3: Changes in pH of distilled water, MEM, and NTE buffer after exposure to the four types of plasma at low and high exposure distances………… Table 2.4: Concentration of H2O2 formed in distilled water after exposure to various types of plasma………………………………………………… Table 2.5: Decimal reduction times (D-values) and estimated time for 4-log10 reduction and complete reduction (5.83 log10) of FCV in different plasma conditions……………………………………………………… Table 2.6: Comparison of known virucidal effects of several non-thermal foodprocessing techniques…………………………………………………… Table 3.1: Rate constants for reactions of scavengers with relevant reactive species 48 53 57 95 96 97 98 99 100 138 Table 3.2: Estimated concentrations of muconic acid as determined by liquid chromatography mass spectrometry…………………………………… 139 Table 3.3: pH values and hydrogen peroxide concentration obtained after plasma exposure of 100 µl distilled water……………………………… Table 3.4: Reported half-life (t ½) of RONS Lifetimes are approximate as are influence by exact solution composition………………………………… Table 3.5: Concentrations of nitrite, nitrate and hydrogen peroxide in water after direct exposure to various plasmas for and 30 minutes……………… Table 3.6: Virucidal effects of RONS generated and their concentrations………… Table 4.1: Forward and reverse primers with size and annealing temperatures…… 140 141 142 143 187 Table 4.2: Peptide fragments of trypsin-digested FCV capsid protein detected by OrbitrapVelos-MS system using the protein gi|692348862 as a reference sequence………………………………………………………………… 188 v Table 4.3: Unique peptide fragments containing oxidized amino acids in CAPexposed FCV capsid protein…………………………………………… 189 Table 5.1: Oligonucleotides for TaqMan-based NoV RT-qPCR used in this study… 226 Table 5.2: Coded levels and actual values of the variables in rotatable central composite design (RCCD)……………………………………………… 227 Table 5.3: The full design, experimental and predicted responses of RCCD……… Table 5.4: Analysis of variance (ANOVA) of RCCD……………………………… Table 5.5: Verification of the second order polynomial model (Equ 4) using random level-combinations of the four CAP parameters: operational power (X1), air flow rate (X2), exposure time (X3) and exposure distance (X4)……………………………………………………………………… Table 6.1: Oligonucleotides for TaqMan-based FCV and NoV RT-qPCR used in this study………………………………………………………………… 228 229 230 258 Table 6.2: D values and estimated times for log10 reduction and complete reduction (5.83 log10) of FCV in presence of fecal impurities under 2DAPMA wet exposure on stainless steel surface………………………… 259 Table 7.1: Inactivation constant (k), D-value (for CAP) or DID (for UV), and estimated times (for Cap) or dose (for UV) for log10 reduction and complete reduction (5.39 log10) of FCV under dry and wet exposure to CAP and UVC on stainless steel surface…………………… ………… 275 vi List of Figures Figure 1.1: Immune electron microscopy image of Norwalk Virus from an infected stool……………………………………………………………………… 11 Figure 1.2: Genome structure of NoVs…………………………………………… 14 Figure 1.3: Classification of noroviruses…………………………………………… 15 Figure 1.4: Capsid structure of HuNoVs…………………………………………… 17 Figure 1.5: Schematic overview of the transmission routes of human and animal… 21 Figure 1.6: Pictorial representation of the four states of matter…………………… 32 Figure 1.7: Paschen ionization curves obtained for helium (He), neon (Ne), argon (Ar), hydrogen (H2), and nitrogen (N2) VB (breakdown voltage, in volts) as a function of pd (pressure × distance, in torr cm−1) Assumes parallel plate electrodes………………………………………………… 35 Figure 1.8: Point-to-plate electrode arrangements for generating a negative dc corona discharge………………… …………………………………… 38 Figure 1.9: Typical electrode arrangements for DBDs……………………………… 39 Figure 1.10: Principle designs for APPJs…………………………………………… 41 Figure 1.11: Categories of UV irradiation according to International Organization for Standardization……………………………………………………… 65 Figure 2.1: Schematic diagram of the CAP system including the plasma jet, treatment of samples, and the electrical and gas inputs…………………………… 101 Figure 2.2: The effect of changes in Ar-based plasma generation power on virucidal activity…………………………………………………………………… 102 Figure 2.3: The effect of plasma exposure distance and gas mixture type on virucidal activity………………………………………………………… 103 Figure 2.4: The effect of virus-suspending media on virucidal activity of CAP…… 104 Figure 2.5: Virucidal effect of liquid hydrogen peroxide against FCV…………… 105 Figure 2.6: The effective FCV-lethal time of plasma exposure…………………… 106 Figure 2.7: The survival kinetic curves of FCV exposed to Ar, Ar+1% O2, Ar+1% air, and Ar+0.27% water plasmas showing the slopes of regression lines using the liner portions of the survival curves…………………… 107 Figure 3.2: Schematic diagram of the experimental plasma setup and treatment condition.………………………………………………………………… 144 Figure 3.2: Inactivation of FCV suspended in distilled water using Ar, Ar+1% O2, Ar+ 1% air and Ar+ 0.27% H2O cold gaseous plasma as a function of plasma exposure time…………………………………………………… 145 Figure 3.3: Effect of various scavengers on the virucidal activity of Ar+1% O2 plasma against FCV suspended in (a) sterile distilled water and (b) NTE buffer…………………………………………………………………… 146 vii Figure 3.4: Effect of various scavengers on the virucidal activity of (a) Ar plasma and (b) Ar+1% air plasma against FCV suspended in sterile distilled water…………………………………………………………………… Figure 3.5: a) Inactivation of FCV by singlet oxygen using photosensitized Rose Bengal (RB) for different concentrations of RB and light exposure durations.…… Figure 3.6: Assessment of virucidal activity of H2O2 and chemically generated hydroxyl radicals mimicking plasma conditions of Ar+0.27% water and Ar+1% O2 in NTE buffer……………………………………………… Figure 3.7: Virucidal activity of chemically generated peroxynitrous acid against FCV at different concentrations………………………………………… 147 148 149 150 Figure 3.8: Virucidal activity of gas phase NO species against FCV suspended in distilled water and hydrogen peroxide solutions at different pH values… 151 Figure 3.9: Virus inactivation activity (bar plot) of plasma treated distilled water by Ar+ 1% O2 plasma for for addition of the virus to the solution before the treatment (direct exposure) and at different delay times after the treatment…………………………………………………………… 152 Figure 3.10: Virus inactivation activity (bar plot) of plasma treated NTE buffer solution by Ar+ 1% O2 plasma for for addition of the virus to the solution before the treatment (direct exposure) and at different delay times after the treatment………………………………………… 153 Figure 3.11: Virus inactivation activity (bar plot) of plasma treated distilled water by Ar+ 1% air plasma for for addition of the virus to the solution before the treatment (direct exposure) and at different delay times after the treatment……………………………………………… 154 Figure 3.12: Virus inactivation activity (bar plot) of plasma treated distilled water by Ar plasma for for addition of the virus to the solution before the treatment (direct exposure) and at different delay times after the treatment……………………………………………………………… 155 Figure 3.13: One-dimension SDS-PAGE (4-15 % gradient gel) picture of Ar+1%O2 plasma-exposed FCV proteins (15 s and vs control)…………… 156 Figure 3.14: Proposed reaction scheme of 1O2 with His after plasma exposure…… 157 Figure 3.15: a) Amino acid sequence of FCV Capsid protein.………………… 158 Figure 4.1: Schematic diagram of the plasma jet including sample treatment and electrical and gas inputs………………………………………………… 190 Figure 4.2: CAP exposure effect on FCV infectivity………………………………… 191 Figure 4.3: Transmission electron microscopic images of FCV…………………… 192 Figure 4.4: Quantification of capsid-destruction as a function of CAP-exposure time 193 viii Niemira BA (2012 b) Cold plasma 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DE NOVO: parent mass error tolerance 20 ppm, fragment mass error tolerance 0.1 Da, enzyme trypsin, fixed modification carbamidomethyl cysteine (57.0215), variable modification oxidized methionine (15.9949), maximum variable mods per peptide, report peptides 325 C- PEAKS Database Search: parent mass error tolerance 50 ppm, fragment mass error tolerance 0.1 Da, enzyme trypsin, precursor monoisotopic, trypsin enzyme, maximum missed cleave sites 2, non-specific cleavage at both ends, fixed mods, variable mods and max number of mods per peptide the same as DE NOVO settings, protein reference database NCBI non-redundant feline calicivirus (taxID 11978)from 12/10/14 merged with NCBI RefSeq Felis (taxID 9682)from 10/12/12 and contaminants database (http://www.thegpm.org/crap/), false discovery rate estimation Enabled D- PEAKS PTM: max number of PTM’s per peptide 3, PTM’s selected: Deamidation NQ (0.9840), Oxidation DFKNPRYHW (15.9949), Formylation KPRY (27.9949), Acetylation N-term (42.0106), Dihydroxy MFKPRWY (31.9898), Carbamylation K, N-term (43.0058), Cysteic acid C (47.9847), HisImid 13.98 H, Pyro-glutamic acid from Q N-term (-17.0265), Aminotyrosine Y (15.0109), Kynurenin W (3.9949), Oxidation to Nitro WY (44.9851), Hydrated imidazolone H (31.9898), 2-Amino-N-formylureido-succinamic acid H (47.9847), Tryptophandione W (29.9742); SPIDER homology match (search for amino acid modifications and de novo sequence homology) invoked We exported peptide summaries with the following PEAKS® parameters: 1% peptide FDR, Protein score (10logP) 20 and unique peptide 3- Quantification Analysis: quantification via RIPPER Optimized Parameters RIPPER allows analysts to optimize analyte information extraction from MS1 data The RIPPER optimized analyte extraction parameters were: Group MZ Distance = 005, Group RT Distance = 120, Minimum Charge to Process = 2, Maximum Charge to Process = 4, 326 Minimum Mass to Process = 100, Minimum Monoisotopic Peak Cluster Size = 2, Minimum Retention Time to Process = 0.0, Maximum Retention Time to Process = 99999, Signal to Noise Ratio = 3.0, Minimum Number of XIC Peaks = 5, XIC mz range = 0.02, XIC Consecutive Peak mz Tolerance = 0.003, XIC Consecutive Peak RT Tolerance = 20 327