DECONTAMINATION OF FOOD AND FOOD-PROCESSING SURFACES FROM NOROVIRUS BY COLD ATMOSPHERICPRESSURE GASEOUS PLASMA

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 viruci

DECONTAMINATION OF FOOD AND FOOD-PROCESSING SURFACES FROM NOROVIRUS BY COLD ATMOSPHERIC-

PRESSURE 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

i

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

ii

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

iii

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

iv

Table of Contents

List of Tables…… ……… V List of Figures……… ……… VII

GENERAL INTRODUCTION………. 1

CHAPTER 1: Literature review………. 9

1.1 NOROVIRUSES……… 10

1.1.1 History, taxonomy and classification and structure……… 10

1.1.2 Infection and clinical symptoms……… 16

1.1.3 Burden of HuNoVs on public health and economy……… 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……… 31

1.2.2 Atmospheric-pressure plasma sources……… 36

1.2.3 Plasma chemistry……… 42

1.2.4 Antimicrobial efficacy of cold atmospheric plasma on foods………… 44

1.2.5 Mechanisms of germicidal efficacy of CAP……… 59

CHAPTER 2: Virucidal effect of cold atmospheric gaseous plasma against feline calicivirus, a surrogate to human norovirus……… 71

CHAPTER 3: Inactivation of virus in solution by cold atmospheric pressure plasma: identification of chemical inactivation pathways………… 108

CHAPTER 4: Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus, a surrogate of human norovirus……… 159

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……… 200

CHAPTER 6: Factors affecting the virucidal efficacy of cold plasma against HuNoV as compared to its surrogate, feline calicivirus……… 241

CHAPTER 7: Comparison of cold atmospheric-pressure plasma and ultraviolet C irradiation on inactivation of feline calicivirus……… 263

CHAPTER 8: General Discussion……… 281

BIBLIOGRAPHY……… 288

APPENDIX 1.……… 325

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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

nozzle and the surface of the virus suspension……… 95

Table 2.2: Changes in the temperature of distilled water after exposure to various

types of atmospheric gaseous plasma jet for various exposure times…… 96

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………… 97

Table 2.4: Concentration of H2O2 formed in distilled water after exposure to

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

chromatography mass spectrometry……… 139

Table 3.3: pH values and hydrogen peroxide concentration obtained after 2 min

plasma exposure of 100 µl distilled water……… 140

Table 3.4: Reported half-life (t ½) of RONS Lifetimes are approximate as are

influence by exact solution composition……… 141

Table 3.5: Concentrations of nitrite, nitrate and hydrogen peroxide in water after

direct exposure to various plasmas for 0 and 30 minutes……… 142

Table 3.6: Virucidal effects of RONS generated and their concentrations………… 143 Table 4.1: Forward and reverse primers with size and annealing temperatures…… 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

vi

Table 4.3: Unique peptide fragments containing oxidized amino acids in

CAP-exposed 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

Table 5.3: The full design, experimental and predicted responses of RCCD……… 228 Table 5.4: Analysis of variance (ANOVA) of RCCD……… 229 Table 5.5: Verification of the second order polynomial model (Equ 4) using

random level-combinations of the four CAP parameters: operational

power (X 1 ), air flow rate (X 2 ), exposure time (X 3) and exposure distance

Table 6.1: Oligonucleotides for TaqMan-based FCV and NoV RT-qPCR used in

Table 6.2: D values and estimated times for 4 log10 reduction and complete

reduction (5.83 log10) of FCV in presence of fecal impurities under APMA wet exposure on stainless steel surface……… 259

2D-Table 7.1: Inactivation constant (k), D-value (for CAP) or DID (for UV), and

estimated times (for Cap) or dose (for UV) for 4 log10 reduction and

complete reduction (5.39 log10) of FCV under dry and wet exposure to

CAP and UVC on stainless steel surface……… ………… 275

vii

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

viii

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

Figure 3.5: a) Inactivation of FCV by singlet oxygen using photosensitized Rose

Bengal (RB) for different concentrations of RB and light exposure

durations.…… 148

Figure 3.6: Assessment of virucidal activity of H2O2 and chemically generated

hydroxyl radicals mimicking plasma conditions of Ar+0.27% water and

Figure 3.7: Virucidal activity of chemically generated peroxynitrous acid against

FCV at different concentrations……… 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 2 min for addition of the virus to the solution

before the treatment (direct exposure) and at different delay times after

Figure 3.10: Virus inactivation activity (bar plot) of plasma treated NTE buffer

solution by Ar+ 1% O2 plasma for 2 min 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 2 min 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 2 min for addition of the virus to the solution before the treatment (direct exposure) and at different delay times after the

Figure 3.13: One-dimension SDS-PAGE (4-15 % gradient gel) picture of Ar+1%O2

plasma-exposed FCV proteins (15 s and 2 min 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

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

ix

Figure 4.5: One dimensional SDS-PAGE (4-15 % gradient gel) image of

CAP-exposed FCV proteins (15 s and 120 s vs control)……… 194

Figure 4.6: Representative LC-MS/MS annotated spectrum (top) with alignment

Figure 4.7: Space-filling model of the structure of the major capsid protein (VP1) and

Figure 4.8: Crystal structure of A/B dimer of the major capsid protein (VP1) using

electrical and air inputs A) without enclosure and B) with enclosure

attached to the discharge electrode……… 231

Figure 5.2: Recombinant plasmid used to construct standard curves for norovirus

Figure 5.3: Reduction in FCV titer as a function to CAP exposure time on dry and

wet stainless-steel surface at 10slm air flow rate, 4mm exposure

distance, and 10.3W plasma discharge power……… 233

Figure 5.4: Reduction of FCV titer as a function to CAP exposure time on wet

stainless-steel surface at different plasma generation powers………… 234

Figure 5.5: Reduction of FCV titer as a function to (A) air flow rate power, and (B)

Figure 5.6: Response surface plot and contour diagram for FCV inactivation by CAP

as a function to discharge power and exposure time at the central levels

of air flow rate (15slm) and exposure distance (6mm)……… 236

Figure 5.7: Response surface plot and contour diagram for FCV inactivation by

CAP as a function to exposure distance and air flow rate at the central levels of discharge power (10.5W) and exposure time (3min)………… 237

Figure 5.8: A) Effect of exposure distance using 2D-APMA with enclosure on FCV

coated stainless steel discs B) Efficacy of 2D-APMA with enclosure against FCV as a function of exposure time……… 238

Figure 5.9: Effect of discharge power, as a function to the exposure time, on FCV

coated stainless steel discs using 2D-APMA with enclosure at 16.4 slm air flow rate, 10cm exposure distance, and exposure time……… 239

Figure 5.10: Efficacy of CAP against HuNoV GII-4 on wet stainless-steel discs and

Romaine lettuce leaves samples at the operational conditions (12.9 W discharge power, 10 cm exposure distance, 16.4 slm air flow rate, andwet exposure) as function to exposure time……… 240

x

Figure 6.1: Measurement of CAP inactivation of FCV by cell culture titration vs

measurement of CAP inactivation of the same FCV sample by

Figure 6.2: Exposure time dependent virucidal efficacy of CAP exposure against

FCV prepared in pure distilled water and in fecal extract ……… 261

Figure 6.3: CAP Inactivation of HuNoV GII-4 (A) and FCV (B) on stainless steel

discs at high, medium and low initial titers…….……… 262

Figure 7.1: Schematic diagram of the 2D-APMA setup including sample platform,

electrical and air inputs (A) Top view (B) and side view (C) of the

Teflon block with plastic shoulders for holding samples in indirect

Figure 7.2: Picture of the entire UVC system (A) Top view (B) and side view (C) of

the Teflon block with plastic shoulders for holding samples in indirect

Figure 7.3: Efficacy of dry and wet direct-exposure of UVC against FCV on stainless

steel discs and regression analysis……… 278

Figure 7.4: Efficacy of dry and wet direct exposure of CAP against FCV on stainless

steel discs Each point is the average value of triplicate samples……… 279

Figure 7.5: Inactivation of FCV on stainless steel discs by direct and indirect

GENERAL INTRODUCTION

Estimates of the global burden of foodborne diseases indicate that consumption of contaminated food results in one illness per 10 people and leads to 420,000 deaths every year (WHO, 2015) Of the known etiologies, human norovirus (HuNoV) is the leading cause of both sporadic cases and outbreaks of acute gastroenteritis due to foodborne diseases in the US and worldwide (CDC, 2016; Ahmed et al., 2014) About 48% of all foodborne disease outbreaks due to a single known cause reported to the Centers for Disease Control and Prevention from 2009 to 2012 were attributed to HuNoV (CDC, 2016) In the European Union, HuNoV was responsible for 97 of 104 strong-evidence

foodborne viral outbreaks that occurred in 2012 (EFSA, 2014) Globally, HuNoV is

responsible for 685 million gastroenteritis cases including 200 million cases among children

<5 years old (CDC, 2016) The annual global economic burden of HuNoV was estimated

to be $4.2 billion in direct health system costs and $60.3 billion in societal costs (Bartsch

et al., 2016) In the United states, HuNoV is one of the top five pathogens with respect to the total cost of foodborne illnesses It causes 19-21 million cases of acute gastroenteritis annually in the U.S including 1.7–1.9 million outpatient visits, 400,000 emergency room visits, 56,000-71,000 hospitalizations, and 570-800 deaths, mostly among young children (CDC, 2016)

Human NoV belongs to the family Caliciviridae and is a well-known cause of

“winter-vomiting disease” or “stomach-flu” (ECDC, 2013) It transmits mainly by close personal contact with an infected person or through the fecal-oral route when a person consumes contaminated food or water Infection can also occur by touching contaminated surfaces, objects, or substances (CDC, 2016)

Fresh produce and leafy vegetables are the most commonly implicated foods in HuNoV outbreaks Between 1998 and 2005, green salad, lettuce, fruits, and vegetable-based HuNoV outbreaks represented 24%, 5.1%, 3.2%, and 2.3% of all produce-based outbreaks, respectively (DeWaal and Bhuiya, 2007) Among 364 HuNoV outbreaks reported in the USA between 2001 and 2008 with a single and simple food implicated, the most frequent implicated commodities were leafy vegetables (33%), fruits/nuts (16%), and mollusks (13%) Among 191 (52%) of these outbreaks, contamination during preparation

or service was more frequent (85%, n = 162) than during food production or processing (15%, n = 29) (Hall et al., 2012b) Between 2004 and 2012, the United States and European Union reported a total of 223 and 108 fresh produce-associated outbreaks, respectively This high number of outbreaks linked to fresh produce was explained by the high risk of surface cross-contamination of such foods through contact with fecally contaminated soil and irrigation water or due to the low sanitation in post-harvest processing environments through infected handlers and/or contaminated equipment including cutting boards, knives, and working surfaces (Koopmans et al., 2004)

Contemporary life style and a consumer desire to eat foods with high nutritive value and high-quality attributes has increased the demand for fresh produce and leafy vegetables (Callejón et al, 2015) Sales of fresh produce increased by two figures in the past few years and is expected to continue growing over the next decade In addition, packaged salad is the second- fastest-selling item in U.S following bottled water (Bhagwat, 2006) This has led to an increased risk of HuNoV outbreaks, which has necessitated the emergence of so-called non-thermal technologies, referring to preservation methods that are effective at ambient or sub-lethal temperatures, thereby minimizing the negative effects of thermal

processing on nutritional and quality attributes of food (Misra et al., 2011) Unfortunately, studies on a variety of foods have revealed that non-thermal and minimal food processing (freezing, cooling, salting, and mild heat treatment) have low inactivation efficiency against foodborne viruses (Baert et al., 2009; FAO/WHO, 2008; Hirneisen et al., 2010, Mormann et al., 2010) This represents a critical barrier to ensuring the safety of fresh produce and leafy vegetables The development of novel and efficient non-thermal technology allowing for simple and effective viral decontamination of foods and food contact surfaces is urgently needed to protect the public from foodborne illnesses

Some of the novel non-thermal technologies e.g., ionizing radiation, UV treatment, pulsed light, and high hydrostatic pressure have showed notable antiviral and antibacterial activities but have their own limitations For instance, food irradiation, although among the high potential and most versatile food preservation technologies, encounters unfavorable public perception and hence its development and commercialization has been hampered (Pereira and Vicente, 2010) High hydrostatic pressure has notable efficacy against foodborne pathogens, but it can change the rheological properties of the food products (Ahmed et al., 2003) High hydrostatic pressure is effective in decontamination of high acidic foods, contrary to low acid, shelf-stable products because of the limitations in killing spores (Pereira and Vicente, 2010) Also, it requires complex equipment and the operating system is batch processing rather than in-line (Karakasiliotis et al., 2006)

The benefits of UV treatment in comparison to other methods of disinfection are clear The process does not require the addition of chemicals and no heat is released during the treatment Compared to other processes, there is low effect on color, flavor, odor, and

pH of food However, a potential problem of using short-wave UV light is that it can

damage human eyes, and prolonged exposure can cause burns and skin cancer in humans (Bintsis,2000; Shama, 1999), which is a concern among industry workers In addition, under certain treatment and storage conditions, the treated pathogen (either bacteria or viruses) may be able to repair the defects produced in their genomes by UV treatment and can then revive themselves and create a risky condition (Hijnen et al., 20016)

Pulsed light [short and intense pulses of light in the Ultraviolet to Near Infrared (UV–NIR) range] is also considered an emerging, non-thermal technology capable of reducing microbial populations on the surface of foods and food-contact surfaces However, poor penetrating power of light, requirement for transparent and surface smoothness of the product to be treated, and high investment costs are the limitations of

this method (Palmieri and Cacace, 2005; Pereira and Vicente, 2010)

The objective of this study was to examine cold atmospheric-pressure gaseous plasma (CAP) as a new non-thermal tool to decontaminate food and food contact surfaces Cold plasmas are ionized gases with neutral net charge which are referred to as “the fourth state of matter” Cold plasmas are generated by high electric fields which accelerate electrons in the gas The energetic collision of electrons with the surrounding gas leads to ionization, dissociation of molecules and the formation of photons (UV), ions, free electrons, and a cocktail of reactive species depending on the process gas used, including atoms (O and N), molecules (e.g., O3, H2O2, and HNO2), and radicals {OH•, NO•, and singlet oxygen [O2(a1∆g)]}, which all have antimicrobial properties (Graves, 2012; Bruggeman and Locke, 2013)

In the last few decades, plasma produced in low pressure gases has been used to achieve major advancements in the semiconductor industry In recent years, technological progress has allowed the creation of cold plasmas in a variety of geometries and configurations at atmospheric pressure This has enabled surface treatment applications of heat sensitive materials and even biological matter and fresh foods The generation of particularly high amounts of reactive species, ions and UV produced by these room temperature plasmas create excellent decontamination capabilities

Cold plasma technology has also attracted the attention of food researchers due to its biocidal effects against spoilage microorganisms and bacterial pathogens at ambient air environment without the use of heat The use of CAP is especially advantageous for inactivating bacteria on the surfaces of fresh foods such as vegetables, fruits, meats, nuts, poultry, and eggs (Azharonok et al., 2009; Deng et al., 2006; Fernandez et al., 2013; Gurol

et al., 2012; Liu et al., 2010; Noriega et al., 2011); and for decontamination of food preparation surfaces However, only a handful of studies are available on the use of cold plasma against viruses including influenza and parainfluenza viruses, adenovirus, corneal herpes simplex virus type 1, and MS-2 bacteriophage (Alekseev et al., 2014; Wu et al., 2015; Zimmermann et al., 2011)

Only three published reports are available on the efficacy of CAP against foodborne viruses Bae et al (2009) inactivated murine norovirus (MNV) and hepatitis A virus (HAV)

on meat samples using nitrogen-based gliding arc plasma setup Ahlfeld et al (2015) observed a significant reduction in HuNoV GII-4 on plastic surface by exposure to air-based plasma generated by a dielectric barrier discharge setup Niemira et al (2015) reported significant decrease in the titer of Tulane virus (TV) and MNV on blueberries by

exposure to air-based gliding arc-plasma The availability of only three reports on the use

of CAP for viral decontamination of food and food-contact surfaces shows that this technology is still in its infancy

There is a real need for enhancing CAP technology to be more cost effective since the cost of CAP technology is one of the major challenges facing its adoption in the food industry (Niemira et al., 2012) Designing new plasma generation setups that use low electric power and only air as a feed gas are suggested since these two items are the determining factors on the cost-effectiveness of CAP to be used on an industrial scale

(Niemira et al., 2012) Therefore, we conducted this research to study the in vitro and in

situ virucidal activity of CAP against HuNoV and its surrogates for decontamination of

food and food-contact surfaces to use this non-thermal technology for enhancing food safety and to protect consumers from the hazards of foodborne viruses

Specific aims:

Aim 1: In vitro testing the virucidal activity against FCV as a surrogate of human NoV

using radio frequency plasma jet setup generating plasma from four gas admixtures

Aim 2: Study the factors affecting virucidal activity of CAP

Aim 3: Understanding the mode of action by which CAP inactivates the virus:

Aim 3A: Identification of the chemical inactivation pathways lead to inactivation

of the virus in solutions

Aim3B: Understanding the biological impact of plasma species of the virus

Aim 4: In situ evaluation and optimization of virus inactivation using a unique

two-dimensional air-based plasma microdischarge array (2D-APMA) that fits surface treatments

Aim 5: Validation of the virucidal effect against HuNoV on real food and food-contact

CHAPTER 1: Literature review

1.1 NOROVIRUSES

1.1.1 History, taxonomy and classification and structure

In 1929, without knowing the causative agent, illness due to a human norovirus (HuNoV) was described as “winter vomiting disease” because of its primary symptom of vomiting (Zahorsky, 1929) At that time, cases of gastroenteritis that could not be attributed

to a bacterial agent were termed as “acute nonbacterial gastroenteritis of unknown etiology” (Greening and Cannon, 2016) In 1968, an outbreak of winter vomiting disease occurred in Bronson elementary school in Norwalk, Ohio (USA) and four years later, using immune electron microscopy on infectious stool filtrates, Kapikian et al (1972) discovered the viral etiology beyond this disease (Figure 1.1) That time the virus got the name of the place where it was first discovered ‘Norwalk virus’ (NV) (Greening and Cannon, 2016) This was the first evidence of a viral etiology for human diarrheal disease Although, noroviruses remained largely unrecognized until about 20 years ago because of the technical difficulty of their detection and because it mostly causes short-lived and mild illness, so it was frequently unreported to public health authorities (Greening and Cannon, 2016) Later, other viruses with a similar morphology were discovered and grouped together as ‘small round structured viruses’ (SRSVs) In the 1990’s, with the advent of molecular techniques such as polymerase chain reaction, further typing and classification

of the SRSVs were made possible; the SRSVs were shown to be members of the

Caliciviridae family (Jiang et al., 1993; Lambdenet al, 1993; Loncke, 2016) The name

"norovirus" (Norovirus for the genus) was approved by the International Committee on Taxonomy of Viruses (ICTV) in 2002 (Mayo, 2002; ICTV, 2006)

Figure 1.1: Immune electron microscopy image of Norwalk Virus from an infected stool

filtrate Bar = 100 nm (Kapikian et al., 1972)

Taxonomically, Norovirus (formerly known as Norwalk-like viruses), Vesivirus,

Lagovirus, Nebovirus and Sapovirus (the former Sapporo-like viruses) are the five genera

that Caliciviridae family consists of Human enteric viruses are contained in Norovirus and

Sapovirus genera with the same names in addition to a number of viruses causing enteric

diseases in animals, such as murine and canine noroviruses (Greening and Cannon, 2016; Robilotti et al., 2015)

Noroviruses are small in virion diameter (28-35 nm), non-enveloped viruses Their genome is composed of a linear, single-stranded positive-sense RNA (+ssRNA) that is ~7.6

kb in length (Buesa and Rodriguez-Díaz, 2016) The genome is polyadenylated at the 3’ end A major feature of the genome of calicivirus (including noroviruses) is the absence of

a methylated cap structure or a ribosomal entry site (IRES) at its 5’ end Instead, the 5’ end of the viral RNA genome is covalently linked to a small viral protein genome (VPg)

It is essential for the infectivity of the RNA (Rahman et al., 2003) and for other circoviruses (Daughenbaugh et al., 2003) It plays also an important role in translation of the norovirus RNA (Buesa and Rodriguez-Díaz, 2016) The genome of noroviruses consists of three open reading frames (ORFs), ORF-1, ORF-2, and ORF-3, encoding eight viral proteins (Figure 1.2) except murine norovirus (MuNoV) which contain one more open reading frame (ORF4) The structural components of the virion [i.e viral protein 1 (VP1) and viral protein

2 (VP2)] are encoded in ORF-2 and ORF-3 (Karst et al., 2014;Robilotti et al., 2015)

The Norovirus genus encompasses at least six distinct genogroups (GI to GVI) and

40 genotypes (Robilotti et al., 2015) Recently, Vinjé (2015) proposed a tentative genogroup (GVII) that infects canines (Figure 1.3) Depending on the amino acid diversity

of the 3 ORFs, the RNA-dependent RNA polymerase (RdRp) and VP1 encoding regions,

or the VP1 encoding region alone, genogroups of Norovirus are defined and classified The current 6 genogroups were distinguished based on the divergence in their VP1 proteins Genogroups I and II (GI and GII) contain the majority of NoVs that are contagious to humans (Buesa and Rodriguez-Díaz; 2016) Human NoVs within one genogroup share at least 60% amino acid sequence identity of the major capsid protein VP1

Each genogroup can be subdivided into “genetic clusters” To be within a specific genetic cluster, NoV strains should share at least 80% of VP1 amino acid sequence identity with the cluster’s reference strain (Hutson et al., 2004) The cluster is defined based on the virus name by a digit located after the Latin number of the genogroup For instance, NoV GII.4 resembles NoV strains from genetic cluster 4 of genogroup II (Loncke, 2016) An overview of all NoVs genogroups and genetic clusters is shown in Figure 1.3 The NoVs that are human infectious are usually referred to as human noroviruses (HuNoV) Genotype

2 (GII), mostly GII.4, is the most predominant NoV strain causing human viral gastroenteritis worldwide Norovirus human infections are caused also by genogroup 1 (GI), and genogroup 4 (GIV) to a very limited extent which has some genotypes infecting pigs (Vinjé, 2015; Robilotti et al., 2015) The most recent study confirmed global spread

of norovirus GII.17, Kawasaki 308 variant (Chan et al., 2016) This new variant has two characteristic amino acid insertions in the most surface-exposed antigenic region of the major capsid protein 1 (VP1) This raises concern about the global spread of this variant and its replacement of GII.4 variants because these changes alter the antigenic properties

of the virus or the virus–host cell binding preference (de Graaf et al., 2014; Chan et al., 2015; Chan et al., 2016)

Figure 1.2: Genome structure of NoVs The genome is comprised of a linear, positive-sense RNA, 7.6 kb in length, covalently linked

to the viral protein genome (VPg) at the 5’ end and polyadenylated at the 3’ end There are three open reading frames (ORFs), designated ORF-1, ORF-2, and ORF-3, encoding 8 viral proteins ORF-1 encodes six non-structural (NS) proteins that are proteolytically processed

by the virally encoded cysteine proteinase (Pro) ORF-2 and ORF-3 encode the structural components of the virion, major viral protein

1 (VP1) and minor viral protein 2 (VP2), respectively The VP1 protein has shell (S) and protruding (P) domains The P domain is further subdivided into P1 stalk domain and hypervariable P2 domain ORF 4 (only in murine NoV genomes) encodes virulence factor

1, or VF1

VP2

S       P2      P1 VP1

VF1

AAAn

NS1/2 p48

(aa296 & other P2 residues) virulence determinant

ORF4 

in MuNoV only

NS1/2 (p48)P Nterm (amino terminal protein) NS7 (RdRp) RNA‐dependent RNA polymerase P1 Protruding domain 1

Figure 1.3: Classification of noroviruses into 7 genogroups (GI to GVII) based on amino acid sequence diversity in the complete VP1

capsid protein To build the phylogenetic tree, capsid sequences from 105 strains representing the spatial and temporal sequence diversity

of noroviruses from diverse geographic regions across the world were selected Viruses belonging to GI, GII, and GIV infect humans, except GII.11, GII.18, and GII.19 viruses, which infect porcine species, and GIV.2 viruses, which infect canine species GII.15 viruses, which have been detected only in humans, form a tentative new genogroup (dotted circle) GIII viruses infect cows and sheep, GIV.2 infects canines, GV.1 and GV.2 infect mice and rats, respectively, and GVI and GVII infect canine species GII.4 viruses (arrow) are responsible for the majority of norovirus infections worldwide The scale bar reflects the number of amino acid substitutions per site (Vinjé, 2015)

The capsid of mature virions of HuNoVs composed mainly from one major structural protein: viral protein 1 (VP1) The VP1-protein is composed of three domains; (i) an N-terminal arm (NTA), (ii) a conserved shell domain (S), (iii) and a protruding domain (P) The latter is divided into P1 and P2 subdomains; the P1 subdomain exists in two polypeptide segments: pps-1 and pps-2 while the P2 subdomain (the outmost protein

of capsid) is inserted between pps-1 and pps-2 (Prasad et al.,1999) The HuNoV capsid consists of 180 VP1arranged in 90 VP1 protein dimers with icosahedral symmetry Such structure forms hollows or cup-like structures on the virus capsid surface Figure 1.4 shows the capsid structure of HuNoV as modeled by the help of cryo-electron microscopy and x-ray crystallography (Prasad et al., 1999) Caliciviruses share the same VP1-protein capsid structure presented in Fig 1.4 (Ossiboff et al., 2010)

1.1.2 Infection and clinical symptoms

Human noroviruses are highly contagious to human The 50% infectious dose of HuNoV (GI.1 strain) in humans was estimates as little as 18 particles (Teunis et al., 2008) and as high as 2800 virus particles (Atmar et al., 2013) The clinical symptomes of infection develop within 12-48 h of exposure and ususally include vomiting and/or non-bloody diarrhea, nausea, abdominal pain and general malaise (Greening and Cannon, 2016) Dehydration is a common complication that can particularly affect the children and elderly The symptoms of HuNoVs are generally mild, transient and usually resolve within 24-60 hours Although the infection with HuNoVs does not show any long-term sequelae, it might lead to death in infected infants and elderly Therefore, hospitalization may be required in

Figure 1.4: Capsid structure of HuNoVs It has been solved by cryo-electron microscopic reconstruction to 22A° (top, surface

representation; bottom, cross-section) and by x-ray crystallography to 3.4A° The NV VLPs have 90 dimers of capsid protein (left, ribbon diagram) assembled in T ¼ 3 icosahedral symmetry Each monomeric capsid protein (right, ribbon diagram) is divided into an N-terminal arm region (green) facing the interior of the VLP, a shell domain (S-domain, yellow) that forms the continuous surface of the VLP, and a protruding domain (P-domain) that emanates from the S-domain surface The P-domain is further divided into subdomains, P1 and P2 (red and blue, respectively) with the P2-subdomain at the most distal surface of the VLPs Adapted by Hutson

et al (2004) for the structure modeled by Prasad et al (1999) and Bertolotti-Ciarlet et al., (2002)

some cases During and after the illness (up to 28 days or longer), the virus particles are excreted in vomit and feces (Atmar et al., 2008)

Immunity against the infecting strain of HuNoVs is normally developed following stimulated production of gut and serum antibodies However, this immunity is strain-specific, short-lived, and does not confer protection against future infection Hence, re-infection with a different strain can occur and people are likely to be re-infected many times during their lifetimes because of the genetic variability of HuNoVs (Greening and Cannon, 2016) The association between susceptibility of infected persons to HuNoVs and histo-blood group antigens (HBGAs) expression on their gastrointestinal tract‘s mucosal epithelial surface was reported (Atmar et al., 2011; Hutson et al., 2002; Lindesmith et al., 2003; Tan and Jiang, 2010) The HuNoV GI.1 (Norwalk virus)-susceptible persons (secretors) are those who can express HBGAs on their gut mucosal epithelial and its secretions including saliva while “non-secretors” are the individuals who do not express HBGAs on their gut mucosa and they are resistant to symptomatic infection The HBGAs work as receptors or co-receptors for virus attachment and entry into cells (Atmar et al., 2011; Hutson et al., 2002; Lindesmith et al., 2003; Tan and Jiang, 2010) Also, susceptibility to infection with HuNoV GII-4 was found to be associated to the expression

of HBGAs (Frenck et al., 2012; Tan and Jiang, 2010) On the other hand, evidences revealed that susceptibility to infection by other HuNoVs genotypes and expression of HBGAs are independent (Lindesmith et al., 2005; Murakami et al., 2013) In addition, susceptibility to infection with HuNoVs might be affected by inherited factors other than HBGAs (Robilotti et al., 2015)

1.1.3 Burden of HuNoVs on public health and economy

In the United states HuNoVs are on average responsible for 19–21 million illnesses, 1.7–1.9 million outpatient visits, 400,000 emergency department visits, 56,000–71,000 hospitalizations, and 570–800 deaths (Hall et al., 2013) The highest rates of deaths associated with HuNoVs occur in adults aged ≥65 years (Hall et al., 2012a) while children aged <5 years recorded the highest rates of HuNoVs-associated medical care visits (Gastañaduy et al., 2013; Hall, 2016; Lopman et Al., 2011) In 2010, the estimated annual cost of HuNoVs in the US was $5.8 million (Scharff, 2010) In 2012, Scharf estimated the total annual burden of HuNoVs on the US economy is approximately $2.9 million using

an enhanced model for calculation (Scharff, 2012)

Globally, HuNoVs are responsible for 685 million cases of gastroenteritis including

200 million cases among children <5 years old (CDC, 2016) One-fifth of all diarrhea cases

in the world are associated with HuNoVs with similar prevalence in both children and adults Over 200,000 deaths annually are caused by HuNoVs in developing countries (Lopman et al., 2016) The estimated annual global economic burden of HuNoV is $4.2 billion in the direct health system costs and $60.3 billion in societal costs (Bartsch et al., 2016)

1.1.4 Transmission routes, point of infection, and implicated food

The HuNoVs are ideal infectious agents because of their very low infectious dose, genomic variability and constant evolving, short-time immunity, and the multiple transmission routes (Loncke, 2016) In addition, three factors facilitate the transmission of HuNoVs (i) the viral shedding by the infected person for prolonged duration even after the end of

symptoms, (ii) Fecal shedding of HuNoVs during asymptomatic infections, and (iii) the high persistence of HuNoVs on environmental surfaces and in water and foods (Mathijs

et al 2012)

The transmission routes of HuNoVs are classified into: (i) direct transmission from person-to-person through the fecal-oral route or by aerosolized vomit, (ii) indirect transmission through consumption of fecally-contaminated food or water, or after contact with contaminated surfaces including high-touch surfaces (i.e door handles, faucets, utensils) (Greening and Cannon, 2016) The schematic overview (Figure 1.5) summarizes the most important HuNoV transmission routes

The primary routes of HuNoVs transmission depend on the characteristics of the community settings in which the infection occurs (Table 1.1) Although the variability of HuNoVs transmission routes, person-to-person (non-foodborne) is the dominant transmission route (Kroneman et al 2008) The HuNoV outbreaks caused by person-to-person transmission route mostly occur in semi-closed community settings (e.g hospitals, cruise ships, day-care centers, and military settings) However, the primary cases in HuNoV outbreaks often have a food or water borne cause (Mathijs et al., 2012) While most reported norovirus outbreaks are non-foodborne, HuNoVs are responsible for ~ 50%

of all foodborne outbreaks in U.S caused by a single known pathogen (Gould et al., 2013; Hall et al., 2014) making it a leading cause of foodborne disease outbreaks The food products can be contaminated by HuNoVs due to the contact with fecal material or vomit

at any point along the farm-to-fork continuum However, in more than 90% of reported outbreaks the contamination happens during final preparation and service (Baert et al., 2011; Hall et al., 2014)

Figure 1.5: Schematic overview of the transmission routes of human and animal infective NoVs Solid and dashed arrows indicate

proven and hypothetical transmission routes, respectively The thickness of the arrows is related to the likeliness of the transmission route (Mathijs et al., 2012)

Contamination of foods by HuNoVs can occur at a pre-harvest level, or at a harvest level (Mathijs et al., 2012) Most of the food products that can be contaminated at the pre-harvest level are fresh produce and shellfish (Baert et al 2011; Lowther et al 2012; Mattison et al 2010; Stals et al 2011) Fresh produce can be contaminated at the pre-harvest stage by polluted irrigation water or contaminated organic fertilizers (Wei and Kniel 2010) and shellfish can be contaminated if grown in fecal-contaminated water (Lowther et al 2008) At post-harvest level, contamination of food products can happen

post-at any point during harvesting, processing, preparing, and packaging of the food (Moe 2008; Todd et al 2009) Infected food handlers have been confirmed to play a major role

in transmission of HuNoVs to food products at post-harvest stages (Baert et al 2009; Widdowson et al 2005)

Table 1.1 Primary transmission routes for noroviruses by setting and by characteristics of

the settings (Hedberg, 2016)

Setting

Primary Transmission Route

Characteristics of Setting That Favor Transmission Route

Resident food handlers provide extended source of contamination during outbreaks

Institutional Person-to-person,

environmental

Resident population with many opportunities for environmental contamination and repeated exposures

An average of 365 foodborne outbreaks of HuNoVs were reported annually to CDC from 2001 to 2008 In 85% of the reported foodborne outbreaks, the identified point of contamination was at the point of service (Hall et al., 2012b) The number of foodborne norovirus outbreaks reported to CDC from 2009-2012 declined to an average of 252 per year However, contamination at the point of service was identified in 92% of outbreaks,

in which one specific food item was implicated (Hall et al., 2014) It was found during these time periods that infected food handlers were the source of contamination for most

of foodborne norovirus outbreaks at the point of service (53% of outbreaks from

2001-2008 and 70% of outbreaks from 2009-2012) as estimated by (Hall et al., 2014)

1.1.5 Role of food and food-contact surfaces in foodborne HuNoV

infection and outbreaks

Fresh produce and leafy vegetables are the most commonly implicated foods in HuNoV outbreaks Several types of fresh produce (e.g green vegetable salads, lettuce, raspberries, cabbage, kimchi, and raw frozen fruit mixes) have been responsible for disease outbreaks after being contaminated by polluted water or by virus-infected food handlers (Table 1.2) Between 1998 and 2005, NoV-contaminated green salads, lettuce, fruits, and vegetables contributed to 24%, 5.1%, 3.2%, and 2.3% of all produce-based outbreaks, respectively (Dewaal and Bhuiya, 2007) Leafy vegetables were the implicated commodity in 33% of the HuNoV outbreaks between 2001 and 2008 in the United States (Hall, 2012) Of the 223 fresh fruit and vegetable-implicated HuNoV outbreaks that occurred in the US between

2004 and 2012, salads and leafy vegetables were implicated in 159 (71%) of them (Callejón

et al., 2015) This is due to the high risk of surface cross-contamination of such foods

Table 1.2: Foodborne outbreaks of NoV transmitted by fresh produce from 2005 to 2016

Adapted from Goyal and Aboubakr, (2016)

Implicated food Virus

(genotype)

Year and Location (location of origin if international)

No of Cases

Reference

Cabbage kimchi NoV (GI.3) 2011, Korea 451 Cho et al., 2014

Frozen raspberries NoV 2005, France 75 Cotterelle et al., 2005

Frozen raspberries NoV 2005, Denmark 466 Korsager et al., 2005

Frozen raspberries NoV 2006, Sweden 43 Hjertqvist et al., 2006

Frozen raspberries NoV (GI.4) 2009, Finland

(Poland) 200 Maunula et al., 2009

Frozen raspberries NoV (GII.4,

GII.b, GII.7, GI.4)

2009, Finland (Poland)

Lettuce (salad and

260 Ethelberg et al., 2010

Salad (Mixed) NoV (GII.4) 2007, U.K 34 Showell et al., 2007

Salad buffet

vegetables NoV (GI.3) 2007, Sweden 413 Zomer et al., 2010 Salad vegetables NoV (GII.4) 2007, Japan 23 Oogane et al., 2008

Salad (Fresh cabbage

and dried radish)

NoV (GII.4) 2008, Korea 131 Yu et al., 2010

Australia 46 Coutts et al., 2017

2004) Discharge of inadequately treated sewage might contaminate the surface and ground water supplies that are used for irrigation (Borchardt et al., 2011) The virus particles transferred to plants through irrigation water may remain viable throughout the distribution and shelf-life of these food commodities (El-Senousy et al., 2013)

Food-contact surfaces play also a key role in HuNoV infection and outbreaks Many studies confirmed the transmission of viruses between hands, environmental surfaces, and food (Bidawid et al., 2004; D’Souza et al., 2006) Work surfaces and utensils can be contaminated with HuNoVs via contaminated hands or foods (Grove et al., 2015) For example, an investigation of HuNoV outbreak found that 29% of kitchen samples and 53%

of bathroom samples were positive for norovirus (Boxman et al., 2011) Contamination of environmental surfaces contributes in HuNoV transmission and infection in institutional settings where resident populations have many opportunities for repeated exposures to contaminated environmental surfaces (Hedberg, 2016) Also, exposure to contaminated bathroom surface was initially found to be a point source of the outbreak (Repp et al., 2013) Another point source outbreak was eating food that was carried in a reusable grocery bag that had been stored in a bathroom used by a person with norovirus-like illness (Repp and Keene, 2012)

1.1.6 Uncultivability and infectivity determination methods of HuNoVs

In environmental and food virus control studies, the discrimination between infectious and noninfectious viral particles is crucial Methods depend on the observation of viral cytopathic effect (CPE) on cell cultures [i.e 50% tissue culture infectious dose (TCID50)

or plaque assays (for viruses that form plaques)] are the gold standard for virus infectivity estimation (Hamza et al., 2011) In which, confluent host cell lines, prepared in several wells of 96-well plates (for TCID50) or 6-well plates (for plaque assay) are infected by 10-fold serial dilutions of the sample After 2 to 7 days of incubation at 37°C under 5% CO2, the development of CPE is observed under an inverted microscope Based on the number

of wells per dilution that show CPE, the virus titer is calculated using one of two formulas (Karber, 1931; Reed and Muench, 1938)

Although frequent attempts by scientists during last few decades, they were unable

to develop a reliable in vitro cultivation method of HuNoV on cell cultures and they failed

to find a simple animal model that supports HuNoV replication Twenty-six cell lines have been tested for growing HuNoV but virus propagation was unsuccessful even with addition

of digestive enzymes and cell differentiation-inducing supplements (insulin, DMSO or butyric acid), or using different inoculation methods (Duizer et al., 2004) In 2005, Malik

et al (2005) evaluated 19 cell lines from different animal sources for susceptibility to HuNoV infection No evidence of HuNoV growth was observed in any one of them even after five blind passages (Malik et al 2005) On the other hand, HuNoV successfully replicate by transfection of its RNA into human hepatoma Huh-7 cells However, a complete viral infection, and viral spread from HuNoV-transfected cells to nạve cells does not occur (Guix et al 2007) A 3-dimentional organoid model of human small intestinal

epithelium has been reported as the first in vitro cell culture method that shows strong

evidence of infection by HuNoV (Straub et al 2007) There was no follow up experiment

to that work until 2011 when the same research group reported HuNoV infectivity to 3-D

model of large intestinal epithelium (Straub et al., 2011) However, this work has not been repeated or verified by other labs and was not enough to provide a simple cell culture method for growing HuNoV and titrating its infectivity on cell culture to be used routinely

in labs Jones et al (2015) reported replication of HuNoV in an in vitro human B cell culture system with commensal bacteria as an infection cofactor, but the system needed further development due to the modest viral output Most recently, multiple strains of HuNoV have been cultivated successfully in stem cell–derived, non-transformed human intestinal enteroid monolayer cultures Bile was required for strain-dependent HuNoV replication (Ettayebi et al., 2016) Despite this outstanding progress, the method is still not

in routine use

In the absence of a simple and reliable in vitro cell culture method for the growth and titration f HuNoV, the use of animal models was suggested In 2010, seronegative chimpanzees inoculated with the HuNoV did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar

to that observed in humans (Bok et al., 2010) Humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV (Taube et al., 2013) A gnotobiotic piglet model was proposed and used to estimate the effect of High Hydrostatic Pressure on HuNoV inactivation (Lou et al., 2015) Although these findings hold promise, the use of animal models suffers from many drawbacks e.g., these models

do not provide accurate log reduction data and their cost is very high Molecular technique such as real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the only available method for detection and titration of HuNoVs in vitro Because

of their very low detection limits (theoretically, a single genome copy per RT-PCR reaction), RT-PCR-based methods are the method of choice for detecting HuNoVs in foods and environmental samples since the concentration of HuNoVs in contaminated samples

is usually low Virus particles with defective capsid having intact or partially intact genomes, and even degraded viral RNA from non-infectious particles that might have intact PCR’s target sequence region (usually short, about 100 nucleotides among the whole genome 7600 nucleotides) So, this method is usually unable to discriminate between infectious and non-infectious virus particles and usually underestimates the titer of inactivated virus in virus-control studies (Knight et al., 2013) Therefore, many modified molecular techniques, in addition to using HuNoV surrogates, have been suggested and used to counteract the problem of discrimination between infectious and non-infectious viruses

The HuNoV surrogates usually share pathological and/or biological characteristics with HuNoVs and are able to propagate in cell cultures For example, Feline calicivirus

(FCV) is a member of Caliciviridae family The primary sequence and genomic

organization of FCV in addition to its capsid structure are similar to HuNoV It grows perfectly in Crandell Reese Feline Kidney cells (CRFK) These features made FCV a frequently used surrogate virus in previous studies (e.g Aboubakr et al., 2014; Lee et al., 2012; Ossiboff et al., 2010; Sosnovtsev et al., 2005) Because FCV is transmitted via respiratory route unlike the gastro-intestinal route of HuNoV, it does not bind to the same receptors (HBGAs) as HuNoV In addition, FCV is sensitive to extremes in pH (low pH of 2.0 to 4.0 and high pH 10.0) unlike HuNoVs (Cannon et al., 2006) For these reasons, FCV

is not the most appropriate surrogate for HuNoV (D’Souza et al., 2016; Richards, 2012)