1 The Effect of Abiotic and Biotic Stress on the Internalization and Dissemination of Human 1 ...

36 Pages·2015·1.14 MB·English

AEM Accepted Manuscript Posted Online 8 May 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.00650-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 The Effect of Abiotic and Biotic Stress on the Internalization and Dissemination of Human
2 Norovirus Surrogates in Growing Romaine Lettuce
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5 Erin DiCaprioa, Anastasia Purgiantob*, Jianrong Lia#,
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7 Department of Veterinary Biosciences, College of Veterinary Medicinea, Department of Food loa
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8 Science and Technology, College of Food Agriculture and Environmental Sciencesb, The Ohio d

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9 State University, Columbus OH m

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12 Running Title: Stress and norovirus internalization in Romaine Lettuce .a
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15 #Address correspondence to Jianrong Li, [email protected] A
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16 *Present Address: Anastasia Purgianto, H.J. Heinz Company, Pittsburg, PA, USA il
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24 ABSTRACT
25 Human norovirus (NoV) is the major causative agent of fresh produce related outbreaks of
26 gastroenteritis; however the ecology and persistence of human NoV in produce systems is poorly
27 understood. In this study, the effects of abiotic and biotic stress on viral internalization and
28 dissemination of two human NoV surrogates (murine norovirus [MNV-1] and Tulane virus
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29 [TV]) in Romaine lettuce was determined. To induce abiotic stress, Romaine lettuce was grown w
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30 under drought and flood conditions that mimic extreme weather events, followed by inoculation a
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31 of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus d

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32 (LMV) to induce biotic stress followed by inoculation TV. Plants were grown for 14 days and m

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33 viral titer in harvested tissues was determined by plaque assay. It was found that drought stress tt
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34 significantly decreased the rate of both MNV-1 and TV internalization and dissemination. In a
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35 contrast, flood stress and biotic stress did not significantly impact viral internalization and .a
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36 dissemination. Additionally, the rate of TV internalization and dissemination in soil grown .
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37 lettuce was significantly higher than that of MNV-1. Collectively, these results demonstrated /
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38 that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and A
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39 leaves of Romaine lettuce grown in soil; (ii) abiotic stress (drought) but not biotic stress (LMV il
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40 infection) affect the rate of viral internalization and dissemination; and (iii) the type of virus 0
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41 affect the efficiency of internalization and dissemination. This study also highlights the need to b
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42 develop effective measures to eliminate internalized viruses in fresh produce. u
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48 IMPORTANCE
49 Human NoV is a leading causative agent of outbreaks associated with leafy greens. In
50 this study two human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV-1),
51 were found to be internalized by the roots of Romaine lettuce grown in soil with subsequent
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52 dissemination to the leaves. Drought stress was found to have a significant negative impact on w
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53 the rate of viral internalization and dissemination while flood conditions had no significant a
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54 effect. In addition, lettuce mosaic virus infection did not alter the rate of TV uptake by lettuce. d

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55 Finally, TV was more efficiently internalized and disseminated in lettuce compared to MNV-1. m

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56 This data indicates that the presence of human NoV in soil can lead to internalization of the virus tt
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57 in leafy greens and may contribute to the high instance of human NoV outbreaks associated with a
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58 these food commodities. .a
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70 INTRODUCTION
71 Human norovirus (NoV) is the major cause of non-bacterial gastroenteritis, contributing
72 to over 95% of the non-bacterial acute gastroenteritis worldwide and over 60% of all foodborne
73 illnesses reported annually in the US (1-4). High risk foods for human NoV contamination
74 include fresh produce, shellfish, and ready to eat foods. As individuals are increasingly striving
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75 to achieve healthier diets, the consumption of fresh produce has increased in recent years and w
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76 fresh produce is now recognized as a leading cause of foodborne illness in the US (5, 6). Human a
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77 NoV alone accounts for over 40% of the fresh produce-related illnesses reported each year in the d

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78 U.S. (1, 2, 4, 6-9). Human NoV has been attributed to outbreaks in many diverse types of m

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79 produce including: fresh cut fruit, lettuce, tomatoes, melons, salads, green onions, strawberries, tt
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80 blueberries, raspberries, salsa as well as many others (6, 7, 10-15). Human NoV is highly a
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81 infectious, resistant to common disinfectants, has a low infectious dose, and is highly stable in .a
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82 the environment which contributes to the high prevalence of foodborne outbreaks associated with .
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83 the virus and its presence and persistence in food commodities (3, 5, 7, 16-19). /
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84 Fresh produce can become contaminated with human NoV at any step from production to A
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85 processing. In a survey of human NoV point of contamination of specific food commodities il
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86 responsible for outbreaks from 2001-2008 in the US, nearly half of the outbreaks in leafy 0
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87 vegetables and fruits/nuts were associated with viral contamination that occurred during b
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88 preparation or service; only few outbreaks were traced to contamination during production and u
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89 processing (2). However, in large number of the outbreaks of human NoV in leafy vegetables
90 and fruits/nuts, the point of contamination could not be determined and it is possible that more
91 contamination occurred during production and processing than could be verified in this survey
92 (2). During production, virus-contaminated irrigation water, water for dilution of agrochemicals
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93 and fertilizers, and water for hydroponic cultures can all introduce virus to fresh produce (20-22).
94 Sewage contaminated irrigation water has been theorized to account for several human NoV
95 outbreaks in berries although not confirmed (11, 12). Producers use various water sources for
96 irrigation, including well water, river, and lake water, human NoV has been detected in surface
97 and also well water (23-30). Infected humans can shed 106-1011 virus particles per gram of feces
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98 and it has been shown that conventional wastewater treatment practices are not sufficient to w
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99 completely inactivate or remove viruses (31-35). Therefore, even treated water may harbor a
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100 human NoV and, when discharged, may contaminate sources of irrigation water. The use of d

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101 human NoV-contaminated irrigation water in the production of fresh produce poses a risk of m

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102 surface contamination of the food and also the potential for viral internalization into the produce tt
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103 during growth. a
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104 Human NoV is a member of the viral family Caliciviridae, and all viruses within this .a
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105 family are non-enveloped with a single-stranded, positive-sense RNA genome (9, 36-38). A .
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106 major hindrance to human NoV research is attributed to fact that the virus cannot be cultivated in /
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107 cell culture and lacks a small animal model. To date, much of the understanding of human NoV A
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108 molecular biology, pathogenesis, and environmental stability has come from the study of other il
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109 caliciviruses, human NoV surrogates (39-43). These surrogate viruses are cultivable in the 0
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110 laboratory and closely resemble human NoV in regards to size, genetic make-up, receptor b
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111 binding, cell tropism, and disease manifestations. Murine norovirus (MNV) has been used u
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112 extensively as a surrogate for the study of human NoV and is currently the only cultivatable
113 member of the genus Norovirus. MNV has the closest genetic relation to human NoVs compared
114 to other surrogate viruses, and also has a similar capsid structure, viral particle size, and buoyant
115 density as human NoVs (44). MNV has been shown to be more stable at low pHs when
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116 compared to other surrogates such as feline calicivirus (FCV), which indicates it may have
117 similar environmental stability as human NoV (40). However, MNV does not cause
118 gastroenteritis in mice and utilizes sialic acid for attachment to cells, which differs from human
119 NoV where histo-blood group antigens (HBGAs) are the cellular attachment moieties. Tulane
120 virus (TV) is a newly recognized surrogate for human NoV and is member of the genus
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121 Recovirus, family Caliciviridae (45). TV is closely related to human NoV in terms of genome w
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122 size and organization, viral capsid structure, and viral particle size (45). TV was isolated from a
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123 the stool of rhesus macaques, which implicates it as a cause of gastrointestinal disease. Further, d

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124 TV utilizes HBGAs as a cellular attachment molecule similar to human NoV (46). m

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125 Several studies have evaluated whether enteric viruses and their surrogates can be tt
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126 internalized in growing produce with varying results based on the virus used, plant growth a
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127 conditions, and plant varieties (47-54). However, no studies have systemically evaluated .a
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128 whether abiotic and biotic stresses on growing produce affect the level of enteric virus .
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129 internalization and dissemination. In this study, the internalization and dissemination of human /
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130 norovirus surrogates, MNV-1 and TV, in Romaine lettuce grown in soil was determined. The A
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131 plants were maintained under normal conditions, and drought and flood conditions to mimic il
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132 extreme weather events to determine whether abiotic stresses affect virus internalization. In 0
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133 addition, phytopathogen infection (lettuce mosaic virus) was introduced in lettuce to determine b
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134 whether biotic stress affected enteric virus internalization and dissemination. u
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139 MATERIALS AND METHODS
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141 Viruses and cell culture. Murine norovirus (MNV) was generously provided by Dr. Herbert W.
142 Virgin IV, Washington University School of Medicine and Tulane virus (TV) was a generous
143 gift from Dr. Xi Jiang at Cincinnati Children's Hospital Medical Center. MNV and TV were
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144 propagated in confluent monolayers of the murine macrophage cell line RAW 264.7 and the w
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145 monkey kidney cell line MK2-LLC (ATCC, Manassas, VA), respectively (47). RAW 264.7 a
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146 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, d

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147 Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), at 37°C under a m

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148 5% CO atmosphere. For growing MNV stock, confluent RAW 264.7 cells in T-150 flasks were tt
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149 infected with MNV at a multiplicity of infection (MOI) of 0.1. After 1 h incubation at 37°C, 15 a
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150 ml of DMEM with 2% FBS was added. The virus was harvested two days post inoculation (PI) .a
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151 by three freeze-thaw cycles and low speed centrifugation at 1000 × g for 30 min. MK2-LLC .
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152 cells were cultured in low serum Eagle’s minimum essential medium (Opti-MEM, Invitrogen), /
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153 supplemented with 2% FBS, at 37°C under a 5% CO atmosphere. For growing TV stock, MK2- A
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154 LLC cells in T-150 flasks were washed with Hank’s balanced salt solution (HBSS) and il
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155 subsequently infected with TV at an MOI of 0.1. After 1 h incubation at 37°C, 15 ml of Opti- 0
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156 MEM with 2% FBS was added. The virus was harvested day two PI and subjected to three b
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157 freeze-thaw cycles, followed by centrifugation at 1000 × g for 30 min to remove cellular debris. u
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159 Virus enumeration by plaque assay. MNV-1 and TV were quantified by plaque assay in RAW
160 264.7 and LLC-MK2 cells, respectively (47, 55). Briefly, cells were seeded into six-well plates
161 (Corning Life Sciences, Wilkes-Barre, PA) at a density of 2 × 106 cells per well. After 24 h
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162 incubation, RAW 264.7 and MK2-LLC cell monolayers were infected with 400 µl of a 10-fold
163 dilution series of MNV-1 or TV, respectively, and the plates were incubated for 1 h at 37°C with
164 gentle agitation every 10 min. The cells were overlaid with 3 ml of Eagle minimum essential
165 medium (MEM) containing 1% agarose, 2% FBS, 1% sodium bicarbonate, 0.1 mg of
166 kanamycin/ml, 0.05 mg of gentamicin/ml, 15 mM HEPES (pH 7.7), and 2 mM L-glutamine.
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167 After incubation at 37°C and 5% CO2 for 2 days, the plates were fixed in 10% formaldehyde. w
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168 The plaques were visualized by staining with 0.05% (w/v) crystal violet. The limit of detection a
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169 for viral plaque assay was determined to be 0.5 log PFU/ml. Viral titer was expressed as mean d
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170 log plaque forming unit (PFU)/ml ± standard deviation. m
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172 Determination of viral survival in conventional potting mix. 1 gram of Fafards 3B potting a
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173 mix was weighed and placed in a sterile petri dish. For the viral inoculated soils, 1mL of 1×107 .a
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174 PFU/ml of virus (MNV-1 or TV) was pipetted onto the soil (control soils received no virus). The .
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175 plates were sealed with parafilm and were maintained in the lab under light conditions of 12 h /
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176 light and 12 h dark, at 20°C and 50% relative humidity (RH). On Day 0 (immediately after A
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177 inoculation), Day 7, and Day 14 three samples from each group (MNV-1 inoculated, TV il
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178 inoculated, or uninoculated control) were processed for the determination of viral survival. 1g of 0
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179 soil contained in each petri dish was transferred to a sterile stomacher bag followed by the b
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180 addition of 10ml sterile diH O to each bag. Each soil sample was stomached for 2 min and then u
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181 the liquid was collected from each stomacher bag and transferred to a sterile 15ml tube. The
182 samples were centrifuged the for 30 min at 1000 × g to pellet soil particles and the supernatant
183 was transferred supernatant to new 15ml collection tube and the volume of the collected
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184 supernatant was recorded. Virus survival was determined using plaque assay and results were
185 reported as log PFU/g of soil.
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187 Plant cultivation under normal or extreme weather conditions. Seeds of Romaine lettuce
188 (Lactuca sativa) were planted in plug trays and grown under greenhouse conditions. Ten days
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189 after germination, plugs were transplanted into 4” pots containing Fafard’s 3B potting mix. w
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190 Thirty days after germination, plants were maintained in the lab under light conditions of 12 h a
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191 light and 12 h dark, at 20°C and 50-65% relative humidity (RH). The plants were separated into d

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192 4 groups: normal conditions receiving no viral inoculation (control), normal conditions receiving m

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193 viral inoculum, drought conditions receiving viral inoculum, and flood conditions receiving viral tt
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194 inoculum. The pots were kept in tray flats and trays were filled with water for delivery to lettuce. a
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195 For normal conditions the trays were filled every 5 days with 1000ml water. For drought .a
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196 conditions the trays were filled every 14 days with 1000ml water. For flood conditions the trays .
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197 were filled every 2 days with 2000ml. Following lettuce harvest, the percent moisture was /
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198 determined as described below. A
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199 il
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200 Virus inoculation, sample collection, and virus internalization. Plants were inoculated at the 0
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201 base of the shoot with 20ml of 1×107 pfu/ml virus, in two separate 10 ml treatments 2 h apart on b
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202 the same day. Following inoculation, the surface of the soil was covered with parafilm to limit u
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203 viral contamination of the aerial portions of the plant. At days 0 (before viral inoculation), 1, 3,
204 7, and 14 the leaves, shoots, and roots were harvested. All tissues were submerged in 2000ppm
205 chlorine following harvest to remove any virus present on the surface of the plant. Following
206 chlorine treatment, lettuce tissues were rinsed for 5 min in diH O and following diH2O rinse
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207 residual chlorine was inactivated by submersion of tissues in 0.25M sodium thiosulfate. The
208 shoot sample consisted of the aerial portion of the lettuce 3 inches above the soil line and 10g
209 samples (total 30-40g) of leaves were randomly selected from each plant for homogenization.
210 The samples were homogenized using liquid nitrogen and mortars and pestles. Homogenized
211 tissue was resuspended in 5ml phosphate buffered saline (PBS) and homogenates were
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212 centrifuged at1000 × g for 30 min to remove cellular debris. The virus containing supernatant w
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213 was transferred to a new collection tube for viral enumeration by plaque assay. a
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214 d

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215 Soil measurements. Soil samples from each time point were collected for pH, salinity, m

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216 conductivity, total dissolved solids (TDS), and percent moisture (%M) measurements. To tt
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217 determine the percent moisture of the soil samples, 1 g of soil was weighed and the wet weight a
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218 was recorded. The soil sample was then dried overnight in a 60°C drying oven followed by 1 .a
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219 hour incubation in a dessicator. The %M was then determined by dividing the dry weight by the .
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220 initial wet weight and then multiplying by 100. To determine the pH, salinity, conductivity, and /
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221 TDS, 1 g samples of soil were suspended in 10 ml of diH2O. The samples were vigorously A
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222 shaken for 2 min to disperse the soil in the water. Samples were allowed to settle at room il
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223 temperature for 1 hour. The measurements were taken using standardized pH (Fisher Brand, 0
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224 Accument® basic pH meter), salinity (Fisher Traceable® salinity meter), conductivity (Fisher b
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225 Traceable® conducitivity meter), and TDS (Fisher Traceable® conducitivity/TDS meter) meters u
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226 (56).
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228 Lettuce inoculation with lettuce mosaic virus (LMV). Seeds of Romaine lettuce (Lactuca
229 sativa) were planted in plug trays and grown under greenhouse conditions. Ten days after
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