AEM Accepted Manuscript Posted Online 29 January 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.03573-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Thermal inactivation of enteric viruses and bioaccumulation of enteric foodborne viruses in live oysters (Crassostrea virginica)
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Elbashir Arauda, Erin DiCaprioa, Yuanmei Maa, Fangfei Loub, Yu Gaod,e, David Kingsleyd, John
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H. Hughesc, and Jianrong Lia#
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Department of Veterinary Biosciencesa, Department of Food Science and Technologyb,
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Department of Molecular Virology, Immunology and Medical Geneticsc, Department of
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Extensiond, The Ohio State University, Columbus, OH 43210; South Centerse, The Ohio State
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University, Piketon, OH 45661; fU.S. Department of Agriculture, Agricultural Research Service,
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Food Safety and Intervention Technologies Research Unit, James W.W. Baker Center, Delaware
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State University, Dover, DE
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Running Title: thermal inactivation of viruses
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#Corresponding author:
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Department of Veterinary Biosciences
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College of Veterinary Medicine
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The Ohio State University
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311 Goss Laboratory
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1925 Coffey Road,
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Columbus, OH, 43210
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Tel: 614-688-2064 1
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Fax: 614-292-6473
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Email:
[email protected] 27 28
Mention of trade names or commercial products in this publication is solely for the purpose of
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providing specific information and does not imply recommendation or endorsement by the U.S.
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Department of Agriculture (USDA). The USDA is an equal opportunity provider and employer
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Abstract
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Human enteric viruses are one of the main causative agents of shellfish associated
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outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stability
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of the most predominant enteric viruses were determined in both tissue culture and in oyster
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tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates [murine norovirus (MNV-
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1); Tulane virus (TV)], hepatitis A virus (HAV), and human rotavirus (RV) were bioaccumulated
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to a high titer within the oyster tissues with different patterns of bioaccumulation for each virus.
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We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses
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can be ranked from the most heat resistant to the least stable as: HAV>RV>TV>MNV-1. In
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addition we found that oyster tissues provided a protective effect to the virus during heat
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treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was
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treated at 80°C for increasing time intervals. It was found that the integrity of viral capsid was
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disrupted whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to
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complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding
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activity of TV as determined by the porcine gastric mucin magnetic bead binding assay.
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Similarly, HuNoV VLPs and a HuNoV GII.4 strain retained some receptor binding ability
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following heat treatment. Although foodborne viruses have variable heat stability, 80oC for >6
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min was sufficient to completely inactivate most enteric viruses in oysters, with the exception of
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HAV.
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Introduction
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Approximately 7.6-14.5 million illnesses in the US are attributed to the consumption of
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contaminated seafood each year, and enteric viruses are responsible for more than 50% of these
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cases (1). In a review of available epidemiological evidence, human norovirus (NoV) and
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hepatitis A virus (HAV) were the leading viruses associated with shellfish, accounting for 83.7%
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and 12.8% of outbreaks, respectively (2). The type of shellfish most frequently associated with
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viral outbreaks was oysters, which was the vehicle in 58.4% of outbreaks (2). In some regions,
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human enteric viruses are rather ubiquitous in harvested shellfish. Keller et al (3) showed that
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100% of shellfish samples collected from Vitória Bay, Espírito Santo, Brazil were positive for
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rotavirus (RV) and adenovirus. However, only 80% of the growing water samples were positive
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for these pathogens. The viral titers were 400 times higher in the shellfish samples compared to
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the levels in the growing water, indicating that high levels of natural bioaccumulation occur (3).
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In the Galician Rias area, the largest shellfish production area in the European Union, 55% of
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mussels, clams, and cockle samples were contaminated by HuNoV GI and GII and HAV (4).
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Thus, understanding the ecology and persistence of enteric viruses in shellfish is needed to help
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prevent future outbreaks.
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The consumption of uncooked contaminated bivalve shellfish continues to pose a public
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health risk. These bivalve filter-feeders sieve many gallons of water a day through their gills
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which can lead to the bioaccumulation of both bacterial and viral pathogens within shellfish
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tissues. Human enteric viruses have been reported as one of the main causative agents associated
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with bivalve mollusk outbreaks. Human norovirus (HuNoV) and hepatitis A virus (HAV)
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account for the majority of these shellfish outbreaks (2, 5-8) . Pioneering studies have shown that
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these enteric viruses persist and can be bioaccumulated to high titers within shellfish tissues (9-
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12). Infectious HAV was detected in oysters held in HAV-contaminated water for up to 3 wk
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and viral RNA was detected in oysters for 6 wk (13). It has also been shown that acid stable
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enteric viruses persist in the hemocytes of Eastern oysters (Crassostrea virginica) (11). Transfer
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of virus-containing hemocytes to naïve oysters leads to virus detection for up to 3 wk (11).
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HuNoV causes severe gastroenteritis characterized by vomiting, diarrhea, and stomach
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cramps (14). In the US, it is estimated that HuNoV accounts for as much as 60% of foodborne
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illnesses and it is the second leading cause of gastroenteritis-related mortality, causing 797
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deaths annually (15, 16). It has been a challenge to study HuNoV because it cannot be grown in a
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cell culture system and lacks a small animal model. Therefore, cultivable viral surrogates that are
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closely related to HuNoV, such as murine norovirus (MNV) and Tulane virus (TV) have been
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used to study the survival of HuNoVs in foods and the environment (17-21). HAV causes
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gastroenteritis, liver damage, and jaundice. HAV outbreaks have declined due to improvements
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in drinking water quality and sanitation practices, as well as the availability of effective vaccines
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in developed countries. However, HAV remains endemic in developing countries (22).
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Although shellfish imported to the US is often cooked and importation is limited to a few
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countries, there is still potential for HAV contamination in imported shellfish.
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Rotaviruses (RVs) are the major etiological agent of acute gastroenteritis in infants
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worldwide and account for 27% of deaths in children according to the WHO (23). RV is highly
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infectious among children due to its low infectious dose (