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Inactivation of a Human Norovirus Surrogate by Chlorine Dioxide Gas and Prediction of Human Norovirus Contamination by a Fecal Indicator System

Yeap, Jia Wei
http://rave.ohiolink.edu/etdc/view?acc_num=osu1366640143

Abstract Details

2013, Master of Science, Ohio State University, Food Science and Technology.
Human norovirus (NoV) is the leading causative agent of foodborne illnesses worldwide, accounting for more than 95% of acute nonbacterial gastroenteritis in humans. Human NoV is highly contagious, stable, and resistant to environmental stresses and common disinfectants. Although human NoV causes significant health and economic burdens, research on human NoV has been hampered due to the lack of an in vitro cell culture method and a small animal model. Currently, there is no effective measure to remove and inactivate human NoV from food contact surfaces. One of the major routes for human norovirus (NoV) transmission is the fecal-oral route. However, there is no rapid indicating system to predict human NoV contamination. The increasing prevalence of human NoV requires the development of novel corrective and preventive interventions to reduce the burden of foodborne viruses in food, water and the environment. The objectives of this thesis are to (i) determine whether murine norovirus (MNV), a cultivable human NoV surrogate, can be effectively inactivated by chlorine dioxide (ClO2) gas; and (ii) explore whether a fecal indicator system can be developed for the prediction of human NoV contamination. To determine the effectiveness of virus inactivation by ClO2, 100 µl of MNV-1 (108 PFU/ml) was inoculated onto the sterile surface of stainless steel coupons (6.5 cm2) to reach an inoculation level of 107 plaque forming unit (PFU)/coupon, and the samples were treated with ClO2 at 1, 1.5, 2, 2.5, and 4 mg/L for up to 5 min at 25°C and relative humidity of 25%. Viral plaque assays were used to quantify surviving viral particles. It was found that MNV-1 was inactivated by ClO2 in a dose and time dependent manner. At least a 3-log reduction in MNV-1 was achieved after treating the stainless steel coupons for 5 min at 1 mg/L. At a concentration of 4 mg/L, no infectious virus was recovered even at 1 min of treatment time. Subsequently, the mechanism of viral inactivation by ClO2 was investigated using transmission electron microscopy, SDS-PADE, Western blotting, and RT-PCR. It was found that ClO2 disrupts the integrity of the virion structure, and also degrades the major capsid protein (VP1) and the genomic RNA. A minimum treatment of ClO2 at 2.5 mg/L for 2 min was required for complete disruption of the virion and subsequent degradation of the primary protein structure of VP1 and the genomic RNA. To develop a fecal indicator system for human NoV contamination, the ratio of Enterococcus sp. and Bacteroides fragilis was used as a human NoV surrogate because it was found that there is an alteration in the gut microbiota following viral infection of the gasterointestinal tract. To determine if the ratio of the two bacteria changes in fecal samples of human NoV-infected patients, a total of 75 human fecal samples were collected from patients (n=36) with symptoms of gastroenteritis and healthy adults (n=39). The levels of human NoV and the two most common bacteria (Enterococcus sp. and B. fragilis) in fecal samples were quantified by real-time RT-PCR or real-time PCR. The 23S rRNA, gyrB, and VP1 gene makers were used to detect Enterococcus sp., B. fragilis, and human NoV, respectively. It was found that among the clinical samples, 100% (36/36) was positive for human NoV while among the samples from healthy adults, 33% (13/39) was positive for human NoV and 67% (26/39) was negative. Enterococcus sp. and B. fragilis levels between human NoV positive and negative fecal samples were not statistically different (p<0.05). Linear regression showed a weak correlation between individual bacterial indicator (Enterococcus sp. or B. fragilis) and human NoV in the human fecal samples. Logistical regression modeling revealed a significant change in the ratio of Enterococcus sp. and B. fragilis in human NoV-infected fecal samples as expected, suggesting a trend where detection of higher Enterococcus sp. and lower B. fragilis levels implys a greater chance of human NoV presence. Therefore, the ratio of Enterococcus sp. and B. fragilis can potentially be used as an indicator for the presence of human NoV contamination. Collectively, we conclude that (i) ClO2 serves as an effective method to inactivate a human norovirus surrogate on stainless steel contact surfaces; (ii) the mechanism of viral inactivation by ClO2 includes disruption of the virion structure and degradation of both viral proteins and viral RNA and (iii) alteration in the ratio of Enterococcus sp. and B. fragilis in human feces can potentially be used as an indicator for human NoV contamination.
Jianrong Li (Advisor)
Richard Linton (Committee Member)
Jiyoung Lee (Committee Member)
Ahmed Yousef (Committee Member)
126 p.
Food Science

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Citations

  • Yeap, J. W. (2013). Inactivation of a Human Norovirus Surrogate by Chlorine Dioxide Gas and Prediction of Human Norovirus Contamination by a Fecal Indicator System [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366640143

    APA Style (7th edition)

  • Yeap, Jia Wei. Inactivation of a Human Norovirus Surrogate by Chlorine Dioxide Gas and Prediction of Human Norovirus Contamination by a Fecal Indicator System. 2013. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1366640143.

    MLA Style (8th edition)

  • Yeap, Jia Wei. "Inactivation of a Human Norovirus Surrogate by Chlorine Dioxide Gas and Prediction of Human Norovirus Contamination by a Fecal Indicator System." Master's thesis, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366640143

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.