Enhanced Removal of Norovirus Surrogates, Murine Norovirus and Tulane Virus, from Aqueous Systems by Zero-Valent Iron

ABSTRACTViral contamination can compromise the safety of water utilized for direct consumption, produce irrigation, and postharvest washing of produce. Zero-valent iron (ZVI) is used commercially for chemical remediation of water and has been demonstrated to remove some biological contaminants from water in laboratory and field studies. This study investigated the efficacy of ZVI to remove human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV), from water and to characterize the reversibility and nature of viral association with ZVI. Genomic material of TV and MNV recovered from the effluent of inoculated water treatment columns containing a 1:1 mixture of ZVI and sand was 2 and 3 log, respectively, less than that recovered from the effluent of treatment columns containing only sand. Elution buffers (citrate buffers, pH 4 and 7, and virus elution buffer, pH 9.5, with and without added 1 M NaCl) did not increase recovery of infectious TV and MNV from ZVI as compared with elution with water alone. TV-inoculated lettuce washed with water in the presence of ZVI yielded 1.5 to 2 log fewer infectious TV from washwater as compared with lettuce washed with water alone or in the presence of sand. These data demonstrate the enhanced removal of human norovirus surrogates, TV and MNV, from water by ZVI and provide indications that unrecovered viruses are not readily disassociated from ZVI by buffers of various pH and ionic strength. These findings warrant further investigation into larger-scale simulations of water remediation of viral contaminants for potential application in the treatment of water used for drinking, irrigation, and food processing.

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Comparing Human Norovirus Surrogates: Murine Norovirus and Tulane Virus

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-216 ◽

2013 ◽

Vol 76 (1) ◽

pp. 139-143 ◽

Cited By ~ 78

Author(s):

KIRSTEN A. HIRNEISEN ◽

KALMIA E. KNIEL

Keyword(s):

Tap Water ◽

Plaque Assay ◽

Heat Treatments ◽

Viral Infectivity ◽

Murine Norovirus ◽

Human Norovirus ◽

Human Noroviruses ◽

Ph Values ◽

Significant Difference ◽

Tulane Virus

Viral surrogates are widely used by researchers to predict human norovirus behavior. Murine norovirus (MNV) is currently accepted as the best surrogate and is assumed to mimic the survival and inactivation of human noroviruses. Recently, a new calicivirus, the Tulane virus (TV), was discovered, and its potential as a human norovirus surrogate is being explored. This study aimed to compare the behavior of the two potential surrogates under varying treatments of pH (2.0 to 10.0), chlorine (0.2 to 2,000 ppm), heat (50 to 75°C), and survival in tap water at room (20°C) and refrigeration (4°C) temperatures for up to 30 days. Viral infectivity was determined by the plaque assay for both MNV and TV. There was no significant difference between the inactivation of MNV and TV in all heat treatments, and for both MNV and TV survival in tap water at 20°C over 30 days. At 4°C, MNV remained infectious over 30 days at a titer of approximately 5 log PFU/ml, whereas TV titers decreased significantly by 5 days. MNV was more pH stable, as TV titers were reduced significantly at pH 2.0, 9.0, and 10.0, as compared with pH 7.0, whereas MNV titers were only significantly reduced at pH 10.0. After chlorine treatment, there was no significant difference in virus with the exception of at 2 ppm, where TV decreased significantly compared with MNV. Compared with TV, MNV is likely a better surrogate for human noroviruses, as MNV persisted over a wider range of pH values, at 2 ppm of chlorine, and without a loss of titer at 4°C.

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Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments

Applied and Environmental Microbiology ◽

10.1128/aem.01532-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5743-5751 ◽

Cited By ~ 100

Author(s):

Theresa Cromeans ◽

Geun Woo Park ◽

Veronica Costantini ◽

David Lee ◽

Qiuhong Wang ◽

...

Keyword(s):

Culture Method ◽

Aichi Virus ◽

Murine Norovirus ◽

Human Norovirus ◽

The Public ◽

Test Parameters ◽

Reverse Transcription Quantitative Pcr ◽

Tulane Virus ◽

Comprehensive Comparison ◽

Identical Test

ABSTRACTHuman norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log10units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log10units by alcohols, in contrast to 2 and 3 log10units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log10units by 1,000 ppm chlorine, in contrast to 1 log10unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.

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Evaluation of Various Methods for Recovering Human Norovirus and Murine Norovirus from Vegetables and Ham

Journal of Food Protection ◽

10.4315/0362-028x-73.9.1651 ◽

2010 ◽

Vol 73 (9) ◽

pp. 1651-1657 ◽

Cited By ~ 13

Author(s):

HYEONJIN PARK ◽

MINJUNG KIM ◽

GWANGPYO KO

Keyword(s):

Molecular Weight ◽

Polyethylene Glycol ◽

Hollow Fiber ◽

Significant Positive Correlation ◽

Recovery Efficiency ◽

Murine Norovirus ◽

Human Norovirus ◽

Ph Values ◽

Elution Buffer ◽

Concentration Methods

We evaluated and optimized each step in an analytical method for detecting norovirus from various foods. We characterized the buffers needed for eluting norovirus from foods such as ham and lettuce. Two different concentration methods, polyethylene glycol (PEG) precipitation and hollow fiber ultrafiltration (HUF), were compared using both murine norovirus (MNV) and human norovirus (HuNoV). For PEG precipitation, an elution buffer containing 3% beef extract (pH 7.1) was more suitable than 0.05 M glycine plus 0.14 M NaCl (pH 7.5), and the recovery efficiency increased with increasing molecular weight of PEG. To determine the optimal buffer for concentrating norovirus by HUF, glycine buffers with different pH values and ionic strengths were examined as elution buffers. Overall, HUF was more efficient for norovirus recovery than was PEG precipitation. Because there was a significant positive correlation between MNV and HuNoV results, MNV could be a useful surrogate for detecting HuNoV in foods.

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Inactivation of Human Norovirus and Its Surrogates on Alfalfa Seeds by Aqueous Ozone

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-029 ◽

2015 ◽

Vol 78 (8) ◽

pp. 1586-1591 ◽

Cited By ~ 3

Author(s):

QING WANG ◽

SARAH MARKLAND ◽

KALMIA E. KNIEL

Keyword(s):

Deionized Water ◽

Murine Norovirus ◽

Food Matrix ◽

Human Norovirus ◽

Viral Genomes ◽

Tulane Virus ◽

Foodborne Outbreaks ◽

Alfalfa Sprouts ◽

All Treatment ◽

Alfalfa Seeds

Alfalfa sprouts have been associated with numerous foodborne outbreaks. Previous studies investigated the effectiveness of aqueous ozone on bacterially contaminated seeds, yet little is known about the response of human norovirus (huNoV). This study assessed aqueous ozone for the disinfection of alfalfa seeds contaminated with huNoV and its surrogates. The inactivation of viruses without a food matrix was also investigated. Alfalfa seeds were inoculated with huNoV genogroup II, Tulane virus (TV), and murine norovirus (MNV); viruses alone or inoculated on seeds were treated in deionized water containing 6.25 ppm of aqueous ozone with agitation at 22°C for 0.5, 1, 5, 15, or 30 min. The data showed that aqueous ozone resulted in reductions of MNV and TV infectivity from 1.66 ± 1.11 to 5.60 ± 1.11 log PFU/g seeds; for all treatment times, significantly higher reductions were observed for MNV (P < 0.05). Viral genomes were relatively resistant, with a reduction of 1.50 ± 0.14 to 3.00 ± 0.14 log genomic copies/g seeds; the reduction of TV inoculated in water was similar to that of huNoV, whereas MNV had significantly greater reductions in genomic copies (P < 0.05). Similar trends were observed in ozone-treated viruses alone, with significantly higher levels of inactivation (P < 0.05), especially with reduced levels of infectivity for MNV and TV. The significant inactivation by aqueous ozone indicates that ozone may be a plausible substitute for chlorine as an alternative treatment for seeds. The behavior of TV was similar to that of huNoV, which makes it a promising surrogate for these types of scenarios.

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Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce

Applied and Environmental Microbiology ◽

10.1128/aem.00650-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4791-4800 ◽

Cited By ~ 11

Author(s):

Erin DiCaprio ◽

Anastasia Purgianto ◽

Jianrong Li

Keyword(s):

Abiotic Stress ◽

Biotic Stress ◽

Fresh Produce ◽

Extreme Weather Events ◽

Murine Norovirus ◽

Romaine Lettuce ◽

Human Norovirus ◽

Abiotic And Biotic Stresses ◽

Biotic Stresses ◽

Tulane Virus

ABSTRACTHuman norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce.

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Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce

Applied and Environmental Microbiology ◽

10.1128/aem.01081-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6143-6152 ◽

Cited By ~ 50

Author(s):

Erin DiCaprio ◽

Yuanmei Ma ◽

Anastasia Purgianto ◽

John Hughes ◽

Jianrong Li

Keyword(s):

Real Time ◽

Fresh Produce ◽

Plaque Assay ◽

Feed Water ◽

Murine Norovirus ◽

Rt Pcr ◽

Romaine Lettuce ◽

Human Norovirus ◽

Genotype 4 ◽

Tulane Virus

ABSTRACTFresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 106RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 106to 2 × 106PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (105to 106RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (101to 103PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (105to 106PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.

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Evidence of the Internalization of Animal Caliciviruses via the Roots of Growing Strawberry Plants and Dissemination to the Fruit

Applied and Environmental Microbiology ◽

10.1128/aem.03867-14 ◽

2015 ◽

Vol 81 (8) ◽

pp. 2727-2734 ◽

Cited By ~ 20

Author(s):

Erin DiCaprio ◽

Doug Culbertson ◽

Jianrong Li

Keyword(s):

Contaminated Soils ◽

Fresh Produce ◽

Plaque Assay ◽

The United States ◽

Epidemiological Studies ◽

Murine Norovirus ◽

Plant Leaves ◽

Human Norovirus ◽

Tulane Virus ◽

Bell Peppers

ABSTRACTHuman norovirus (NoV) is the leading cause of foodborne disease in the United States, and epidemiological studies have shown that fresh produce is one of the major vehicles for the transmission of human NoV. However, the mechanisms of norovirus contamination and persistence in fresh produce are poorly understood. The objective of this study is to determine whether human NoV surrogates, murine norovirus (MNV-1) and Tulane virus (TV), can attach and become internalized and disseminated in strawberries grown in soil. The soil of growing strawberry plants was inoculated with MNV-1 and TV at a level of 108PFU/plant. Leaves and berries were harvested over a 14-day period, and the viral titer was determined by plaque assay. Over the course of the study, 31.6% of the strawberries contained internalized MNV-1, with an average titer of 0.81 ± 0.33 log10PFU/g. In comparison, 37.5% of strawberries were positive for infectious TV, with an average titer of 1.83 ± 0.22 log10PFU/g. A higher percentage (78.7%) of strawberries were positive for TV RNA, with an average titer of 3.15 ± 0.51 log10RNA copies/g as determined by real-time reverse transcriptase quantitative PCR (RT-qPCR). In contrast, no or little virus internalization and dissemination were detected when TV was inoculated into bell peppers grown in soil. Collectively, these data demonstrate (i) virally contaminated soils can lead to the internalization of virus via plant roots and subsequent dissemination to the leaf and fruit portions of growing strawberry plants and (ii) the magnitude of internalization is dependent on the type of virus and plant.

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Testing for Human Norovirus and Recovery of Process Control in Outbreak-Associated Produce Items

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-134 ◽

2017 ◽

Vol 81 (1) ◽

pp. 105-114 ◽

Cited By ~ 3

Author(s):

Kaoru Hida ◽

Efstathia Papafragkou ◽

Michael Kulka

Keyword(s):

Limit Of Detection ◽

Detection Methods ◽

Murine Norovirus ◽

Human Norovirus ◽

Elution Buffer ◽

Alternative Approach ◽

Reverse Transcription Quantitative Pcr ◽

Concentration Step ◽

Food Commodity ◽

Better Than

ABSTRACT The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris–glycine–beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 103 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.

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NMR Experiments Shed New Light on Glycan Recognition by Human and Murine Norovirus Capsid Proteins

Viruses ◽

10.3390/v13030416 ◽

2021 ◽

Vol 13 (3) ◽

pp. 416

Author(s):

Robert Creutznacher ◽

Thorben Maass ◽

Patrick Ogrissek ◽

Georg Wallmann ◽

Clara Feldmann ◽

...

Keyword(s):

Blood Group ◽

Protein Interactions ◽

Capsid Proteins ◽

Blood Group Antigens ◽

Biological Processes ◽

Murine Norovirus ◽

Human Norovirus ◽

Head Groups ◽

Significant Difference

Glycan–protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

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Application of chitosan microparticles against human norovirus

Journal of Food Protection ◽

10.4315/jfp-21-220 ◽

2021 ◽

Author(s):

Candace Barnes ◽

Rebecca Barber ◽

Keith R. Schneider ◽

Michelle D. Danyluk ◽

Anita C. Wright ◽

...

Keyword(s):

Environmental Water ◽

The United States ◽

Virus Infectivity ◽

Log10 Reduction ◽

Human Norovirus ◽

Virus Particles ◽

Chitosan Microparticles ◽

Natural Treatment ◽

Fecal Suspension ◽

Tulane Virus

Human norovirus (HuNoV) is the leading causative agent of foodborne outbreaks and is associated with the second most prevalent cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CM) against HuNoV GII.4 Sydney and its cultivable surrogate, Tulane virus (TuV), in suspensions mimicking fecally-contaminated water. CM was prepared by crosslinking chitosan molecules with sodium sulfate, and then its anti-noroviral activity was assessed using infectivity assay on TuV and RT-qPCR on TuV and HuNoV. A 3% CM suspension in PBS (pH 7.2) showed binding to TuV particles but with a negligible impact on virus infectivity (p>0.05). TuV and HuNoV suspended in fecal suspensions showed a 1.5-log10 reduction in genomic copies per ml following a 10-min contact time (p<0.05). Despite the negligible impact on viral infectivity, CM moderately binds to virus particles and helps purify environmental water by removing infectious virus particles. In this study, TuV served as a suitable surrogate for HuNoV by showing a similar log10 reduction in fecal suspension. Overall, the outcomes of thisresearch highlight the potential application of CM as a novel, natural treatment to minimize the spread of water-transmitted viral pathogens.

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