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

2012 ◽  
Vol 78 (17) ◽  
pp. 6143-6152 ◽  
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.

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

2015 ◽  
Vol 81 (14) ◽  
pp. 4791-4800 ◽  
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.

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

2015 ◽  
Vol 81 (8) ◽  
pp. 2727-2734 ◽  
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.

Tenacity of Human Norovirus and the Surrogates Feline Calicivirus and Murine Norovirus during Long-Term Storage on Common Nonporous Food Contact Surfaces

2015 ◽  
Vol 78 (1) ◽  
pp. 224-229 ◽  
Author(s):  
SASCHA MORMANN ◽  
CATHRIN HEIßENBERG ◽  
JENS PFANNEBECKER ◽  
BARBARA BECKER
Keyword(s):  
Stainless Steel ◽  
Real Time ◽  
Room Temperature ◽  
Plaque Assay ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Food Contact Surfaces ◽  
Contact Surfaces

The transfer of human norovirus (hNV) to food via contaminated surfaces is highly probable during food production, processing, and preparation. In this study, the tenacity of hNV and its cultivable surrogates feline calicivirus (FCV) and murine norovirus (MNV) on two common nonporous surface materials at two storage temperatures was directly compared. Virus titer reduction on artificially inoculated stainless steel and plastic carriers was monitored for 70 days at room temperature and at 7°C. Viruses were recovered at various time points by elution. Genomes from intact capsids (hNV, FCV, and MNV) were quantified with real-time reverse transcription (RT) PCR, and infectivity (FCV and MNV) was assessed with plaque assay. RNase treatment before RNA extraction was used to eliminate exposed RNA and to assess capsid integrity. No significant differences in titer reduction were found between materials (stainless steel or plastic) with the plaque assay or the real-time quantitative RT-PCR. At room temperature, infectious FCV and MNV were detected for 7 days. Titers of intact hNV, FCV, and MNV capsids dropped gradually and were still detectable after 70 days with a loss of 3 to 4 log units. At 7°C, the viruses were considerably more stable than they were at room temperature. Although only MNV infectivity was unchanged after 70 days, the numbers of intact capsids (hNV, FCV, and MNV) were stable with less than a 1-log reduction. The results indicate that hNV persists on food contact surfaces and seems to remain infective for weeks. MNV appears to be more stable than FCV at 7°C, and thus is the most suitable surrogate for hNV under dry conditions. Although a perfect quantitative correlation between intact capsids and infective particles was not obtained, real-time quantitative RT-PCR provided qualitative data about hNV inactivation characteristics. The results of this comparative study might support future efforts in assessment of foodborne virus risk and food safety.

Comparing Human Norovirus Surrogates: Murine Norovirus and Tulane Virus

2013 ◽  
Vol 76 (1) ◽  
pp. 139-143 ◽  
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.

scholarly journals Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses

2016 ◽  
Vol 82 (19) ◽  
pp. 6037-6045 ◽  
Author(s):  
Fangfei Lou ◽  
Erin DiCaprio ◽  
Xinhui Li ◽  
Xianjun Dai ◽  
Yuanmei Ma ◽  
...  
Keyword(s):  
High Pressure ◽  
Real Time ◽  
Receptor Binding ◽  
High Pressure Processing ◽  
Blood Group Antigens ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Genotype 4 ◽  
Processing Technologies

ABSTRACTHuman norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagatedin vitro. In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1 > GII.6 > GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H2O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters.IMPORTANCEHuman norovirus (HuNoV) is a leading cause of foodborne disease worldwide. Noroviruses are highly diverse, both antigenically and genetically. Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivablein vitro. Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix.

scholarly journals Inactivation and UV Disinfection of Murine Norovirus with TiO2 under Various Environmental Conditions

2008 ◽  
Vol 74 (7) ◽  
pp. 2111-2117 ◽  
Author(s):  
JungEun Lee ◽  
KyungDuk Zoh ◽  
GwangPyo Ko
Keyword(s):  
Real Time ◽  
Salt Concentration ◽  
Environmental Conditions ◽  
Plaque Assay ◽  
Surface Characteristics ◽  
Uv Disinfection ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Stool Suspension

ABSTRACT We studied inactivation and UV disinfection of murine norovirus (MNV) as a surrogate for human norovirus. We investigated the effects of different surface characteristics, temperatures, and NaCl concentrations on MNV survival using both a plaque assay and a real-time TaqMan reverse transcription (RT)-PCR assay. MNV survived more than 40 days on diaper material, on gauze, and in a stool suspension. Compared to inactivation at lower temperatures (−20 and 4°C), inactivation of MNV was greater at higher temperatures (18 and 30°C). On the surface of both gauze and diaper material, there was a <2-log10 reduction in the amount of infectious MNV in 40 days after incubation at both −20 and 4°C, compared to a >5-log10 reduction after incubation at 30°C in 24 days. MNV survived better in a stool suspension than on the surface of gauze or diaper material. A higher salt concentration increased the rate of inactivation of MNV. In 72 h, <0.3-, 1.5-, and 2.5-log10 reductions in the amount of infectious MNV occurred in distilled water and 0.5 and 1 M NaCl, respectively. We observed only minor reductions in the numbers of viral RNA copies as quantified by real-time TaqMan RT-PCR regardless of the temperature, the salt concentration, or the suspending medium. We also evaluated UV disinfection of infectious MNV with and without TiO2. The amount of MNV was significantly reduced by 254-nm UV with and without TiO2. When 25 mJ/cm2 UV was used, 3.3- and 3.6-log10 reductions in the amounts of infectious MNV occurred with and without TiO2, respectively. Our results demonstrate that MNV can persist in various environmental conditions and can be efficiently controlled by UV disinfection.

Effect of Temperature, pH, and NaCl on the Inactivation Kinetics of Murine Norovirus

2012 ◽  
Vol 75 (3) ◽  
pp. 533-540 ◽  
Author(s):  
KYEONGJIN SEO ◽  
JUNG EUN LEE ◽  
MI YOUNG LIM ◽  
GWANGPYO KO
Keyword(s):  
Real Time ◽  
Environmental Conditions ◽  
Low Ph ◽  
Effect Of Temperature ◽  
Inactivation Kinetics ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Norovirus ◽  
Pcr Assays ◽  
Kinetics Of

We investigated the resistance of murine norovirus (MNV) and coliphage MS2, a culturable human norovirus surrogate, to temperature, salt, and pH. Virus inactivation was measured by plaque, real-time TaqMan reverse transcription (RT) PCR, and long-template RT-PCR assays. Both MNV and MS2 were rapidly inactivated at temperatures above 60°C. Similarly, MNV tolerated low salt concentrations (0.3% NaCl) to a greater degree than high salt concentrations (3.3 to 6.3% NaCl). MNV was relatively resistant to strong acidic conditions (pH 2) and was more tolerant of slightly acidic (pH 4) or neutral (pH 7) conditions. In contrast, MS2 was resistant to high salinity. Overall, temperature had a greater effect on infectivity than salt or low pH. Additionally, temperature and low pH had a synergistic effect on MNV infectivity. Both real-time and long-template RT-PCR assays significantly underestimated the inactivation by temperature, salt, and pH. The inactivation kinetics of both MNV and MS2 under various environmental conditions gave a good fit by the Weibull model (R2 &gt; 0.9). This study suggests both the capacity of infectious human norovirus to persist in the face of various environmental conditions and its sensitivity to high temperatures, which may provide a mechanism of protection against this virus.

scholarly journals Characterization of Ozone Disinfection of Murine Norovirus

2009 ◽  
Vol 76 (4) ◽  
pp. 1120-1124 ◽  
Author(s):  
Mi Young Lim ◽  
Ju-Mi Kim ◽  
Jung Eun Lee ◽  
GwangPyo Ko
Keyword(s):  
Public Health ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Plaque Assay ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Human Noroviruses ◽  
Pcr Assays ◽  
Ozone Disinfection

ABSTRACT Despite the importance of human noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using murine norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
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