scholarly journals Detection of Murine Norovirus-1 by Using TAT Peptide-Delivered Molecular Beacons

2011 ◽  
Vol 77 (15) ◽  
pp. 5517-5520 ◽  
Author(s):  
Payal S. Ganguli ◽  
Wilfred Chen ◽  
Marylynn V. Yates
Keyword(s):  
Molecular Beacon ◽  
Plaque Assay ◽  
Molecular Beacons ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Murine Norovirus ◽  
Infective Virus ◽  
Tat Peptide ◽  
Human Norovirus ◽  
Time Required

ABSTRACTA TAT peptide-delivered molecular beacon was developed and utilized to enumerate murine norovirus 1, a human norovirus (NoV) surrogate, in RAW 264.7 cells. This allowed the detection of a single infective virus within 6 h, a 12-fold improvement in time required for viral detection and quantification compared to that required by the conventional plaque assay.

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.

Evaluation of Murine Norovirus Persistence in Environments Relevant to Food Production and Processing

2011 ◽  
Vol 74 (11) ◽  
pp. 1847-1851 ◽  
Author(s):  
S. FALLAHI ◽  
K. MATTISON
Keyword(s):  
Stainless Steel ◽  
Food Production ◽  
Room Temperature ◽  
Potable Water ◽  
Plaque Assay ◽  
The Other ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Log Reduction ◽  
Virus Survival

Human norovirus (NoV) causes outbreaks of acute gastroenteritis associated with many ready-to-eat foods, including fresh produce. Effective inactivation procedures must consider virus survival under conditions of produce production and processing. This study aimed to investigate the persistence of NoV in a variety of environments, using murine NoV (MNV) as a surrogate for NoV. MNV was incubated for up to 42 days at room temperature on stainless steel disks, on lettuce, on soil, and in potable water and titers determined by plaque assay. A 1-log reduction of MNV infectivity was observed after 29 days in water, 4 days on lettuce, 12 days on soil, and 15 days on stainless steel disks. MNV survived longer in water than in any of the other environments, indicating that drying may contribute to NoV inactivation. MNV genomes were not significantly reduced for up to 42 days, suggesting that genomic detection is not a reliable indicator of viability. Overall, our findings provide valuable information regarding the potential for NoV transmission in the food supply.

scholarly journals Viral Population Changes during Murine Norovirus Propagation in RAW 264.7 Cells

2017 ◽  
Vol 8 ◽  
Author(s):  
Takuya Kitamoto ◽  
Reiko Takai-Todaka ◽  
Akiko Kato ◽  
Kumiko Kanamori ◽  
Hirotaka Takagi ◽  
...  
Keyword(s):  
Viral Population ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Murine Norovirus ◽  
Population Changes

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.

Recovery and Disinfection of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, from Hard Nonporous and Soft Porous Surfaces

2015 ◽  
Vol 78 (10) ◽  
pp. 1842-1850 ◽  
Author(s):  
THOMAS YEARGIN ◽  
ANGELA FRASER ◽  
GUOHUI HUANG ◽  
XIUPING JIANG
Keyword(s):  
Sodium Hypochlorite ◽  
Limit Of Detection ◽  
Plaque Assay ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Surface Type ◽  
Log Reduction ◽  
Porous Surfaces

Human norovirus is a leading cause of foodborne disease and can be transmitted through many routes, including environmental exposure to fomites. In this study, both the recovery and inactivation of two human norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), on hard nonporous surfaces (glass) and soft porous surfaces (polyester and cotton) were evaluated by both plaque assay and reverse transcription quantitative PCR method. Two disinfectants, sodium hypochlorite (8.25%) and accelerated hydrogen peroxide (AHP, at 4.25%) were evaluated for disinfection efficacy. Five coupons per surface type were used to evaluate the recovery of FCV and MNV by sonication and stomaching and the disinfection of each surface type by using 5 ml of disinfectant for a contact time of 5 min. FCV at an initial titer of ca. 7 log PFU/ml was recovered from glass, cotton, and polyester at 6.2, 5.4, and 3.8 log PFU/ml, respectively, compared with 5.5, 5.2, and 4.1 log PFU/ml, respectively, for MNV with an initial titer of ca. 6 log PFU/ml. The use of sodium hypochlorite (5,000 ppm) was able to inactivate both FCV and MNV (3.1 to 5.5 log PFU/ml) below the limit of detection on all three surface types. AHP (2,656 ppm) inactivated FCV (3.1 to 5.5 log PFU/ml) below the limit of detection for all three surface types but achieved minimal inactivation of MNV (0.17 to 1.37 log PFU/ml). Reduction of viral RNA by sodium hypochlorite corresponded to 2.72 to 4.06 log reduction for FCV and 2.07 to 3.04 log reduction for MNV on all three surface types. Reduction of viral RNA by AHP corresponded to 1.89 to 3.4 log reduction for FCV and 0.54 to 0.85 log reduction for MNV. Our results clearly indicate that both virus and surface types significantly influence recovery efficiency and disinfection efficacy. Based on the performance of our proposed testing method, an improvement in virus recovery will be needed to effectively validate virus disinfection of soft porous surfaces.

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.

scholarly journals Bacteriophage inhibits murine norovirus replication after co-incubation in RAW 264.7 cells

2021 ◽  
Author(s):  
Lili Zhang ◽  
Khashayar Shahin ◽  
Ran Wang
Keyword(s):  
Cellular Response ◽  
Antimicrobial Effect ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Murine Norovirus ◽  
Rna Seq ◽  
Qpcr Analysis ◽  
Ifn Γ ◽  
Phage Treatment

AbstractBacteriophages (phages) are viruses of bacteria. Despite the growing progress in research on phage interactions with eukaryotic cells, our understanding of the roles of phages and their potential implications remains incomplete. The objective of this study was to investigate the effects of the Staphylococcus aureus phage vB_SauM_JS25 on murine norovirus (MNV) replication. Experiments were performed using the RAW 264.7 cell line. After phage treatment, MNV multiplication was significantly inhibited, as indicated by real-time quantitative PCR (RT-qPCR) analysis, western blot, and immunofluorescence assay. Furthermore, we demonstrated the transcriptional changes in phage–MNV co-incubated RAW 264.7 cells through RNA sequencing (RNA-Seq) and bioinformatic analysis. Our subsequent analyses revealed that the innate immune response may play an important role in the restriction of MNV replication, such as the cellular response to IFN-γ and response to IFN-γ. In addition, the gene expression of IL-10, Arg-1, Ccl22, GBP2, GBP3, GBP5, and GBP7 was proven to increase significantly by RT-qPCR, showing a strong correlation between RT-qPCR and RNA-Seq results. Furthermore, phage treatment activated guanylate binding proteins (GBPs), as revealed by RT-qPCR analysis, western blotting, and confocal microscopy. Taken together, these data suggest that the phage affects the innate response (such as the IFN-inducible GTPases and GBPs), reflecting their direct antimicrobial effect on the membrane structure of the MNV replication complexes, and therefore, exerts an antiviral effect in vitro. Collectively, our findings provide insights into the interactions of immune cells and phages, which improve our understanding of the actual role and potential of phages.

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.

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