Inactivation Kinetics and Mechanism of a Human Norovirus Surrogate on Stainless Steel Coupons via Chlorine Dioxide Gas

Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO₂) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO₂ gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 10⁷ PFU/coupon. The samples were treated with ClO₂ gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO₂ gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO₂ gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO₂ gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log₁₀ reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log₁₀ gc/liter; 95% credible interval [CI], 3.5, 4.3 log₁₀ gc/liter) is larger than the value for NoV GI (1.5 log₁₀ gc/liter; 95% CI, 0.4, 2.4 log₁₀ gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log₁₀ reductions were −2.4 log₁₀ gc/liter (95% CI, −3.9, −1.1 log₁₀ gc/liter) for NoV GI, −2.7 log₁₀ gc/liter (95% CI, −3.6, −1.9 log₁₀ gc/liter) for NoV GII, and −2.9 log₁₀ PFU per liter (95% CI, −3.4, −2.4 log₁₀ PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were −1.4 log₁₀ gc/liter (95% CI, −3.3, 0.5 log₁₀ gc/liter) for NoV GI, −1.7 log₁₀ gc/liter (95% CI, −3.1, −0.3 log₁₀ gc/liter) for NoV GII, and −3.6 log₁₀ PFU per liter (95% CI, −4.8, −2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log₁₀ reduction in NoV GII and the mean log₁₀ reduction in MSCs.     

Inactivation of murine norovirus 1, coliphage phiX174, and Bacteroides [corrected] fragilis phage B40-8 on surfaces and fresh-cut iceberg lettuce by hydrogen peroxide and UV light

In this study, the inactivating properties of liquid hydrogen peroxide (L-H(2)O(2)), vaporized hydrogen peroxide (V-H(2)O(2)), UV light, and a combination of V-H(2)O(2) and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H(2)O(2) (2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log(10) units within 10 min of exposure, whereas for B40-8, 15% of L-H(2)O(2) was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log(10) unit after 5 min of exposure) by V-H(2)O(2) (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log(10)-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H(2)O(2) and a combination of V-H(2)O(2) and UV light can be used for norovirus inactivation on surfaces; V-H(2)O(2) (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.     

Inactivation of Murine Norovirus on a Range of Copper Alloy Surfaces Is Accompanied by Loss of Capsid Integrity

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome.     

Antiviral Properties of Silver Nanoparticles on a Magnetic Hybrid Colloid

Silver nanoparticles (AgNPs) are considered to be a potentially useful tool for controlling various pathogens. However, there are concerns about the release of AgNPs into environmental media, as they may generate adverse human health and ecological effects. In this study, we developed and evaluated a novel micrometer-sized magnetic hybrid colloid (MHC) decorated with variously sized AgNPs (AgNP-MHCs). After being applied for disinfection, these particles can be easily recovered from environmental media using their magnetic properties and remain effective for inactivating viral pathogens. We evaluated the efficacy of AgNP-MHCs for inactivating bacteriophage ϕX174, murine norovirus (MNV), and adenovirus serotype 2 (AdV2). These target viruses were exposed to AgNP-MHCs for 1, 3, and 6 h at 25°C and then analyzed by plaque assay and real-time TaqMan PCR. The AgNP-MHCs were exposed to a wide range of pH levels and to tap and surface water to assess their antiviral effects under different environmental conditions. Among the three types of AgNP-MHCs tested, Ag30-MHCs displayed the highest efficacy for inactivating the viruses. The ϕX174 and MNV were reduced by more than 2 log₁₀ after exposure to 4.6 × 10⁹ Ag30-MHCs/ml for 1 h. These results indicated that the AgNP-MHCs could be used to inactivate viral pathogens with minimum chance of potential release into environment.     

Inactivation of Caliciviruses

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100°C. UV-B radiation from 0 to 150 mJ/cm² caused dose-dependent inactivation, with a 3 D (D = 1 log₁₀) reduction in infectivity at 34 mJ/cm² for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.     

Sorting through the Wealth of Options: Comparative Evaluation of Two Ultraviolet Disinfection Systems

Background

Environmental surfaces play an important role in the transmission of healthcare-associated pathogens. Because environmental cleaning is often suboptimal, there is a growing demand for safe, rapid, and automated disinfection technologies, which has lead to a wealth of novel disinfection options available on the market. Specifically, automated ultraviolet-C (UV-C) devices have grown in number due to the documented efficacy of UV-C for reducing healthcare-acquired pathogens in hospital rooms. Here, we assessed and compared the impact of pathogen concentration, organic load, distance, and radiant dose on the killing efficacy of two analogous UV-C devices.

Principal Findings

The devices performed equivalently for each impact factor assessed. Irradiation delivered for 41 minutes at 4 feet from the devices consistently reduced C. difficile spores by ∼ 3 log₁₀CFU/cm², MRSA by>4 log₁₀CFU/cm², and VRE by >5 log₁₀CFU/cm². Pathogen concentration did not significantly impact the killing efficacy of the devices. However, both a light and heavy organic load had a significant negative impacted on the killing efficacy of the devices. Additionally, increasing the distance to 10 feet from the devices reduced the killing efficacy to ≤3 log₁₀CFU/cm² for MRSA and VRE and <2 log₁₀CFU/cm² for C.difficile spores. Delivery of reduced timed doses of irradiation particularly impacted the ability of the devices to kill C. difficile spores. MRSA and VRE were reduced by >3 log₁₀CFU/cm² after only 10 minutes of irradiation, while C. difficile spores required 40 minutes of irradiation to achieve a similar reduction.

Conclusions

The UV-C devices were equally effective for killing C. difficile spores, MRSA, and VRE. While neither device would be recommended as a stand-alone disinfection procedure, either device would be a useful adjunctive measure to routine cleaning in healthcare facilities.     

Effect of food residues on norovirus survival on stainless steel surfaces

Background

In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated.

Methodology/Principal Findings

Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons.

Conclusions/Significance

This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line

Aim: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean‐in‐place regimes. Methods and Results: Exposing sessile spores to 1% NaOH at 65°C for 30 min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from ?20 to ?32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l^?1 KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log₁₀ CFU cm^?2) than untreated spores (3·6 log₁₀ CFU cm^?2). Conclusions: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. Significance of Study: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.     

Growth, greening, and phytochrome in etiolated spirodela (lemnaceae)

Two species of Spirodela were grown aseptically in a simple mineral medium containing sucrose. Weak red light (15 erg cm⁻² sec⁻¹) enhanced dark growth of S. oligorrhiza, whereas weak far red light (15 erg cm⁻² sec⁻¹) when given after the red light reduced this effect.     

Effects of Light, Carbon Dioxide, and Temperature on Photosynthesis, Oxygen Inhibition of Photosynthesis, and Transpiration in Solanum tuberosum

Individual leaves of potato (Solanum tuberosum L. W729R), a C₃ plant, were subjected to various irradiances (400-700 nm), CO₂ levels, and temperatures in a controlled-environment chamber. As irradiance increased, stomatal and mesophyll resistance exerted a strong and some-what paralleled regulation of photosynthesis as both showed a similar decrease reaching a minimum at about 85 neinsteins·cm⁻²·sec⁻¹ (about ½ of full sunlight). Also, there was a proportional hyperbolic increase in transpiration and photosynthesis with increasing irradiance up to 85 neinsteins·cm⁻²·sec⁻¹. These results contrast with many C₃ plants that have a near full opening of stomata at much less light than is required for saturation of photosynthesis.     

The effect of light on the tricarboxylic Acid cycle in green leaves: I. Relative rates of the cycle in the dark and the light

Excised green leaves of mung bean (Phaseolus aureus L. var. Mungo) were used to determine the effect of light on the rate of endogenous respiration via the tricarboxylic acid cycle. Illumination with white light at an intensity of 0.043 gram calories cm⁻²min⁻¹ (approximately 8600 lux) of visible radiation (400-700 nm) gave a rate of apparent photosynthesis, measured as net CO₂ uptake, of 21 mg CO₂ dm⁻²hr⁻¹ which was about 11-fold greater than the rate of dark respiration. The feeding of ¹⁴CO₂ or ¹⁴C-labeled acids of the tricarboxylic acid cycle in the dark for 2 hours was established as a suitable method for labeling mitochondrial pools of cycle intermediates.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

Control of Listeria spp. by Competitive-Exclusion Bacteria in Floor Drains of a Poultry Processing Plant

In previous studies workers determined that two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis C-1-92 and Enterococcus durans 152 (competitive-exclusion bacteria [CE]), which were originally obtained from biofilms in floor drains, are bactericidal to Listeria monocytogenes or inhibit the growth of L. monocytogenes both in vitro and in biofilms at 4 to 37°C. We evaluated the efficacy of these isolates for reducing Listeria spp. contamination of floor drains of a plant in which fresh poultry is processed. Baseline assays revealed that the mean numbers of Listeria sp. cells in floor drains sampled on six different dates (at approximately biweekly intervals) were 7.5 log₁₀ CFU/100 cm² for drain 8, 4.9 log₁₀ CFU/100 cm² for drain 3, 4.4 log₁₀ CFU/100 cm² for drain 2, 4.1 log₁₀ CFU/100 cm² for drain 4, 3.7 log₁₀ CFU/100 cm² for drain 1, and 3.6 log₁₀ CFU/100 cm² for drain 6. The drains were then treated with 10⁷ CE/ml in an enzyme-foam-based cleaning agent four times in 1 week and twice a week for the following 3 weeks. In samples collected 1 week after CE treatments were applied Listeria sp. cells were not detectable (samples were negative as determined by selective enrichment culture) for drains 4 and 6 (reductions of 4.1 and 3.6 log₁₀ CFU/100 cm², respectively), and the mean numbers of Listeria sp. cells were 3.7 log₁₀ CFU/100 cm² for drain 8 (a reduction of 3.8 log₁₀ CFU/100 cm²), <1.7 log₁₀ CFU/100 cm² for drain 1 (detectable only by selective enrichment culture; a reduction of 3.3 log₁₀ CFU/100 cm²), and 2.6 log₁₀ CFU/100 cm² for drain 3 (a reduction of 2.3 log₁₀ CFU/100 cm²). However, the aerobic plate counts for samples collected from floor drains before, during, and after CE treatment remained approximately the same. The results indicate that application of the two CE can greatly reduce the number of Listeria sp. cells in floor drains at 3 to 26°C in a facility in which fresh poultry is processed.