Efficacy of Chlorine Dioxide Gas Against Hepatitis A Virus on Blueberries, Blackberries, Raspberries, and Strawberries.

Seeking a means of sanitizing berries, the effectiveness of steady state levels of gaseous chlorine dioxide (ClO2) against hepatitis A virus (HAV) on laboratory-contaminated berries was determined. The generated ClO2 was maintained with 1 or 2 mg/l air inside a 269-l glove box to treat 50 g batches of blueberries, raspberries, and blackberries, and 100 g batches of strawberries that were immersion coated with HAV. Normalized data for ClO2 (ppm-h/g product) is reported as a function of ClO2 concentration, treatment time, and weight of treated product. Treatments of ClO2 ranging from 1.00 to 6.27 ppm-h/g berry were evaluated. When compared to untreated HAV-contaminated berries, log reductions of HAV were > 2.1 for all berry types and conditions tested indicating the gaseous ClO2 was effective. The average log reduction with strawberries, raspberries, blueberries and blackberries treated with 1.00 ppm-h/g, the lowest ClO2 treatment tested, were 2.44, 2.49, 3.23, and 3.45, respectively. The highest treatment of 6.27 ppm-h/g was applied at two different gas concentrations of 1 mg/l and 2 mg/l. Average log reductions for blueberries and strawberries treated with 6.27 ppm-h/g were 4.34 and 4.42, and 4.03 and 3.51, applied at 1 mg/l and 2 mg/l, respectively. For blackberries and raspberries 3.20 and 3.24, and 3.23 and 3.97 log reductions were observed for 6.27 ppm-h/g treatments applied at 1 mg/l and 2 mg/l, respectively. Results indicate that HAV contamination of berries can be substantially reduced by gaseous ClO2 and offer industry a waterless means of sanitizing berries against HAV.

Detection of Hepatitis A Virus and Other Enteric Viruses in Shellfish Collected in the Gulf of Naples, Italy.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015-2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Evaluation of Steady-State Gaseous Chlorine Dioxide Treatment for the Inactivation of Tulane virus on Berry Fruits.

The effectiveness of steady-state levels of gaseous chlorine dioxide (ClO2) against Tulane virus (TV), a human norovirus surrogate, on berries was determined. The generated ClO2 was maintained at 1 mg/L inside a 269 L glove box to treat two 50 g batches of blueberries, raspberries, and blackberries, and two 100 g batches of strawberries that were immersion coated with TV. The standardized/normalized treatment concentrations of ClO2 ranging from 0.63 to 4.40 ppm-h/g berry were evaluated. When compared to untreated TV contaminated berries, log reductions of TV were in excess of 2.9 log PFU/g for all berry types and conditions tested, indicating that ClO2 was highly effective. In general, the efficacy of all ClO2 treatments on log reductions of TV on all berries was not significantly different (p < 0.05). The average log reduction with strawberries, raspberries, blueberries, and blackberries, treated with the lowest ClO2 concentration, 0.63 ppm-h/g, were 2.98, 3.40, 3.82, and 4.17 log PFU/g, respectively. Overall results suggest that constant levels of ClO2 could be quite effective against foodborne viruses.

Evaluation of a Male-Specific DNA Coliphage Persistence Within Eastern Oysters (Crassostrea virginica).

Male-specific coliphages (MSCs) are currently used to assess the virologic quality of shellfish-growing waters and to assess the impact of sewage release or adverse weather events on bivalve shellfish. Since MSC can have either DNA or RNA genomes, and most research has been performed exclusively on RNA MSCs, persistence of M13, a DNA MSC, was evaluated for its persistence as a function of time and temperature within Eastern oysters (Crassostrea virginica). Oysters were individually exposed to seawater containing a total of 1010 to 1012 pfu of M13 for 24 h at 15 °C followed by maintenance in tanks with as many as 21 oysters in continuously UV-sterilized water for up to 6 weeks at either 7, 15, or 22 °C. Two trials for each temperature were performed combining three shucked oysters per time point which were assayed by tenfold serial dilution in triplicate. Initial contamination levels averaged 106.9 and ranged from 106.0 to 107.0 of M13. For oysters held for 3 weeks, log10 reductions were 1.7, 3.8, and 4.2 log10 at 7, 15, and 22 °C, respectively. Oysters held at 7 and 15 °C for 6 weeks showed average reductions of 3.6 and 5.1 log10, respectively, but still retained infectious M13. In total, this work shows that DNA MSC may decline within shellfish in a manner analogous to RNA MSCs.

Evaluation of gaseous chlorine dioxide for the inactivation of Tulane virus on blueberries.

To determine the effectiveness of gaseous chlorine dioxide (gClO2) against a human norovirus surrogate on produce, gClO2 was generated and applied to Tulane virus-coated blueberries in a 240 ml-treatment chamber. gClO2 was produced by an acidifying sodium chlorite solution. Initial assessments indicated that blueberries treated with gClO2 generated from ≤1 mg acidified sodium chlorite in the small chamber appeared unaffected while gClO2 generated from ≥10 mg of acidified sodium chlorite solution altered the appearance and quality of the blueberries. Treatments of inoculated blueberries with gClO2 generated from 0.1 mg sodium chlorite reduced the virus populations by >1 log after exposure for 30 to 330 min. For the 1 mg sodium chlorite treatments, the virus populations were reduced by >2.2 log after 15 min exposure and to non-detectable levels (>3.3 logs reductions) after 180 min exposure. Measured concentrations of gClO2 peaked in the treatment chamber at 0.9 µg/l after 10 min for 0.1 mg treatments and 600 µg/l after around 20 min for 1 mg treatment. Overall results indicate that gClO2 could be a feasible waterless intervention for blueberries and other produce.

Surfactant-Enhanced Organic Acid Inactivation of Tulane Virus, a Human Norovirus Surrogate.

Combination treatments of surfactants and phenolic or short-chain organic acids (SCOA) may act synergistically or additively as sanitizers to inactive foodborne viruses and prevent outbreaks. The purpose of this study was to investigate the effect of gallic acid (GA), tannic acid, p-coumaric acid, lactic acid (LA), or acetic acid (AA), in combination with sodium dodecyl sulfate (SDS), against Tulane virus (TV), a surrogate for human norovirus. An aqueous stock solution of phenolic acids or SCOA with or without SDS was prepared and diluted in a twofold dilution series to 2× the desired concentration with cell growth media (M119 plus 10% fetal bovine serum). The solution was inoculated with an equal proportion of 6 log PFU/mL TV with a treatment time of 5 min. The survival of TV was quantified using a plaque assay with LLC-MK2 cells. The minimum virucidal concentration was 0.5:0.7% (v/v) for LA-SDS at pH 3.5 (4.5-PFU/mL reduction) and 0.5:0.7% (v/v) AA-SDS at pH 4.0 (2.6-log PFU/mL reduction). GA and SDS demonstrated a minimum virucidal concentration of 12.5 mM GA-SDS at pH 7.0 (0.2:0.3% GA-SDS) with an 0.8-log PFU/mL reduction and 50 mM GA-SDS (0.8:1.4% GA-SDS at pH 7.0) increased log reduction to 1.6 log PFU/mL. The combination treatments of AA or LA with SDS at pH 7.0 did not produce significant log reduction, nor did individual treatments of tannic acid, GA, p-coumaric acid, AA, LA, or SDS. This study demonstrates that a surfactant, such as SDS, aids in the phenolic acid and SCOA toxicities against viruses. However, inactivation of TV by combination treatments is contingent upon the pH of the sanitizing solution being lower than the pKa value of the organic acid being used. This information can be used to develop sanitizing washes to disinfect food contact surfaces, thereby aiding in the prevention of foodborne outbreaks.

Persistence of MS-2 Bacteriophage Within Eastern Oysters.

Male-specific bacteriophages have been proposed as human enteric virus indicators for shellfish. In this study, Eastern oysters (Crassostrea virginica) were individually exposed to 5.6 × 1010 PFU of MS-2 for 48 h at 15 °C followed by collective maintenance in continuously UV-sterilized seawater for 0-6 weeks at either 7, 15, or 24 °C. Initial contamination levels of MS-2 were >6 log PFU. Assessment of weekly declines of viable MS-2 indicated that cooler temperatures dramatically enhanced the persistence of MS-2 within oyster tissues. At 3 weeks, the average log PFU reductions for MS-2 within oysters were 2.28, 2.90, and 4.57 for oysters held at 7, 15, and 24 °C, respectively. Fitting temporal survival data with linear and nonlinear Weibull models indicated that the Weibull model best fit the observed reductions. In total, these data can serve as a guideline for regulatory agencies regarding the influence of water temperature on indicator phage after episodic sewage exposure.

Evaluation of Chlorine Treatment Levels for Inactivation of Human Norovirus and MS2 Bacteriophage during Sewage Treatment.

This study examined the inactivation of human norovirus (HuNoV) GI.1 and GII.4 by chlorine under conditions mimicking sewage treatment. Using a porcine gastric mucin-magnetic bead (PGM-MB) assay, no statistically significant loss in HuNoV binding (inactivation) was observed for secondary effluent treatments of ≤25 ppm total chlorine; for both strains, 50 and 100 ppm treatments resulted in ≤0.8-log10 unit and ≥3.9-log10 unit reductions, respectively. Treatments of 10, 25, 50, and 100 ppm chlorine inactivated 0.31, 1.35, >5, and >5 log10 units, respectively, of the norovirus indicator MS2 bacteriophage. Evaluation of treatment time indicated that the vast majority of MS2 and HuNoV inactivation occurred in the first 5 min for 0.2-µm-filtered, prechlorinated secondary effluent. Free chlorine measurements of secondary effluent seeded with MS2 and HuNoV demonstrated substantial oxidative burdens. With 25, 50, and 100 ppm treatments, free chlorine levels after 5 min of exposure ranged from 0.21 to 0.58 ppm, from 0.28 to 16.7 ppm, and from 11.6 to 53 ppm, respectively. At chlorine treatment levels of >50 ppm, statistically significant differences were observed between reductions for PGM-MB-bound HuNoV (potentially infectious) particles and those for unbound (noninfectious) HuNoV particles or total norovirus particles. While results suggested that MS2 and HuNoV (measured as PGM-MB binding) behave similarly, although not identically, both have limited susceptibility to chlorine treatments of ≤25 ppm total chlorine. Since sewage treatment is performed at ≤25 ppm total chlorine, targeting free chlorine levels of 0.5 to 1.0 ppm, these results suggest that traditional chlorine-based sewage treatment does not inactivate HuNoV efficiently.IMPORTANCE HuNoV is ubiquitous in sewage. A receptor binding assay was used to assess inactivation of HuNoV by chlorine-based sewage treatment, given that the virus cannot be routinely propagated in vitro Results reported here indicate that chlorine treatment of sewage is not effective for inactivating HuNoV unless chlorine levels are above those routinely used for sewage treatment.

Nonthermal inactivation of norovirus surrogates on blueberries using atmospheric cold plasma.

Viruses are currently the leading cause of foodborne outbreaks, most of which are associated with foods consumed raw. Cold plasma (CP) is an emerging novel nonthermal technology that can be used to surface decontaminate foods. This study investigated CP technology for the nonthermal inactivation of human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV), on the surface of blueberries. Blueberries (5 g) were weighed into sterile 4 oz. glass jars and inoculated with TV, 5 log PFU/g. Samples were treated with atmospheric CP for 0, 15, 30, 45, and 60 s at a working distance of 7.5 cm with 4 cubic feet/minute (cfm) of CP jet. Temperature readings were taken with an infrared camera prior to, and immediately following, CP treatments. In order to establish the impact of air flow during CP treatment (4 cfm), an additional 7 cfm jet of room temperature air was introduced from a separate nozzle. The experiment was repeated with 90 and 120 s as additional treatment time points. Viral titers were measured immediately after each treatment with a plaque assay using LLC-MK2 cells (TV) or RAW 264.7 cells (MNV). TV was significantly reduced 1.5 PFU/g compared to the control after treatment time of 45s, which was achieved regardless of temperature conditions. With the addition of 7 cfm of ambient air, the maximum log reduction for TV was 3.5 log PFU/g after 120s of treatment. MNV was significantly reduced by 0.5 log PFU/g compare to the control at 15s, and further treatment of MNV with ambient air brought the log reduction to greater than 5 log PFU/g at 90 s of treatment (Fig. 3). These results demonstrate that CP viral inactivation does not rely on thermal inactivation, and is therefore nonthermal in nature. With further optimization, CP may be used by food processors as a means of nonthermal inactivation of foodborne viruses.

Emerging Foodborne and Agriculture-Related Viruses.

Viruses rapidly evolve and can emerge in unpredictable ways. Transmission pathways by which foodborne viruses may enter human populations and evolutionary mechanisms by which viruses can become virulent are discussed in this chapter. A majority of viruses emerge from zoonotic animal reservoirs, often by adapting and infecting intermediate hosts, such as domestic animals and livestock. Viruses that are known foodborne threats include hepatitis E virus, tick-borne encephalitis virus, enteroviruses, adenovirus, and astroviruses, among others. Viruses may potentially evolve and emerge as a result of modern agricultural practices which can concentrate livestock and bring them into contact with wild animals. Examples of viruses that have emerged in this manner are influenza, coronaviruses such as severe acute respiratory syndrome and Middle East respiratory syndrome, and the Nipah virus. The role of bats, bush meat, rodents, pigs, cattle, and poultry as reservoirs from which infectious pathogenic viruses emerge are discussed.

Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses.

UNLABELLED: Human 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 propagated in 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. IMPORTANCE: Human 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 uncultivable in 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.

Temperature-Dependent Persistence of Human Norovirus Within Oysters (Crassostrea virginica).

This study characterizes the persistence of human norovirus in Eastern oysters (Crassostrea virginica) held at different seawater temperatures. Oysters were contaminated with human norovirus GI.1 (Norwalk strain 8FIIa) by exposing them to virus-contaminated water at 15 °C, and subsequently holding them at 7, 15, and 25 °C for up to 6 weeks. Viral RNA was extracted from oyster tissue and hemocytes and quantitated by RT-qPCR. Norovirus was detected in hemocytes and oysters held at 7 and 15 °C for 6 weeks and in hemocytes and oysters held at 25 °C for up to 2 and 4 weeks, respectively. Results confirm that NoV is quite persistent within oysters and demonstrate that cooler water temperatures extend norovirus clearance times. This study suggests a need for substantial relay times to remove norovirus from contaminated shellfish and suggests that regulatory authorities should consider the effects of water temperature after a suspected episodic norovirus-contamination event.

Pathogen reduction in human plasma using an ultrashort pulsed laser.

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma.

Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser.

BACKGROUND: Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses--non-enveloped, icosahedral viruses remains unknown. RESULTS AND DISCUSSIONS: We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. CONCLUSION: We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future.

Inactivation of human norovirus in contaminated oysters and clams by high hydrostatic pressure.

Human norovirus (NoV) is the most frequent causative agent of food-borne disease associated with shellfish consumption. In this study, the effect of high hydrostatic pressure (HHP) on inactivation of NoV was determined. Genogroup I.1 (GI.1) or genogroup II.4 (GII.4) NoV was inoculated into oyster homogenates and treated at 300 to 600 MPa at 25, 6, and 1°C for 5 min. After HHP, samples were treated with RNase and viral particles were extracted with porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs). Viral RNA was then quantified by real-time reverse transcription (RT)-PCR. Since PGM contains histo-blood group-like antigens, which can act as receptors for NoV, deficiency for binding to PGM is an indication of loss of infectivity of NoV. After binding to PGM-MBs, RT-PCR-detectable NoV RNA in oysters was reduced by 0.4 to >4 log10 by HHP at 300 to 600 MPa. The GI.1 NoV was more resistant to HHP than the GII.4 NoV (P < 0.05). HHP at lower temperatures significantly enhanced the inactivation of NoV in oysters (P < 0.05). Pressure treatment was also conducted for clam homogenates. Treatment at 450 MPa at 1°C achieved a >4 log10 reduction of GI.1 NoV in both oyster and clam homogenates. It is therefore concluded that HHP could be applied as a potential intervention for inactivating NoV in raw shellfish. The method of pretreatment of samples with RNase, extraction of viral particles using PGM-MB binding, and quantification of viral RNA using RT-PCR can be explored as a practical means of distinguishing between infectious and noninfectious NoV.

Inactivation of human norovirus using chemical sanitizers.

The porcine gastric mucin binding magnetic bead (PGM-MB) assay was used to evaluate the ability of chlorine, chlorine dioxide, peroxyacetic acid, hydrogen peroxide, and trisodium phosphate to inactivate human norovirus within 10% stool filtrate. One-minute free chlorine treatments at concentrations of 33 and 189 ppm reduced virus binding in the PGM-MB assay by 1.48 and 4.14 log10, respectively, suggesting that chlorine is an efficient sanitizer for inactivation of human norovirus (HuNoV). Five minute treatments with 5% trisodium phosphate (pH~12) reduced HuNoV binding by 1.6 log10, suggesting that TSP, or some other high pH buffer, could be used to treat food and food contact surfaces to reduce HuNoV. One minute treatments with 350 ppm chlorine dioxide dissolved in water did not reduce PGM-MB binding, suggesting that the sanitizer may not be suitable for HuNoV inactivation in liquid form. However a 60-min treatment with 350 ppm chlorine dioxide did reduce human norovirus by 2.8 log10, indicating that chlorine dioxide had some, albeit limited, activity against HuNoV. Results also suggest that peroxyacetic acid has limited effectiveness against human norovirus, since 1-min treatments with up to 195 ppm reduced human norovirus binding by <1 log10. Hydrogen peroxide (4%) treatment of up to 60 min resulted in minimal binding reduction (~0.1 log10) suggesting that H2O2 is not a good liquid sanitizer for HuNoV. Overall this study suggests that HuNoV is remarkably resistant to several commonly used disinfectants and advocates for the use of chlorine (sodium hypochlorite) as a HuNoV disinfectant wherever possible.

Temperature Effects for High-Pressure Processing of Picornaviruses.

Investigation of the effects of pre-pressurization temperature on the high-pressure inactivation for single strains of aichivirus (AiV), coxsackievirus A9 (CAV9) and B5 (CBV5) viruses, as well as human parechovirus-1 (HPeV) was performed. For CAV9, an average 1.99 log10 greater inactivation was observed at 4 °C after a 400-MPa-5-min treatments compared to 20 °C treatments. For CBV5, an average of 2.54 log10 greater inactivation was noted after 600-MPa-10-min treatments at 4 °C in comparison to 20 °C treatments. In contrast, inactivation was reduced by an average of 1.59 log10 at 4 °C for HPeV. AiV was resistant to pressure treatments of 600 MPa for as long as 15 min at 4, 20, and 30 °C temperatures. Thus, different pre-pressurization temperatures result in different inactivation effects for picornaviruses.

High Pressure Processing of Bivalve Shellfish and HPP's Use as a Virus Intervention.

Bivalve shellfish readily bioconcentrate pathogenic microbes and substance, such as algal and dinoflagulate toxins, fecal viruses and bacteria, and naturally present vibrio bacteria. High pressure processing (HPP) is currently used as an intervention for Vibrio vulnificus bacteria within molluscan shellfish and its potential to inactivate food-borne viruses and bacteria are discussed. Mechanisms of action of high pressure against bacteria and viruses, as well as how time of pressure application, pressure levels, and pre-pressurization temperature influence inactivation are described. Matrix influences such as ionic strength are noted as important additional considerations. The potential of HPP to influence spoilage and enhance shelf-life of shucked shellfish is also discussed.

The influence of temperature, pH, and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses.

Detection of human norovirus (HuNoV) usually relies on molecular biology techniques, such as qRT-PCR. Since histo-blood group antigens (HBGAs) are the functional receptors for HuNoV, HuNoV can bind to porcine gastric mucin (PGM), which contains HBGA-like antigens. In this study, PGM-conjugated magnetic beads were used to collect and quantify potentially infectious HuNoV strains GI.1 and GII.4 treated by high hydrostatic pressure (HHP). Both GI.1 and GII.4 strains used in this study showed increasing pressure sensitivity as judged by loss of PGM binding with decreasing temperature over a range of 1 to 35 °C. Both GI.1 and GII.4 were more resistant to pressure at pH4 than at neutral pH. Because GI.1 was significantly more resistant to pressure than GII.4, it was used to evaluate HuNoV pressure inactivation in blueberries. GI.1 on dry blueberries was very resistant to pressure while immersion of blueberries in water during pressure treatments substantially enhanced the inactivation. For example, a 2 min-600 MPa treatment of dry blueberries at 1 and 21 °C resulted in <1-log reductions while a 2.7-log reduction of GI.1 was achieved by a treatment at 500 MPa for 2 min at 1 °C when blueberries were immersed in water. In total, this novel study provides unique information for designing pressure processing parameters (pressure, temperature, and time) and product formulations (such as pH) to inactivate HuNoV in high-risk foods such as berries.

Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.