Disinfection efficiency of chlorine dioxide and peracetic acid against MNV-1 and HAV in simulated soil-rich wash water.

Wash water from fresh vegetables and root vegetables is an important vehicle for foodborne virus transmission. However, there is lack of assessing rapid viral inactivation strategies in wash water characterized by a high soil content at the post-harvest stage. Considering the significance of food safety during the washing stage for fresh and root vegetable produce prior to marketing, we assessed the inactivation efficacy by using chlorine dioxide (ClO2) and peracetic acid (PAA) against a surrogate of human norovirus (murine norovirus 1, MNV-1) and hepatitis A virus (HAV), in wash water containing black soil and clay loam. The results indicated that MNV-1 and HAV were reduced to the process limit of detection (PLOD), with reductions ranging from 4.89 to 6.35 log10 PFU, and 4.63 to 4.96 log10 PFU when treated with ClO2 at 2.5 ppm for 10 mins. Comparatively, when treated with 500 ppm of PAA for 10 mins, MNV-1 and HAV were maximum reduced to 1.75 ± 0.23 log10 PFU (4.50 log10 PFU reduction) and 2.13 ± 0.12 log10 PFU (2.72 log10 PFU reduction). This demonstrated the efficacy of ClO2 in eliminating foodborne viruses in soil-rich wash water. When we validated the recovery of the virus from two types of wash water, the pH (9.24 ± 0.33 and 5.95 ± 0.05) had no impact on the recovery of MNV-1, while the recovery of HAV was less than 1 %. By adjusting the pH to a neutral level, recovery of HAV and its RNA levels was increased to 15.94 and 3.89 %. Thus, this study emphasized the critical role of pH in the recovery of HAV from the complex soil-rich aqueous environment, and the efficacy of ClO2 serving as a pivotal reference for the development of control strategies against foodborne viruses in the supply chain of fresh and root vegetables.

Comparison of the virucidal efficacy of essential oils (cinnamon, clove, and thyme) against hepatitis A virus in suspension and on food-contact surfaces.

Essential oils (EOs) have been used for centuries as flavor enhancers in foods, and owing to their antimicrobial properties, they have potential as natural food preservatives. However, their effect on food-borne viruses is unknown. Therefore, in this study, the virucidal effects of three EOs (cinnamon, clove, and thyme) on the infectivity of the hepatitis A virus (HAV) were investigated. Different concentrations of each EO (0.05, 0.1, 0.5, and 1%) were mixed with viral suspensions in accordance with ASTM E1052-11:2011 and incubated for 1 h at room temperature. The EOs exhibited a concentration-dependent effect in the suspension tests, and HAV titers decreased by approximately 1.60 log PFU/mL when treated with EOs at the highest concentration of 1%. The antiviral effect of EOs treated at 1% for 1 h was also evidenced in surface disinfection tests according to the OECD:2013, as approximately 2 log PFU/mL reduction on hard food-contact surfaces (stainless steel and polypropylene) and approximately 2 and 1.4 log PFU/mL reduction on low-density polyethylene and kraft (soft food-contact surfaces), respectively. Moreover, RT-qPCR results revealed that HAV genome copies were negligibly reduced until treated with a high concentration (1%) in suspension and carrier tests. Overall, our findings highlighted the potential of cinnamon, clove, and thyme EOs as natural disinfectants capable of limiting HAV (cross-) contamination conveyed by food-contact surfaces. These findings advance our knowledge of EOs as antimicrobials and their potential in the food sector as alternative natural components to reduce viral contamination and improve food safety.

Quantitative Risk Assessment of Hepatitis a Virus Infection Arising from the Consumption of Fermented Clams in South Korea.

This study estimated the risk of hepatitis A virus (HAV) foodborne illness outbreaks through the consumption of fermented clams in South Korea. HAV prevalence in fermented clams was obtained from the Ministry of Food and Drug Safety Report, 2019. Fermented clam samples (2 g) were inoculated with HAV and stored at -20-25 °C. Based on the HAV titer (determined using plaque assay) in fermented clams according to storage, the Baranyi predictive models provided by Combase were applied to describe the kinetic behavior of HAV in fermented clams. The initial estimated HAV contamination level was -3.7 Log PFU/g. The developed predictive models revealed that, when the temperature increased, the number of HAV plaques decreased. The Beta-Poisson model was chosen for determining the dose-response of HAV, and the simulation revealed that there was a 6.56 × 10-11/person/day chance of contracting HAV foodborne illness by eating fermented clams. However, when only regular consumers of fermented clams were assumed as the population, the probability of HAV foodborne illness increased to 8.11 × 10-8/person/day. These results suggest that, while there is a low likelihood of HAV foodborne illness from consuming fermented clams across the country, regular consumers should be aware of the possibility of foodborne illness.

Assessing the Removal Efficiency of Murine Norovirus 1, Hepatitis A Virus, and Human Coronavirus 229E on Dish Surfaces Through General Wash Program of Household Dishwasher.

The performance of dishwashers in removing live viruses is an important informative value in practical applications. Since foodborne viruses are present in contaminated food surfaces and water environments. Insufficient washing of dishes typically makes a carrier of foodborne viruses. Dishwashers have shown excellent performance in removing bacterial pathogens, but very limited reports related to eliminate foodborne viruses on contaminated dish surfaces. Here, murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and human coronavirus 229E (HCoV-229E) were experimentally inoculated on the dish surfaces (plate, rice bowl, and soup bowl). Plaque assay, 50% tissue culture infectious dose (TCID50), and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to determine their removal efficiency of them through the general wash program of household dishwashers. Using titration assay, MNV-1 and HAV were reduced by 7.44 and 6.57 log10 PFU/dish, and HCoV-229E was reduced by 6.43 log10 TCID50/dish through the general wash program, achieving a ≥ 99.999% reduction, respectively. Additionally, RT-qPCR results revealed that viral RNA of MNV-1 and HCoV-229E reduced 5.02 and 4.54 log10 genome copies/dish; in contrast, HAV was not detected on any dish surfaces. This study confirmed the performance of household dishwashers in removing pathogenic live viruses through the general wash program. However, residual viral RNA was not sufficiently removed. Further studies are needed to determine whether the viral RNA can be sufficiently removed using combination programs in household dishwashers.

Viability of SARS-CoV-2 on lettuce, chicken, and salmon and its inactivation by peracetic acid, ethanol, and chlorine dioxide.

Since the first SARS-CoV-2 outbreak in Wuhan, China, there has been continued concern over the link between SARS-CoV-2 transmission and food. However, there are few studies on the viability and removal of SARS-CoV-2 contaminating food. This study aimed to evaluate the viability of SARS-CoV-2 on food matrices, depending on storage temperature, and inactivate the virus contaminating food using disinfectants. Two SARS-CoV-2 strains (L and S types) were used to contaminate lettuce, chicken, and salmon, which were then stored at 20,4 and -40 °C. The half-life of SARS-CoV-2 at 20 °C was 3-7 h but increased to 24-46 h at 4 °C and exceeded 100 h at -40 °C. SARS-CoV-2 persisted longer on chicken or salmon than on lettuce. Treatment with 70% ethanol for 1 min inactivated 3.25 log reduction of SARS-CoV-2 inoculated on lettuce but not on chicken and salmon. ClO2 inactivated up to 2 log reduction of SARS-CoV-2 on foods. Peracetic acid was able to eliminate SARS-CoV-2 from all foods. The virucidal effect of all disinfectants used in this study did not differ between the two SARS-CoV-2 strains; therefore, they could also be effective against other SARS-CoV-2 variants. This study demonstrated that the viability of SARS-CoV-2 can be extended at 4 and -40 °C and peracetic acid can inactivate SARS-CoV-2 on food matrices.

Stability and inactivation of SARS-CoV-2 on food contact surfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 269 million people and killed more than 5.3 million people worldwide. Although fomite transmission of SARS-CoV-2 has been continuously reported, few studies have been conducted on food contact surfaces. Therefore, this study aimed to investigate the viability of coronaviruses on food contact surfaces and to remove SARS-CoV-2 contaminated on food contact surfaces with disinfectants. At 20 °C, SARS-CoV-2 was inactivated within 48 h on all food contact surfaces. At 4 °C, it was inactivated at 48 h on kraft paper and 96 h on parchment paper, but it was viable up to 5 days in low-density polyethylene (LDPE). At -20 °C, SARS-CoV-2 did not decrease by even 1 log on all food contact surfaces until 5 days. Treatment with 70% ethanol or 1000 ppm sodium hypochlorite for 5 min was sufficient to completely remove SARS-CoV-2 from 6 food contact surfaces. Similarly, UV-C irradiation at 60 mJ/cm2 eliminated SARS-CoV-2 contaminated on food contact surfaces. Also, the wiping test showed that even wiping an area contaminated with SARS-CoV-2 with a cloth moistened with 70% ethanol or 1000 ppm sodium hypochlorite, it took 5 min to inactivate the virus. Our findings suggested that SARS-CoV-2 contaminated on food contact surfaces in local retail may be viable enough to be transported home. However, if the type and method of use of the disinfectant suggested in this study are followed, it is possible to sufficiently control the fomite transmission of SARS-CoV-2 through food contact surfaces at home.

Combination of paper membrane-based filtration and ultrafiltration to enhance the detection of MNV, HAV, and HCoV from soil-rich post-washing water.

Risk-assessing and controlling virus transmission from soil-rich post-washing water (PWW) are crucial during harvesting raw vegetables. However, viruses are normally difficult to concentrate because of their low concentrations and complex backgrounds. Here, ultrafiltration (UF), virus adsorption-elution (VIRADEL), and optimized paper filtration-coupled ultrafiltration (PFC-UF) methods were employed to evaluate the recovery of non-enveloped murine norovirus (MNV-1), hepatitis A virus (HAV), and enveloped human coronavirus 229E (HCoV-229E) from soil-rich PWW. Among the three methods, PFC-UF outperformed the other methods in the recovery of viruses from PWW with soil content. Under the highest soil condition with virus seeded at a titer of 102 plaque-forming unit (PFU) or TCID50, the PFC-UF method exhibited an exceedingly consistent recovery rate of 78.8 ± 13.3 (MNV-1) and 44.4 ± 25.2% (HAV). However, the recovery of enveloped HCoV-229E was inferior to non-enveloped viruses. Overall, PFC-UF provided a reliable method for recovering viruses in soil-rich PWW.

Prevalence and phylogenetic analysis of human enteric emerging viruses in porcine stool samples in the Republic of Korea.

Emerging infectious diseases (EID) in humans and animals are proving to be a serious health concern. This study investigated the prevalence of emerging or re-emerging human enteric viruses in porcine stools and swabs. Eleven enteric EID viruses were selected as target viruses for the current study and ranked based on their impact on public health and food safety: enterovirus (EV), hepatitis E virus, norovirus GI and GII, sapovirus (SaV), adenovirus (AdV), astrovirus, rotavirus, hepatitis A virus, aichivirus, and bocavirus. Using real-time RT-PCR or real-time PCR, EID viruses were detected in 129 (86.0%) of 150 samples. The most prevalent virus was EV, which was detected in 68.0% of samples, followed by AdV with a detection rate of 38.0%. In following sequencing and phylogenetic analyses, 33.0% (58/176) of the detected viruses were associated with human enteric EID viruses, including AdV-41, coxsackievirus-A2, echovirus-24, and SaV. Our results show that porcine stools frequently contain human enteric viruses, and that few porcine enteric viruses are genetically related to human enteric viruses. These findings suggest that enteric re-emerging or EID viruses could be zoonoses, and that continuous monitoring and further studies are needed to ensure an integrated "One Health" approach that aims to balance and optimize the health of humans, animals, and ecosystems.

Improvement of nested reverse transcription-polymerase chain reaction (RT-PCR) with high specificity and sensitivity detection of sapovirus in food matrix.

Sapovirus (SaV) is a causative agent of human gastroenteritis in both community outbreaks and sporadic cases worldwide. Shellfish accumulate a variety of pathogens during filter feeding. In particular, the contamination of shellfish by SaV has caused several outbreaks. As reported previously, nested RT-PCR (nRT-PCR) has been widely used in clinical samples, but has not proven suitable for food samples, such as oysters. This study aimed to identify a primer set for the detection of SaV with high specificity and sensitivity in food samples. To accomplish this, primers were improved in RNA-dependent RNA polymerase (RdRp) regions of SaV whole genome sequences. The sensitivity of the improved nRT-PCR was 100-1000 times higher than that of previous nRT-PCR and > 10 times higher than that of the previous real-time RT-PCR assay. Notably, cross-reaction with other viruses or food matrices was not observed by the specificity test. This study improved the reliable primer set to detect SaV in various food matrices with high sensitivity.

Comparison of virucidal efficacy of sodium hypochlorite, chlorine dioxide, peracetic acid, and ethanol against hepatitis A virus by carrier and suspension tests.

A carrier (stainless steel disc as a default carrier) testing method is very needed for use in the actual food-processing fields by following the standard guideline. Here, we aimed to compare the virucidal efficacy of four commercial liquid disinfectants, including sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), and peracetic acid (PAA) against hepatitis A virus (HAV) following the OECD guideline protocol based on the quantitative carrier testing method and compared carrier testing results with the suspension testing results. The OECD method specifies a test for establishing whether a chemical disinfectant or a microbicide has a virucidal activity on hard non-porous surfaces. The antiviral efficacy was evaluated by plaque assays, and disinfectants were considered effective if the virus reduction was greater than or equal to 3 log10 (99.9% decrease) for carrier or 4 log10 (99.99% decrease) for suspension tests. Results indicated that ClO2 above 500 ppm and 50% ethanol were effective in the carrier test method. In contrast, more than 200 ppm NaOCl and 50 ppm ClO2 for all exposure times and 70% ethanol with contact for more than 5 min were effective in suspension tests. Treatment with PAA (80-2500 ppm) were not effective in carrier or suspension tests. Therefore, we recommend the use of more than 500 ppm ClO2 or 50% ethanol with exposure for 10 min to disinfect surfaces that may be contaminated with HAV. Thus, these results could be effective in establishing official antiviral efficacy testing methods and basic data.

Effects of Weather and Environmental Factors on the Seasonal Prevalence of Foodborne Viruses in Irrigation Waters in Gyeonggi Province, Korea.

This study aimed to investigate the prevalence of foodborne viruses in reservoirs (an important resource of irrigation water) and its correlation with environmental and weather factors. From May 2017 to November 2018, we visited ten reservoirs and a river in the Anseong region of South Korea and collected a total of 192 samples in accordance with the environment protection agency guidelines. We recorded the weather factors (temperature, humidity, and accumulated precipitation) and investigated the surrounding environment factors (livestock, fishing site, the catchment area of reservoirs, etc.). Our research results show that from the river and reservoirs, the detection rates of human norovirus GII, adenovirus, rotavirus, human norovirus GI, and astrovirus were 27.1, 10.4, 10.4, 4.16, and 3.1%, respectively. Their viral load ranged from -1.48 to 1.55 log10 genome copies/l. However, hepatitis A virus was not detected in any irrigation water sample. Although no sampling was performed in winter, foodborne viruses and male-specific coliphages were frequently found during spring (40.78%) and autumn (39.47%). Interestingly, the significant correlation between the accumulative precipitation and the number of detected norovirus and adenovirus was confirmed by linear regression analysis. Furthermore, when the accumulative precipitation ranged from 20 to 60 mm, it significantly affected the viral load and prevalence. Among the environmental factors, recreational facilities such as fishing sites and bungalow fishing spots were identified as contamination sources by correlation analysis. Our research results confirmed the correlations between environmental contamination factors in the reservoir and weather factors with the prevalence of foodborne viruses in the reservoir. These facilitates the assessment of potential foodborne virus contamination during crop irrigation. In addition, predictive models including environmental and weather factors should be developed for monitoring and controlling the safety of irrigation waters in reservoirs.

Experimental infection of hepatitis E virus induces pancreatic necroptosis in miniature pigs.

Infection by hepatitis E virus (HEV) via the oral route causes acute hepatitis. Extra-hepatic manifestations of HEV infection may stem from various causes; however, its distribution in organs such as the liver, as well as the mechanisms underlying HEV-induced cell injury, remain unclear. The objective of this study was to determine the chronological distribution of HEV in various tissues of HEV-challenged miniature pigs and to investigate the mechanisms underlying HEV-induced cell death in the pancreas and liver. Virological and serological analyses were performed on blood and faecal samples. Histopathology of the liver and extra-hepatic tissues was analysed. Cell death pathways and immune cell characterisation in inflammatory lesions were analysed using immunohistochemistry. The liver and pancreas displayed inflammation and cellular injury, and a large amount of HEV was observed in the lesions. The liver was infiltrated by T and natural killer cells. HEV was identified in all organs except the heart, and was associated with immune cells. Although the liver and the pancreas strongly expressed TNF-α and TRAIL, TUNEL assay results were negative. RIP3 and pMLKL were expressed in the pancreas. RIP3, but not pMLKL, was expressed in the liver. Pancreatitis induced in HEV-infected miniature pigs is associated with necroptosis.

Foodborne Viruses Detected Sporadically in the Fresh Produce and Its Production Environment in South Korea.

Contamination of fresh vegetables and berries with human enteric viruses is a major cause of food poisoning. The aim of this study was to investigate the prevalence of norovirus GI, norovirus GII, hepatitis A virus (HAV), adenovirus, astrovirus, rotavirus, and male-specific coliphage systematically in fresh fruit and vegetables and associated agricultural environmental samples, including irrigation water, soil, and worker's gloves. Enteric viruses were detected by international standard methods (ISO/TS 15216), and male-specific coliphages were isolated using US EPA Method 1601. For the study, 773 samples were collected from June 2016 to April 2017, including Chinese cabbage (n = 244), cucumber (n = 98), lettuce (n = 73), strawberry (n = 120), soil (n = 191), irrigation water (n = 14), and gloves (n = 27). Two cucumber and two irrigation water samples were positive for norovirus GI, and one cucumber and two irrigation water samples were positive for norovirus GII. HAV was detected in one strawberry sample and one glove sample. The other tested foodborne viruses were not detected in any of the samples. Sixteen male-specific coliphages were isolated from Chinese cabbage, cucumber, lettuce, cherry tomato, soil, and irrigation water. The isolation of male-specific coliphage would be more practical to investigate the fecal contamination in produce rather than pathogenic viruses.