A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

Fresh produce is often a high-risk food for norovirus contamination because it can become contaminated at both preharvest and postharvest stages and it undergoes minimal or no processing. Currently, there is no effective method to eliminate the viruses from fresh produce. This study systematically investigated the effectiveness of high-pressure processing (HPP) on inactivating murine norovirus (MNV-1), a surrogate for human norovirus, in aqueous medium and fresh produce. We demonstrated that MNV-1 was effectively inactivated by HPP. More than a 5-log-PFU/g reduction was achieved in all tested fresh produce when it was pressurized at 400 MPa for 2 min at 4°C. We found that pressure, pH, temperature, and food matrix affected the virus survival in foods. MNV-1 was more effectively inactivated at 4°C than at 20°C in both medium and fresh produce. MNV-1 was also more sensitive to HPP at neutral pH than at acidic pH. We further demonstrated that disruption of viral capsid structure, but not degradation of viral genomic RNA, is the primary mechanism of virus inactivation by HPP. However, HPP does not degrade viral capsid protein, and the pressurized capsid protein was still antigenic. Overall, HPP had a variable effect on the sensorial quality of fresh produce, depending on the pressure level and type of product. Taken together, HPP effectively inactivated a human norovirus surrogate in fresh produce with a minimal impact on food quality and thus can provide a novel intervention for processing fruits intended for frozen storage and related products such as purees, sauces, and juices.

Human norovirus (HuNoV) is the leading causative agent of foodborne disease outbreaks worldwide. HuNoV is highly stable, contagious, and only a few virus particles can cause illness. However, HuNoV is difficult to study because of the lack of an efficient in vitro cell culture system or a small animal model. To date, there is very limited information available about the biology of HuNoV, with most data coming from the study of surrogates, such as HuNoV virus-like particle (VLP), murine norovirus (MNV), and feline calicivirus (FCV). High-risk foods for HuNoV contamination include seafood, fresh produce, and ready-to-eat foods. Currently, there is no effective measure to control HuNoV outbreaks; thus, development of food-processing technologies to inactivate HuNoV in these high-risk foods is urgently needed. Although a VLP-based vaccine induces humoral, mucosal, and cellular immunities in animals and currently is in human clinical trials, development of other new vaccine candidates, such as live vectored vaccines, should be considered. Recent evidence suggests that blockage of virus-receptor interaction may be a promising antiviral target. To enhance our capability to combat this important agent, there is an urgent need to develop multidisciplinary, multi-institutional integrated research and to implement food virology education and extension programs nationwide.

Foodborne outbreaks of viral origin have become increasingly a serious public health concern. High-pressure processing (HPP), a nonthermal technology, has come to the forefront for food processing given its minimal effects on food quality. Recent studies have revealed encouraging results for the inactivation of several human viruses by HPP. This review provides comprehensive information on the use of HPP to eliminate viruses in model systems and foods. We address the influences of various parameters, including pressure level, holding time, pH, temperature, and food matrix on the efficacy of pressure inactivation of viruses, as well as insight into the mechanisms for inactivation of enveloped and nonenveloped viruses. HPP is a promising technology for mitigating virus contamination of foods, thus it is essential to identify the optimal parameters for enhancing virus inactivation while ensuring sensory and nutritional quality retention of foods.

Human norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food-and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.

f Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin-magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.A pproximately 7.6 million to 14.5 million illnesses in the United States are attributed to the consumption of contaminated seafood each year, and enteric viruses are responsible for more than 50% of these cases (1). In a review of the available epidemiological evidence, human norovirus (HuNoV) and hepatitis A virus (HAV) were the leading viruses associated with shellfish, accounting for 83.7% and 12.8% of outbreaks, respectively (2). The type of shellfish most frequently associated with viral outbreaks was oysters, which were the vehicle in 58.4% of outbreaks (2). In some regions, human enteric viruses are practically ubiquitous in harvested shellfish. Keller et al. (3) showed that 100% of shellfish samples collected from Vitória Bay, Espírito Santo, Brazil, were positive for rotavirus (RV) and adenovirus. However, only 80% of the growing water samples were positive for these pathogens. Viral titers were 400 times higher in the shellfish samples than in the growing water, indicating high levels of natural bioaccumulation (3). In the Galician Rias area, the largest shellfish production area in the European Union, 55% of mussel, clam, and cockle samples were contaminated by HuNoV genogroup I (GI) and GII and HAV (4). Thus, understanding of the ecology and persistence of enteric viruses in shellfish is needed to help prevent future outbreaks.The consumption of uncooked contaminated bivalve shellfish continues to pose a public ...

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.High-pressure processing (HPP) is a nonthermal pasteurization technology that uses pressure instead of thermal energy to inactivate harmful pathogens. In the food industry, HPP has been used to inactivate food-borne infectious agents (such as bacteria, viruses, fungi, protozoa, and prions) and proteins (such as enzymes, allergens, and toxins) (2, 3, 6, 9). The primary advantage of HPP is that it has minimal effect on the organoleptic and nutritional properties of foods compared to other processing methods, since the treatment does not disrupt the covalent bonds stabilizing the structure of micronutrients as well as color and flavor compounds (3,5,23,39). In addition, since pressure acts instantaneously and uniformly throughout the pressure vessel, the structure and texture of many high-moisture foods are minimally affected. In today's modern society, consumers are increasingly demanding food products that are minimally processed and free of preservatives. Thus, HPP is becoming a widely used nonthermal processing technology that can ensure food safety, food quality, shelf-life extension, and nutritional value.Despite the extensive research on the inactivation of bacterial pathogens by HPP, the inactivation of viruses by HPP is less well understood. With regard to applications in the area of food safety, HPP studies have focused on food-and waterborne virus...

e 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 (ClO 2 ) 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 ClO 2 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 10 7 PFU/coupon. The samples were treated with ClO 2 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 ClO 2 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 ClO 2 gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO 2 gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces. Human norovirus (NoV) is the most prevalent cause of foodborne illnesses worldwide (1-3). This etiological agent accounts for more than 58% of all foodborne illnesses, causing 5.5 million cases of acute gastroenteritis in the United States annually (1). It is estimated that human NoV is responsible for more than 95% of nonbacterial acute gastroenteritis. Human NoV transmission occurs primarily through the fecal-oral route, either via person-to-person contact or contaminated food, water, fomites, and environmental surfaces (4, 5), and airborne transmission of viral particles may also be possible due to aerosolized vomitus or fecal material (6). Human NoV is highly contagious, with an infectious dose as low as 10 particles, and outbreaks often occur in confined settings, including restaurants, coach buses, hotels, nursing homes, hospitals, and cruise ships (7-11). Although human NoV causes significant health and emotional burdens, research on human NoV has been hampered due to the lack of an in vitro cell culture method and a small animal model (12). Therefore, we must rely on proper surrogates to study the survival of human NoV. Currently, cultivable animal caliciviruses, such as murine norovirus (MNV), feline calicivirus (FCV), and Tulane virus (TV), are used as surrogates for the study of human NoV (13,14). Studies have shown that MNV is more resistant to acid, heat, and environmental stresses than FCV (15). While MNV and TV have similar long-term storage stability and resistance to heat ...