ABSTRACT

Human noroviruses are enteric pathogens that cause a substantial proportion of acute gastroenteritis cases worldwide regardless of background variables such as age, ethnicity, and gender. Although person-to-person contact is the general route of transmission, foodborne infections are also common. Thorough cooking eliminates noroviruses, but several food products such as berries, leafy vegetables, and mollusks undergo only limited heat treatment, if any, before consumption. Novel applications of nonthermal processing technologies are currently being vigorously researched because they can be used to inactivate pathogens and extend product shelf life with limited effects on nutrient content and perceived quality. These technologies, adopted from several industrial fields, include some methods already approved for food processing that have been applied in the food industry for years. However, a majority of the research has been conducted with bacteria and simple matrixes or surfaces. This review focuses on elimination of norovirus in food matrixes by use of nonthermal technologies in four categories: high hydrostatic pressure, light, irradiation, and cold atmospheric plasma. We discuss the properties of noroviruses, principles and inactivation mechanisms of select technologies, and main findings of relevant studies. We also provide an overview of the current status of the research and propose future directions for related work.

HIGHLIGHTS
• High pressure processing is the most promising nonthermal treatment for noroviruses.

• High pressure processing, ionizing radiation, and UVC light can reduce noroviruses in foods.

• Treatments used to eliminate viruses can impair food product quality.

• Optimal virus elimination strategies should be validated independently for each food product.

Since its identification in 1972, norovirus (NoV) has been identified, particularly after the development of applicable molecular methods, as the cause of outbreaks and sporadic cases of acute gastroenteritis worldwide (78). Globally 18% (95% confidence interval, 17 to 20%) of all diagnosed cases of acute gastroenteritis are caused by NoV, and the burden of these illnesses is similar in developed and developing countries, resulting in a universal health challenge (4, 59, 61). Bartsch et al. (11) estimated that NoV infections result in annual total losses of $60.3 billion worldwide, which can be further divided to$4.2 billion in health care costs and \$56.2 billion in productivity losses. This division of losses is similar across regions, reinforcing the universal magnitude of the societal and economic burdens associated with NoV. Although the principal mode of NoV transmission is person to person, water- and foodborne routes are also important methods of transmission (58, 93). Foodborne infections, causing an estimated 14 to 15 million illnesses and 400 deaths annually in Europe, typically result from contamination along the production line, for example, during primary processing of irrigation water and during later stages where workers or processing surfaces may be the sources of foodborne pathogens (97). Typical foods at high risk for NoV contamination are those that are not subjected to heat treatments, are manually processed, or are derived from plants or animals that live in water environments. Hence, common sources of foodborne NoV outbreaks include but are not limited to vegetables, berries, and mollusks (9, 78).

The importance of diet quality and nutrition for a healthy lifestyle have been recognized for decades, but only after the beginning of the 21st century have these attitudes shaped consumer trends dramatically by increasing the demand for nutrient-rich, minimally processed, and preservative-free products (7). The food industry, which operates in the consumer-driven market, follows trends closely and has responded to these consumer desires. However, food safety, attractive sensory properties, and long shelf life remain equally important factors for both consumers and industry. Currently, the only generally recognized method for eliminating NoV is heating a product thoroughly to >70°C for several minutes because the virus can survive for long periods at 60°C (15). Hence, the food industry and researchers have been working to develop new approaches for ensuring safe products because thorough heating is not an option for a majority of the at-risk products.

Viruses in general are less susceptible than bacteria (excluding spores and molds) to processing conditions, limiting the effectiveness of traditional techniques such as modification of pH or water activity for eliminating NoV contamination (15). Nonthermal processing techniques are potential options for controlling pathogens in products that are meant to be consumed in a minimally processed form. Although many technologies are still in the development, their benefits go beyond pathogen inactivation, because product shelf life can be extended without significant alterations in nutrient composition or sensory attributes (56). Such technologies employ stress factors such as pressure, light, or irradiation that have already been verified as effective against various bacteria and have been used for sterilization or disinfection in numerous industries. The use of technologies such as high pressure processing (HPP) and UVC and gamma irradiation have been approved and applied in food products such as salsa, juices, and spices. Nevertheless, data on NoV elimination are less available, especially in food matrixes with complex compositions and structures. To overcome the methodological issues associated with culturing NoV, a majority of the studies in food matrixes have used surrogate viruses to model NoV.

This review focuses on the recent findings regarding inactivation of NoVs or surrogates by nonthermal treatments based on the following four methodologies: pressure, light, irradiation, and cold plasma. Pressure applications were based on HPP. Identified light applications utilized different wavelengths including UVC light (253 to 260 nm), blue light (405 nm), and pulsed light (200 to 1,100 nm). Recognized irradiation applications were gamma and electron beam (e-beam) radiation, which differ by the source of the radiation. Cold atmospheric plasma (CAP) was generated by plasma jets and dielectric barrier discharge (DBD) technologies. In addition to the key findings of various studies, we also explored the properties and proposed mechanisms of inactivation of these technologies and summarized the characteristics of NoVs.

## NOROVIRUS: VIROLOGY AND RESEARCH

### Characteristics of NoVs

NoVs belong to the family Caliciviridae and the genus Norovirus, which has only one species, the Norwalk virus (95). NoVs are nonenveloped, single-stranded, positive-sense RNA viruses with a 7.5- to 7.7-kb genome that includes either three open reading frames (ORF1, ORF2 and ORF3) or four ORFs, as in the murine NoV (MNV). ORF1 encodes a polyprotein involved in the replication cycle. ORF2 encodes the major structural protein VP1, and ORF3 encodes a minor structural protein called VP2, which together form the viral capsid. The capsid protecting the viral RNA consists of 180 VP1 proteins organized into 90 dimers with a few copies of VP2 protein located inside the capsid forming a symmetrical icosahedral shape with a diameter of 27 to 40 nm (75). VP1 consists of a protruding domain P linked by a flexible hinge to the shell domain (S) surrounding the RNA. The P domain of VP1 is made up of P1 and P2, which differ among virus species and due to their binding activity are likely to be involved in determining the virus-host interaction. VP2 is likely to play a part in RNA encapsidation and capsid assembly.

The classification of NoVs is based on dual nomenclature utilizing either the amino acid sequence of the complete gene of capsid protein VP1 or the nucleotide sequence of the RNA-dependent RNA polymerase region of ORF1, marked by capital P for “polymerase” in the label, allowing a more precise description of types, variants, and recombinant forms (14). NoVs can be divided into 10 genogroups (GI through GX), and these groups have a variable number of distinct genotypes (49 in total) such as GI.1 or GII.4. Genotypes that replicate in humans belong to groups GI, GII, GIV, GVIII, and GIX although GII and GIV also have genotypes that infect porcine and feline or canine hosts. In the other groups, GIII replicates in bovine hosts, GV in murine hosts, GVI and GVII in canid hosts, and GX in bats. Some predominant variants responsible for epidemic outbreaks surfacing every 2 to 3 years due to mutations and recombination may have the geographical location of the first identified outbreak attached to the virus name. The recently proposed dual-typing nomenclature for NoV strains would first list the genotype followed by P type in brackets, for example, GII.4 Sydney [P16].

### NoV surrogates

Until recently research on the mechanistic features of human NoV (HuNoV) has been hampered by the lack of reproducible culture systems that can support a high level of replication, although successful efforts in B cells and stem cell–derived human enteroids have been recently published (20, 37). However, the increases in virus titers in these cultures are relatively modest, at 101 to 102 compared with 105 to 106 in well-established surrogate cultures that allow extensive passaging. Hence, culturable surrogate viruses resembling HuNoVs are likely to remain a mainstay for research until HuNoV culture systems permit production of high-titer stocks. The most commonly utilized HuNoV surrogates are feline calicivirus (FCV), MNV, bacteriophage MS2, and the Tulane virus (TV), a calicivirus recently discovered in rhesus macaques. MNV is genetically closest to HuNoVs, whereas FCV belongs to the genus Vesivirus and TV to Recovirus, both genera in the Caliciviridae family. Bacteriophage MS2 infects bacteria from the family Enterobacteriaceae but has also been utilized for virus research because of its structural similarities to NoV. In addition to culturable surrogates, virus-like particles assembled in infected insect cells are also commonly used for studying capsid stability and interactions (74). The virus-like particles consist of VP1 proteins forming a capsid that is morphologically and antigenically comparable to that of HuNoV but lacks the genome and VP2 proteins, both of which could contribute to capsid stability. Despite resemblance to HuNoVs, surrogates rarely have similar survival rates when challenged with physical or chemical treatments used in industrial settings (43). For example, based on real-time quantitative PCR (RT-qPCR) signals, HuNoV is significantly more persistent than MNV-1 and FCV F-9 in response to heat and chlorine treatments, respectively (43). Hence, extrapolating results from surrogate studies to HuNoVs is likely to underestimate the measures required for complete inactivation in virus-contaminated foods. Because of these differences, including those in genetic and host-virus interaction properties, no surrogate has been recognized as an adequate replica of HuNoV.

### Assessment of infectivity

The RT-qPCR assay is currently considered the “gold standard” for detecting and assessing the viral loads of HuNoVs from clinical and environmental samples because of the high sensitivity and specificity of this assay (95). However, genome-based molecular methods cannot be used to differentiate between infectious and noninfectious viruses because capsid structures needed for host-virus interactions may be rendered defective without a noticeable effect on viral RNA derived from functional or partially or completely degraded viruses. Evaluation of infectivity is the key determinant when investigating the efficacy of a virus inactivation approach in fresh foods. The lack of a well-established HuNoV cell culture model has prevented the evaluation of infectivity via plaque assays or the 50% tissue culture infectious dose (TCID50) assay, which are routinely applied with the surrogates. Hence, auxiliary approaches to discriminate functional from nonfunctional HuNoVs before use of the RT-qPCR assay involve binding infectious viruses to ligands or use of nucleic acid intercalators to prevent amplification of RNA from viruses with damaged capsids. Several studies with fresh foods have used porcine gastric mucin–conjugated magnetic beads (PGM-MBs) containing histo-blood group antigens that bind TV and HuNoV strains from the genogroups I and II (88, 89). The PGM-MBs are mixed with contaminated samples and capture infectious viruses, which can then be quantified with an RT-qPCR assay. Compared with standard detection by RT-PCR assays, a 2-log increase in detection sensitivity was obtained for PGM-MBs in phosphate-buffered saline (PBS) and in food matrixes such as lettuce, oysters, and strawberries (88). Another advantage of the PGM-MB assay is the presence of sialic acid, an attachment factor for MNV, enabling the parallel use for another surrogate with TV, in addition to HuNoV strains, within the same experimental model. Assay specificity might be further improved by pretreatment with such reagents as RNase or protease K to remove inactivated virions with remaining binding activity (36, 99). Quantification of FCV inactivation by RT-qPCR coupled with the nucleic acid intercalator ethidium monoazide (EMA) was comparable to results obtained with the RNase RT-qPCR method, yet both underestimated inactivation by ∼2 log units when compared with the cell culture method (3). These findings highlight the importance of further development and validation of methods for assessing HuNoV infectivity. In the publications reviewed here (apart from a single study utilizing EMA), only RNase and PGM-MB binding were used to augment the performance of nucleic acid–based methods for assessing virus infectivity.

## NONTHERMAL PROCESSING METHODS

### HPP

The properties of each technology are summarized in Table 1. The essential advantage of HPP is based on the isostatic principle that pressure is uniformly distributed throughout the product independent of the size or geometry. HPP is particularly useful for high-moisture foods such as vegetables and beverages because HPP does not affect covalent bonds but alters noncovalent interactions governing the secondary and tertiary structure of proteins (56). Thus, HPP preserves sensory attributes and low-molecular-mass particles contributing to the flavor and nutritional profile of the treated foods. However, optimal processing parameters for balancing pathogen inactivation with minimal changes in quality are product dependent because HPP can change the color and texture of berries or vegetables and cause berry purees to lose their thickness (55). The potential uses and descriptions of HPP have been reviewed extensively (10, 12, 96).

TABLE 1

Reviewed food processing technologies, with characteristics, mechanism of inactivation, advantages, and disadvantages

Pressure inactivation of HuNoVs and surrogates has been studied since the turn of the 21st century, yet only recently has the research shed light on the mechanism of inactivation (40). Tang et al. (87) evaluated MNV-1 capsid integrity and antigen capture with RT-PCR assays and receptor binding with enzyme-linked immunosorbent assays after HPP treatment at 400 MPa and 0°C. Virus reduction of 8.22 log PFU was obtained, but viral RNA remained intact and the remaining viruses were still capable of binding to the specific antibody. However, binding to the cell receptors was significantly affected, indicating interference with the binding proteins on the viral capsid. Capsid integrity was sustained throughout HPP, but when challenged with proteinase K, the capsid proteins were more prone to enzyme digestion than were those of untreated controls. The authors concluded that inactivation is primarily achieved through changes in the function of capsid binding proteins without significant effect on RNA or capsid integrity. These results were expanded by Lou et al. (55), who used transmission electron microscope imaging to reveal that at 350 MPa the structure of MNV-1 was partly ruptured and at >500 MPa protein debris was the primary result, indicating complete capsid degradation and loss of infectivity. However, despite the degradation of capsid structure, RNA integrity remained and capsid proteins VP1 and VP2 retained their form and antigenic properties, as indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis. This result was expected because HPP does not affect covalent bonds of nucleic acids or proteins.

Dancho et al. (16) extended these previous studies to HuNoV strains of GI.1 and GII.4 by assessing their binding to PGM-MBs after HPP, UVC, and heat treatments. These researchers also assessed the binding efficacy by dividing the PGM-MB–bound RNA equivalents by the total bound and unbound RNA equivalents in the sample. The efficacy for the untreated GII.4 strain was 69%, whereas that for the GI.1 strain was 68 to 84%. The binding to PGM-MBs was consistently reduced with increasing heat, light, or pressure. Although for HPP at 300 MPa no reductions in binding affinity were evident, 400 MPa was the threshold for significant reduction of binding by >3 log units. At 500 to 600 MPa, binding was further reduced by around 5 log units, whereas total RNA decline was around 1.5 log units, mirroring the results at 400 MPa. Such binding reductions also indicated clinical relevance in the only clinical human volunteer study reported, which was conducted by Leon et al. (48), although reduction rates were not measured. These researchers found that GI.1-seeded oysters treated with HPP at 400 MPa were not rendered safe for consumption, whereas participants who consumed oysters treated at 600 MPa had no signs of infection. Thus, these findings further support the hypothesis that the primary inactivation mechanism is modification of binding site integrity of the virus capsid rather than degradation of the whole capsid structure or the virus RNA.

### UVC light

UVC light is short-wavelength (100 to 280 nm) electromagnetic radiation with mutagenic properties attributed to photodimerization of pyrimidines and formation of other photoproducts such as pyrimidine adducts and DNA-protein cross-links (27). In addition to germicidal effects, factors such as low cost, easy handling, and absence of toxic residues have encouraged wide industrial application of UVC light for sanitizing food contact surfaces, water, and solid and liquid foods. However, effectivity is limited to surfaces and rapidly lost in turbid liquids. For RNA viruses, the nucleic acids are the primary site of inactivation, although higher levels of UVC irradiation (>1,000 mJ/cm2) can also initiate capsid denaturation (63). For FCV, several UVC wavelengths at <40 mJ/cm2 induced >3-log reductions in virus infectivity (86). Although UVC irradiation induced capsid protein oxidation, morphological analysis revealed that capsid structures were not significantly affected. However, RNA copy numbers assessed by RT-PCR assay decreased by approximately 50% after treatment. In another study, the binding of HuNoV GI.1 to PGM-MBs was reduced by 1.8 and 3.8 log units following UVC treatment with 1,000 and 2,000 mJ/cm2, respectively (16). The loss in binding was not followed by a similar reduction in RNA copies, as measured by RT-PCR assay. Hence, these results indicate that the loss of infectivity resulting from UVC treatment is due to damage to RNA and capsid proteins.

### Pulsed light

Another application utilizing the electromagnetic energy carried by light is pulsed light treatments, where lamp units produce short, rapid bursts of light over a wide range of wavelengths (200 to 1,100 nm), including UV, visible, and infrared (64). The greatest advantage of pulsed light technology is that it delivers high amounts of energy in a short time (seconds). In extended treatments, the heating effect can make this technology unsuitable for delicate products unless a cooling protocol is adopted (33). Disinfection is attributed to the UV light and energy (0.1 to 50,000 mJ/cm2), which is greater than that of treatment with continuous light delivery. However, the U.S. Food and Drug Administration has ruled that a cumulative fluence of 12,000 mJ/cm2 is the upper limit for pulsed light treatments in foods. The mechanism of virus inactivation by pulsed light was elucidated by Vimont et al. (94), who found that in an MNV-1 suspension the main targets of treatment were capsid structure, major capsid proteins, and virus RNA. Imaging revealed a mixture of debris and empty and intact virus particles, indicating degradation of the main structure of the capsid. The amount of VP1 proteins decreased by half, suggesting disintegration of the primary protein structure, and the amounts of undamaged and total RNA were decreased. These effects resulted in a total loss of infectivity and were achieved below the total fluence level of 12,000 mJ/cm2.

### Blue light

The effect of visible blue light (405 nm) on multiple microbes, such as bacteria and molds, is based on the induction by photosensitizers (molecules excited by light wavelengths) of production of oxidative agents that damage and kill cells (90). Photosensitizers may be present in the surrounding medium in exogenous or endogenous form, as for bacteria in which porphyrin rings absorb light generating singlet oxygen and other reactive oxygen species (ROS). However, viruses contain mainly proteins and nucleic acids that do not directly absorb such wavelengths, so findings from other microorganisms cannot be necessarily extrapolated to viruses. However, exogenous photosensitizers can damage virus capsids and alter the DNA-protein interactions and the DNA secondary structure, together predisposing viruses to inactivation (19). Tomb et al. (91) found that blue light inactivated bacteriophages suspended in nutrient broth; 2.7- to 7.1-log reduction was evident depending on the initial virus level and light dose. However, the reduction was only 0.3 log PFU/mL in simple PBS under otherwise similar conditions. The same authors also obtained similar results for FCV; a blue light dose of 421 J/cm2 yielded a 4.8-log reduction in nutrient-rich medium, whereas a blue light dose of 2,804 J/cm2 yielded a 3.9-log reduction in PBS (90). These findings suggest that the composition of the surrounding medium has a critical effect on inactivation; for FCV inactivation, a 5-h treatment was required in PBS and a 45-min treatment was required in nutrient-rich medium.

During irradiation, food products are subjected to ionizing energy that removes electrons from atoms or molecules (84). Reason to use of irradiation for food products include delay of ripening, inhibition of sprouting, extension of shelf life, control of pathogens, and sterilization. Gamma radiation is emitted from a radioactive source in the process of radioactive decay. Gamma rays from cobalt-60, the radiation source mainly used in food applications, carry 1.17 and 1.33 MeV and are photons without mass, permitting high penetrability as they can pass through food matrixes of various densities. The mechanism of virus inactivation by gamma radiation was elucidated by Feng et al. (21), who determined that for MNV-1 irradiation affects several factors, resulting in the loss of infectivity. The major capsid protein VP1 gradually degraded with increasing radiation dose, and by 22.4 kGy the VP1 proteins were undetectable. Similar to the VP1 proteins, the amount of intact virion structures was reduced with increasing gamma ray dose, and at 22.4 kGy only debris was visible. Doses of 2.8 and 5.6 kGy decreased RNA concentrations and virus titers, and complete degradation occurred at 22.4 kGy. The capsid stability of HuNoV-like particles was comparable to that of MNV-1. However, doses of >11.2 kGy were required to produce significant reductions (>3 log units) in infectivity in buffer solutions.

Under current guidelines, adsorbed radiation doses for assuring microbiological safety of foods may exceed 10 kGy because such doses have been proven safe and not nutritionally detrimental (5). Nevertheless, authorities have stated that irradiation should not be a substitute for hygienic production protocols, applied doses should be the lowest possible to meet the technological need, and treated products must be clearly labeled. In the European Union (EU), irradiation of dried aromatic herbs, spices, and vegetable seasonings are authorized in every member state, and some member states have temporary permission to treat additional foodstuffs. In the United States, regulations are more permissive for doses in specific food categories and labeling. Limits for absorbed radiation in food categories at high risk for NoV are 1 to 4 kGy in the United States and 0.15 to 2 kGy in the EU for vegetables or berries and up to 5.5 kGy in the United States and 3 kGy in the EU for shellfish (5, 71).

With e-beam technology, irradiation is produced electrically by generating electrons and accelerating them by use of electromagnetic fields (84). The perks of e-beams are their controllability; they can be targeted to a small area and switched off when necessary. Accelerated electrons typically are capable of reaching energy levels up to 10 MeV, generating 10 times more energy than generated by gamma rays, making e-beams more cost-efficient and enabling rapid treatment times. However, decontamination by e-beams is limited to the outer surfaces because effective penetration depths are 3 to 10 cm depending on the food material. As ionizing irradiation, e-beams share the inactivation mechanism with other technologies that produce radiation. DiCaprio et al. (17) studied e-beam inactivation in viruses and found that HuNoV-like particles were reduced by 90% after e-beam treatment at 28.3 kGy, and the remaining particles retained binding to PGM-MBs. This finding suggests that the degradation of tertiary protein structures is the primary method of inactivation. Compared with HuNoV GII.4, TV seemed more susceptible to e-beams but not to gamma radiation, although complete degradation of RNA was not achieved. Predmore et al. (77) also concluded that virus elimination likely resulted from the degradation of the capsid protein structure and to a lesser degree from the breakdown of capsid protein VP1, as found for e-beam–treated MNV-1 and TV. A dose of >32 kGy rendered the genetic material of MNV-1 but not TV undetectable.

### CAP

CAP is generated by subjecting inert gas, such as a noble gas or a mixture of gases such as air, to strong electromagnetic fields (62). Electrically produced microwaves, radio frequency currents, and direct or alternating currents can ionize the gas, forming a vast number of excited reactive species as determined by the feed gas. CAP devices operate at pressures of 1 atm at ambient temperatures, are chemical free, and have antimicrobial qualities, all of which make them attractive for food processing. The technologies applied in the studies reviewed here were plasma jet and DBD, where plasma is generated directly on the products by radio frequency electrodes. Apart from their antimicrobial effects, these technologies can also be used to extend shelf life and expand the functions of foods (81). The antivirus properties are associated with the variation of ionic, atomic, radical, and molecular species within the plasma (22). Singlet oxygen and perioxynitrous acid, ROS, and nitrogen species produced by CAP inactivated FCV via modification of capsid proteins (1). The role of these molecules in FCV inactivation was later affirmed by Yamashiro et al. (98), who exposed viruses to concentrations of perioxynitrous acid similar to those generated in CAP treatment and achieved a comparable loss in infectivity. The presence of scavengers of singlet oxygen and perioxynitrous acid decreased inactivation efficiency. A 15-s CAP treatment effectively rendered FCV levels of >6 log TCID50 noninfectious but not due to capsid degradation (2). Infectivity was lost through oxidization of capsid proteins at the attachment and entry sites of the virus capsid. However, a 2-min CAP treatment disintegrated the majority of the virus capsid structure and exposed the RNA to damage.

## HPP OF FRESH PRODUCTS

### Berries

Blueberries, raspberries, and strawberries have been used as substrates in several studies either in their natural form (31, 32, 51, 52, 55) or as puree or juice (18, 32, 44, 55, 65). A detailed overview of the effect of HPP on inactivation of NoV surrogates in berries and other food commodities is given in Table 2. Core features of effective treatments irrespective of virus model and berry type or form were pressure, temperature, and pH. Although the relationship between pressure magnitude and inactivation is linear, the effect of temperature is inverse, with greater inactivation at subambient temperatures of 0 to 4°C. For pH, the optimal range for inactivation was 6.5 to 7.4 compared with acidic conditions of pH 2.5 to 4.0 in surrogates MNV-1 and TV (18, 51, 52, 55). Similar findings were reported for HuNoVs GI.1 and GII.4 (32). The pH of the surrounding water and viral reductions were significantly lower for more acidic strawberries and raspberries than for blueberries after treatment (Table 3). In addition to the pH, the surface structure of whole blueberries compared with strawberries and raspberries was proposed as a reason for significantly higher inactivation in whole blueberries (32). Blueberries have a smooth surface, whereas strawberries and raspberries have an irregular surface with cavities and pouches. HuNoV GI.1 was effectively reduced in blueberries by >3.2 log genome copies per g after 2 min of HPP at 550 MPa and 0°C, but pressures up to 650 MPa resulted in only a 2.5-log reduction in raspberries and a 1.7-log reduction in strawberry quarters. A similar effect was obtained for HuNoV GII.4, albeit greater reductions of this virus suggested increased susceptibility to HPP. No differences in inactivation of GI.1 were found among the berry purees, but GII.4 was more susceptible to HPP in strawberry and raspberry than in blueberry puree. To achieve a ≥3-log reduction of MNV-1 and TV, the pressures required were on average 400 MPa, irrespective of the form of the berry matrix (31, 44, 52, 55, 65). Direct contact with water, i.e., a wet state, was superior to the dry state for virus inactivation, and lower pressures yielded greater inactivation regardless of berry or virus type (31, 51, 52). This finding was thought to be associated with the pressure forcing water into the viral capsid protein structures and disrupting the molecular folding of the protein domains associated with binding and attachment.

TABLE 2

Summary of model viruses, food matrixes, treatment parameters, and inactivation outcomes in studies of the effect of HPP treatment on NoV surrogates

TABLE 3

HuNoV and Tulane virus (TV) reduction based on RT-PCR assays following high pressure processing (HPP), electron-beam, and cold atmospheric plasma (CAP) treatments in various food matrixes

Several factors could account for the significance of matrix composition irrespective of the virus model, indicating that the food matrix protects viruses during HPP. Pressures required for inactivation increase gradually from the simple aqueous to more complex media and were systematically higher in actual food matrixes (18, 44, 51, 52). The degree of processing also influences affects. Pan et al. (65) found that MNV-1 was more easily inactivated in strawberry juice than in strawberry puree, in agreement with findings reported by Kovač et al. (44) and Lou et al. (55). Differences in inactivation associated with the berry type in whole berries were absent in purees, even with more resistant HuNoV GI.1 (32). HPP also has less impact on the sensory qualities of purees than on those of whole berries, in which undesirable changes in the texture and color limit application of pressure levels necessary for achieving satisfactory inactivation (32, 55).

### Vegetables

NoV surrogates have been used in experiments with various fresh and pickled vegetables such as lettuce, green onions, salsa, and cabbage kimchi (26, 29, 55, 68, 83). To reach >3-log inactivation of MNV-1, pressures of 300 to 400 MPa were required in salsa and 400 to 500 MPa were required in lettuce, green onions, and kimchi (29, 55, 68, 83). Hirneisen and Kniel (29) also found that HPP was equally effective against MNV-1 inocula in internal tissues or on the outer surface, underlining the uniformity of HPP. FCV was significantly more susceptible in salsa, in which it was reduced by ∼6.5 log TCID50/g at 250 MPa (26). The only results with HuNoVs were provided by Sido et al. (83), who found that GI.1 was more resistant in salsa. Treatment at 500 MPa and 1°C for 2 min was required to induce a reduction of >3 log genome copies per g, whereas 300 MPa under otherwise equivalent conditions for GII.4 resulted in a 3.31-log reduction. Green onions required even higher pressures for an inactivation of similar magnitude; GI.1 and GII.4 required 600 and 500 MPa, respectively, under otherwise similar conditions. However, these pressure ranges for effective inactivation of most viruses, excluding FCV, negatively affected the sensory qualities of lettuce, green onions, and kimchi (26, 29, 55, 68).

### Seafood

Clams and oysters are particularly associated with microbiological hazards because they are filter feeders that bioconcentrate seaborne human pathogens and can be consumed raw or lightly cooked. Several studies with HPP and NoV surrogates have been conducted with shellfish (6, 50, 52, 85), various seafood salad products (28), and sea squirts (69). On average, inactivation at ambient temperatures occurred at 250 MPa, but to achieve a 4-log reduction in MNV-1 titers at least 400 MPa of pressure and temperatures close to 0°C were required. To reach such reductions at suboptimal temperatures of 25 to 28°C, 500 MPa was need for both Manila clams (6) and sea squirts (69) independent of exposure duration. Such reductions in infectivity were not followed by declines in viral RNA concentrations, denoting the inadequacy of simple PCR assays (50).

The individual effects of various matrixes on the effectiveness of HPP were evaluated by Hirneisen et al. (28) for FCV-inoculated samples of cod, tuna, shrimp, and clams, each with or without mayonnaise. Virus declines after treatment at 200 MPa and 5°C were significantly lower in cod (−1.15 log TCID50/g) than in tuna (−4.54 log TCID50/g) or shrimp (−4.46 log TCID50/g). Addition of mayonnaise to the seafood meats did not significantly affect virus inactivation compared with the meats alone. However, compared with simple cell medium, mayonnaise as a more complex matrix provided significant protection from pressure treatment; FCV and MNV-1 titers were reduced by only 1.09 and 1.39 log units, respectively. Takahashi et al. (85) also observed the protective effect of matrix complexity, noting that increasing pressures were required to achieve satisfactory virus inactivation in salt- and pH-matched buffer, oyster homogenate, and finally whole oysters. HPP treatments did not negatively alter but rather improved the sensory quality of oysters and sea squirts (50, 69, 100).

Imamura et al. (35) assessed the naturally present HuNoV genogroups GI and GII in aquacultured Pacific oysters that had a prevalence of roughly 17% and an average total RNA concentration of 2.95 log genome copies per g. These viruses were inactivated to below the theoretical limit of detection of 2.36 log genome copies per g after treatment at 400 MPa and 10°C (Table 3). Slightly improved yet modest outcomes were obtained in oysters, with −1.87- and −1.99-log reductions of GII.4 and GII.17 in two separate batches after treatment with 400 MPa at 25°C (36). However, these results were obtained with RT-PCR assays after RNase treatment without information on infectivity or binding potential. Like studies of bioaccumulation in water tanks, studies of artificially inoculated oysters or clams have resulted in significantly higher contamination levels of 4 to 7 log RNA copies per g or mL and exclusively applied to genotypes GI.1 and GII.4 (57, 99, 100).

Results obtained with PGM-MB and RT-PCR assays with prior RNase treatment are in agreement with previous findings that GI.1 is more pressure resistant; 450 to 500 MPa produced a ∼4-log reduction in RNA at 1 or 6°C, whereas only 350 to 400 MPa was sufficient to obtain reduction of GII.4, even at 25°C (99, 100). In the only human study of pressure-treated oysters spiked with 4 log units of GI.1 8FIIb, 5 min at 600 MPa but not at 400 MPa, and 6°C protected volunteers from infection, suggesting complete inactivation (48). Lou et al. (57) expanded this research into animal models by feeding oyster homogenates inoculated with HuNoV GII.4 strain 765 to 2-day-old gnotobiotic piglets because swine have histo-blood group antigen phenotypes similar to those of humans. The homogenates had been treated or not treated with HPP, and the aim of the study was to determine whether HPP protected the piglets from NoV infection during a 7-day period. Inactivation of virus by HPP at 350 MPa and 35°C (∼1-log reduction) or 0°C (∼3.7-log reduction) was verified by PGM-MB and RT-PCR assays. Only piglets fed with homogenate treated with 350 MPa at 0°C were protected from infection; signs of virus shedding in feces, mild diarrhea, histologic lesions, and viral antigens in the small intestine were absent in this group in contrast with the other groups. These findings highlight the potential transferability of PGM-MB binding assay results to biological systems when estimating optimal treatment parameters for ensuring food safety.

### Meat products

In a recent survey, although only 3.3% of meat and meat product samples were contaminated with NoV, mishandled meat products represent a health risk (79). Sharma et al. (82) immersed pork meat deli sausages in sterile water for 5 min, dried them, then inoculated them with FCV or bacteriophage MS2. HPP treatment at 500 MPa and ca. 4°C decreased FCV titers by 2.89 log TCID50/mL and MS2 titers by 1.47 log PFU/g but could not completely remove all virus; recoveries of FCV and MS2 were 4.00 log TCID50/mL and 5.34 log PFU/g, respectively. Immersion of the samples in 100 ppm of EDTA or 2% lactoferrin did not significantly alter virus or phage attachment. Reductions were significant in all samples, but no differences were associated with the additives for FCV. In contrast, MS2 was more resistant to pressure, and for products in water and EDTA significantly greater elimination was found compared with that for products in lactoferrin. The authors speculated that the coarse surface of ground meat protected the NoV surrogates from HPP, as has been discussed previously (47). However, the protein, fat, and salt concentrations in the product and their interactions may also protect viruses from inactivation (28, 41).

Overall, HPP has been tested with multiple food matrixes and virus models over the last decade to address the potential uses of this technology. The use of HuNoV strains, generally GI.1 and GII.4, was a particular asset of HPP research, although surrogate use, mainly MNV-1, was equally common. Observed outcomes confirmed the higher resistance of GI.1 than GII.4 based on the binding assay, whereas of all the surrogates FCV was the most susceptible to HPP and MNV-1 and TV share similar resistance to pressure. Although the comparability of surrogates to HuNoVs in terms of HPP susceptibility is questionable, these surrogates mimicked HuNoV response to temperature, pH, and the presence of water (18, 51, 52, 83). From the wide range of treatment parameters (Tables 2 and 3), the best outcomes were achieved with temperatures <5°C and pressures of 400 to 600 MPa, and in berries direct water contact augmented inactivation and limiting matrix damage to some extent (51, 52). The only product-related features observed to favor inactivation were smooth surface and neutral pH of both the product and the surrounding water (18, 32). However, product composition could overcome the effect of low pH, as demonstrated with lemon juice (55). Rather than a single factor accounting for matrix effects, the complexity and interactions between components likely determine the protective effect (28). A disadvantage of HPP is possible texture changes at high pressures for whole berries and leafy greens, making this technology unsuitable for produce sold fresh in their original form (32, 55). However, for purees, juices, and oysters HPP is a viable option. Treatment durations can be reduced by determining the optimal conditions because inactivation occurs rapidly at adequate pressures (26, 68). HPP investments are high because the technology is expensive and requires maintenance throughout its lifespan. However, several HPP-treated products such as salsa and juices are available in retail markets and HPP is used during oyster shucking to extend the shelf life and reduce the levels of Vibrio, but the pressures currently applied are ≤300 MPa (99).

## UVC, PULSED, AND BLUE LIGHT FOR FRESH PRODUCTS

### Berries

UVC light was the most frequent light source used to treat inoculated blueberries, raspberries, or strawberries (13, 23, 53) followed by pulsed light (33, 34) and monochromatic blue light (42) (Table 4). Fino and Kniel (23) found that UVC fluence of 50 to 75 mJ/cm2 inactivated FCV by up to 7 log TCID50/mL in cell culture medium, but reductions reached only 1.13 to 2.28 log TCID50/mL in strawberries at fluences of 40 to 240 mJ/cm2. Similar maximum reductions were achieved with MNV strain S99 in fresh strawberries (1.27 log TCID50/g) and raspberries (1.52 log TCID50/g), but reductions were significantly higher in blueberries (3.12 log TCID50/g) with fluences of ∼200 to 1,300 mJ/cm2 for 20 to 120 s (13). Alterations in sensory characteristics of frozen strawberries were noted after extended exposure to UVC light (4,000 mJ/cm2). Some panelists noted off-flavors or slightly darker colors, likely due to the degradation of anthocyanin pigments. Concurrent water wash could enhance UVC inactivation. Liu et al. (53) obtained MNV-1 reductions of >3.20 log PFU/mL in blueberries at 600 mJ/cm2 increasing to >4.32 log PFU/mL with a fluence of 1,200 mJ/cm2, and both treatment were more effective in wet than in dry conditions. Fluences were substantially higher, 63,200 mJ/cm2 in strawberries and 53,900 mJ/cm2 in raspberries, in pulsed light treatments, resulting in MNV-1 reductions of 1.8 log PFU/g in strawberries and 3.6 log PFU/g in raspberries (33). Cutting the light fluences to 5,900 to 22,500 mJ/cm2 yielded only modest MNV-1 inactivation of 0.7 to 0.9 log PFU per sample in strawberries, but results with blueberries were more marked with mean reductions of 3.1 to 3.8 log PFU per sample (34). Blue light treatment in blueberries was not effective against TV; after treatment with 1,260, 3,780, and 7,560 mJ/cm2, only the highest fluence produced a nonsignificant reduction of 0.06 log PFU (42).

TABLE 4

Overview of model viruses, food matrixes, fluence levels, and inactivation outcomes in the studies of the effects of UVC, blue, and pulsed light on NoV surrogates

Observed differences in virus inactivation among different berries likely stem from the variations in surface topography. In contrast to the smooth exterior of blueberries, strawberries and raspberries have an irregular exterior with cavities that can shield virus particles and permit shadowing (13, 33, 34). The observed saturation effect at a UVC fluence of 200 mJ/cm2 was hypothesized to depend on these factors despite the application of mirror reflectors (13). The surface-related differences could be compensated for by coupling a water wash with UVC treatment (53). A 5-min wash alone reduced viral titers by 1.73 log units but resulted in high virus counts in the wash water. The total reduction of >4.36 log units and the absence of virus in the water after water-assisted UVC treatment suggests that virus particles removed by washing were effectively killed by UVC light. For blueberry juice (2%, v/v) but not crushed berries (5% of sample mass), inactivation outcomes were ∼2-log lower than those for clear water–assisted UVC treatment. Differences likely derive from >3 times higher water turbidity and >20 times higher chemical oxygen demand in the water containing blueberry juice. The presence of water was vital for pulsed light treatments because of the heating effect of fluences up to 63,200 mJ/cm2, which adversely affected the appearance of fresh blueberries (33). Posttreatment surface temperatures were reduced in raspberries from 59.9 to 31.5°C and in strawberries from 53.5 to 33.3°C when water was present. In the single trial with blue light, the light alone was ineffective against TV, but coupling the fluence of 7,560 mJ/cm2 with riboflavin or rose bengal reduced virus titers by approximately 0.5 and 1.0 log PFU, respectively (42). Riboflavin and rose bengal are enhancers of singlet oxygen formation with structural similarities to porphyrins; hence, they absorb light energy and generate ROS that induce inactivation. These compounds alone were effective for inactivating TV, with 0.13-log reductions of virus titers with riboflavin and 0.66-log reductions with rose bengal. Thus, blue light induced ROS formation, allegedly the key mechanism of inactivation, from appropriate molecules.

### Vegetables

UVC fluences of 40 to 240 mJ/cm2 reduced FCV counts by 3.48 to 4.62 log TCID50/mL in lettuce and 2.44 to 3.92 log TCID50/mL in green onions (23). However, MNV-1 was reduced by 0.2 log PFU per plant in internal and 1.2 log PFU per plant in external parts of green onions following an identical 240 mJ/cm2 UVC treatment (29). Li et al. (49) obtained 0.6- to 0.8-log reductions in MNV-1–inoculated lettuce following a 5-min UVC treatment, and the combination of UVC and vaporized 2.52% H2O2 did not improve the outcome. However, the authors did not report the total light dosage, complicating direct comparisons with results of other studies. Despite a surface structure similar to that of lettuce, green onions contain mucus-like compounds exposed during cutting that can affect virus attachment or recovery, possibly explaining observed differences between these food matrixes. Differences between internal and external virus counts in green onions were expected given the low penetrability of UVC light.

### Seafood

Pilotto et al. (72) evaluated whether UVC light as an adjunct could assist in Pacific oyster depuration. Oysters that had bioaccumulated MNV-1 were placed in water tanks, and the standard depuration was compared with that in which UVC light (fluence rate of 44 mW s/cm2) was constantly applied to circulating water. After 48 h, the reduction rate was approximately 1 log PFU/g of digestive tissue, reaching 1.2 log PFU/g by the end of the 120-h period irrespective of the method. Hence, depuration with or without UVC light was not sufficient to ensure the safety of contaminated oysters because virus attachment to oyster digestive tissue reduced pathogen release back to the water.

### Meat products

Research is limited for nonseafood commodities of animal origin. In the only study, Park and Ha (66) applied UVC light to MNV-1–contaminated fresh raw chicken breast, and fluences of 60 to 3,600 mJ/cm2 resulted in declines of 0.14 to 1.23 log PFU/mL from an initial titer of 4.34 log PFU/mL. However, negative impacts on sensory properties were confirmed after treatment at 1,800 mJ/cm2, and the products were deemed unacceptable for human consumption. Hence, fluences ≤1,200 mJ/cm2 (0.58-log reduction) were defined as optimal for preserving consumer acceptability and achieving some virus reduction. Nevertheless, this treatment was far from optimal with regard to food safety and requires additional decontamination steps.

Among the studies, UVC light was the common choice, whereas pulsed and blue light treatments played minor roles. Only two studies included FCV or TV, and none of the studies evaluated HuNoVs, with MNV as the preferred surrogate. In general, the light treatments produced <2-log reductions except for blueberries, and effectiveness was strongly associated with surface structure (13, 29, 34, 53). Irregular surfaces provide more attachment sites and shelter for viruses, with the resulting light shadowing effect, and light treatment had limited to no penetration in solid products and liquids, where the effect was rapidly lost with increasing turbidity. Virus inactivation can be augmented by agitation, free-floating the product in water, and using reflective surfaces and multiple instead of single lights (23, 53). Such augmentations of light treatments are needed because a saturation or plateau of effectivity limits the potential of increasing fluence levels (13, 23, 34). UVC light also can induce fat oxidation or mutagenicity but not generally in the commonly used treatment fluence ranges (13, 66). Much of the information provided here is based on UVC or pulsed light because only a single study included blue light and the results were far from encouraging (42). Thus, the potential of blue or pulsed light technologies for controlling NoV contamination in food products remains inconclusive.

## GAMMA AND E-BEAM IRRADIATION OF FRESH PRODUCTS

### Berries

MNV-1–inoculated strawberries required 8 kGy of e-beam radiation for a decline of 1.56 log PFU/g, and increasing the dose to 12 kGy yielded a 2.21-log reduction from the initial level of 5.37 log PFU/g (Table 5) (80). Gamma radiation was more effective for MNV-1 in strawberries; 2.8 kGy produced a decline of 1.31 log PFU/mL, and gradually increasing the dose to 11.2 and 16.8 kGy produced 4- and 5-log reductions, respectively, from the initial levels of 7 log PFU/mL (21). Pimenta et al. (73) estimated that a gamma ray dose of 3.7 kGy reduced MNV-1 counts by 2.2 log units in strawberries, and a similar reduction in raspberries required a dose of 3.4 kGy, whereas 7 kGy produced a roughly 3-log decline in both berry types. The D10-values were estimated to be 3.0 kGy in strawberries and 3.2 kGy in raspberries.

TABLE 5

Summary of model viruses, food matrixes, radiation doses, and inactivation outcomes in studies in which gamma radiation or electron beams (e-beams) were used to inactivate NoV surrogates

Outcomes for strawberries with the surrogate TV were comparable to those with MNV-1; 4.1 kGy delivered by e-beam resulted in a decrease of 1.4 log PFU/mL, 8.2 kGy resulted in a decrease of 2.6 log PFU/mL, and above 16.3 kGy no virus was detected (77). DiCaprio et al. (17) assessed the susceptibility of TV and HuNoV GII.4 in strawberries to e-beam radiation with the PGM-MB assay (Table 3). GII.4 required 12.2 kGy for a decline of 1 log genome copies per g and 16.3 kGy for a 2.46-log reduction. For TV, the same doses resulted in around a 2-log reduction, indicating comparable resistance to irradiation. After 28.7 kGy, levels for both viruses were below the detection limit. Overall, the doses for efficient inactivation were over the current legal limits, and doses >10 kGy caused notable deterioration in the sensory qualities, making the use of higher doses impractical for such products (77, 80).

### Vegetables

Zhou et al. (101) found that 3 kGy of gamma radiation reduced FCV titers by 1 log PFU/g in lettuce, whereas the highest dose of 5 kGy resulted in a ca. 2-log reduction. The estimated D10-value was approximately 2.95 kGy. For leafy greens, 2.8 kGy of gamma radiation decreased titers in spinach and romaine lettuce by 1.77 and 1.40 log PFU/g, respectively, and for both greens, reductions were around 2 log PFU/g after 5.6 kGy and around 4 log PFU/g after 11.2 kGy (21). Spinach seemed to protect MNV-1 from gamma radiation; after treatment at 22.4 kGy elimination was complete in romaine lettuce but 2.4 log PFU/mL was still detectable in spinach. Better survival on spinach than on lettuce could be attributed to differences in leaf surface texture or binding to carbohydrate moieties on the leaf surface (24, 30). However, in shredded cabbage, e-beams had little effect on MNV-1; 4, 6, and 12 kGy produced reductions of 0.5, >1.0, and 2.8 log PFU/g, respectively (80). A more processed form of cabbage, commercial kimchi, was evaluated by Park and Ha (67), who subjected MNV-1–inoculated samples to gamma irradiation, and the results were similar to those obtained for fresh cabbage. Doses of 5, 7, and 10 kGy produced declines of 0.98, 1.45, and 1.76 log PFU/mL, respectively, and the D10-value was 5.75 kGy based on the linear regression analysis. Identical doses of gamma radiation administered to MNV-1–contaminated green alga Capsosiphon fulvescens and brown alga Hizikia fusiforme, which are widely consumed in eastern Asia, was more effective; 5, 7, and 10 kGy produced 1.41-, 1.94-, and 2.46-log reductions in C. fulvescens and 1.26-, 2.60-, and 2.21-log reductions in H. fusiforme, respectively (70). The D10-values calculated with the Weibull model were 2.89 and 3.93 kGy for C. fulvescens and H. fusiforme, respectively, both lower than that for cabbage kimchi. The authors also estimated that 3-log inactivation would be achieved with 13.83 kGy in green algae and 14.93 kGy in brown algae. Kimchi and algae retained their sensory qualities after average irradiation levels of 10 kGy, and no changes in the quality of cabbage were observed at doses of 4 kGy (67, 70, 80).

### Seafood

Praveen et al. (76) subjected MNV-1–contaminated whole oysters and oyster meat homogenates to various e-beam doses and estimated D10-values of 4.05 and 4.97 kGy, respectively, by linear regression analysis. The required radiation dose for complete MNV inactivation in homogenates from the initial 4.898 log PFU/mL was 31.9 kGy, a value almost six times higher than the approved limit of 5.5 kGy for U.S. shellfish. In another model of contaminated seafood, abalones were inoculated with MNV-1 and irradiated with e-beams by Kim et al. (39), who used a first-order model to determine D10-values of 6.26 and 5.23 kGy for the sliced meat and minced viscera, respectively. At the maximum of 10 kGy, reductions were 1.45 log PFU/mL for meat and 1.56 log PFU/mL for viscera. In gwamegi, a half-dried Pacific herring or saury, and semidried squid contaminated with MNV-1, 10 kGy reduced titers by 1.66 and 1.81 log PFU/mL, respectively (38). A dose of 7 kGy was required in both food samples for a >1-log reduction. These results reveal that the radiation levels currently applied in seafood do not guarantee food safety and an increase of the upper limit to 10 kGy would not yield marked improvements. However, up to 10 kGy of radiation did not undermine the product qualities irrespective of the higher risk of lipid peroxidation within the product (38, 39).

Apart from single studies with HuNoV GII.4 and FCV, e-beam and gamma irradiation were mainly applied to the surrogates MNV-1 and TV. For doses within current limits, inactivation was generally 1 to 2 log units regardless of the virus model or radiation technology. Efficacy may be limited because the production of oxidative molecules is reduced by other scavenger components present in the food. Hence, to obtain satisfactory results, i.e., >3-log reduction of HuNoVs, the applied doses may need to be well over 10 kGy, although berries could be an exception (73, 77). Unfortunately, even if regulations permitted the use of higher doses, organoleptic qualities such as texture and flavor would be affected (38, 77, 80). Thus, product properties, safety aspects, and industrial scale setup are more likely to determine the choice of irradiation source. The complexity and structure of the matrix are more important determinants for e-beam effectivity because of relatively poor penetrability compared with gamma radiation. Delivery of e-beams to irregularly shaped products is challenging because adjacent areas of the same product could receive too much or too little exposure (77). E-beam treatment is more rapid and longer exposure to radiation could improve the inactivation result through extended water radiolysis and ROS generation, but the outcomes occurred at very high exposure levels (17). The cleavage of water molecules to ROS by ionizing radiation induces oxidative stress that can further augment virus inactivation. Thus, the effect of product water activity on outcome should also be investigated (38).

## CAP IN FRESH PRODUCTS

### Berries

Only four published studies included assessment of the potential of CAP for treatment of food products contaminated with NoV or its surrogates (Tables 3 and 6). Lacombe et al. (46) subjected blueberries inoculated with TV and MNV-1 to a CAP jet for 0 to 120 s. Product temperatures rose to 70°C when treatment durations were >60 s. To mitigate the thermal effect, an air stream at ambient temperature was channeled onto the berries during treatment, limiting the rise in berry temperatures to roughly 47°C by the end of the 120-s treatment. MNV-1 was significantly more susceptible to CAP jet with a reduction of 0.5 log PFU/g after 15 s and 5 log PFU/g or the limit of detection after 90 s. In contrast, significant decreases in TV of ca. 1.5, 2.5, and 3.5 log PFU/g were achieved after 45, 90, and 120 s, respectively. Such differences were thought to result from intrinsic differences between MNV-1 and TV such as in the histo-blood group antigen binding properties. Another study from the same authors (45) with a comparable setup revealed that blueberry texture softened significantly after 60 s, anthocyanins degraded significantly after 90 s, and color darkening was apparent after 120 s.

TABLE 6

Summary of cold atmospheric plasma (CAP) technologies, model viruses, food matrixes, treatment durations, and inactivation outcomes in studies on HuNoV surrogates inoculated into food products

### Vegetables

In two studies, lettuce was used as a food matrix and CAP was generated with the DBD technology (3, 60). Min et al. (60) treated TV-inoculated lettuce either in a closed petri dish or in modified atmosphere packaging with 5 or 10% O2 (the rest N2) for 5 min. In the petri dish, total inactivation of the initial 1.3 ± 0.2 log PFU/g was achieved in the 5 and 10% O2 modified atmosphere packaging, with reductions of 0.7 ± 0.3 and 0.2 ± 0.2 log PFU/g, respectively. Variations in effectiveness may be connected to the concentration of available oxygen because the low oxygen concentration in modified atmosphere packaging can limit the generation of ROS that contribute to inactivation. Treatment did not induce any significant changes in the color or appearance of the lettuce. Sample temperatures remained <29°C throughout the treatment, and no significant weight loss was observed. Aboubakr et al. (3) inoculated lettuce with HuNoV GII.4 or FCV and treated it with CAP produced by a two-dimensional array of integrated coaxial-microhollow DBD units. Inactivation of HuNoV GII.4 was determined by the EMA–RT-qPCR method, and inactivation of FCV was determined by the cell culturing method. After 1-, 3-, and 5-min treatments, reductions of GII.4 were ca. 0.5, 1.8, and 2.6 log genome copy number per sample, respectively. After 1, 2, and 3 min, FCV was reduced by ca. 2.7, 3.9, and >5 log TCID50 per sample, respectively. No observable changes were detected in lettuce after treatment. Treatment outcomes did not differ from those on stainless steel surfaces, which is surprising because pathogen elimination is usually impaired in the presence of organic material. Nevertheless, observed discrepancies should be compared cautiously because of differences in the methodological approaches and because the EMA–RT-qPCR method underestimated FCV inactivation by about 2 log units compared with the cell culture assay.

### Meat products

Bae et al. (8) applied CAP jet to MNV-1–contaminated fresh beef loin, pork shoulder, and chicken breast as model matrixes. From the initial inoculum of 5 log PFU/mL, after 5 min of treatment 2.05-, 2.11-, and 2.01-log reductions were obtained for beef, pork, and chicken, respectively. Extending treatment to 20 min did not produce additional benefits, with 2.09-, 2.15-, and 2.07-log reductions for beef, pork, and chicken, respectively. No significant differences in results were detected between meats or between the 5- and 20-min treatments. Changes in the surface color and moisture were insignificant for treatments <5 min but not >5 min. Lipid peroxidation increased gradually with treatment duration but remained under the threshold of critical change. Hence, a 5-min CAP jet treatment of meats preserved the quality and produced significant virus inactivation, but the products still had virus titers of 3 log units.

Overall, the number of publications focusing on decontamination of HuNoV or surrogates in food products by CAP technologies is sparse. Hence, the effect of matrix composition and form on virus inactivation remains unclear. The appropriate virus model, technology, and application also are yet to be determined. In the published studies, CAP was generated by either jet or DBD technology, and treatment durations were 0.5 to 20 min. A time-dependent increase in inactivation was evident in all studies when treatment duration for meats was <5 min, but no additional benefit was realized by treatment for >5 min (8). Significant reductions of HuNoV and surrogates were produced in all treated matrixes with minimal impact on sensory characteristics. However, CAP jet treatment did increase the temperature of blueberry samples to ca. 70°C without parallel cooling, but no heating effect was reported with CAP jet treatment of meats or DBD CAP treatment of lettuce (3, 8, 46, 60). Nevertheless, differences between these technologies, such as gas type, power, and distance complicate direct comparisons.

## CONCLUDING REMARKS AND FUTURE PERSPECTIVES

In a recent review, Hall et al. (25) concluded that annually in the United States alone NoV infections result in 19 to 21 million illnesses, 1.7 to 1.9 million outpatient visits, 400,000 emergency visits, 56,000 to 71,000 hospitalizations, and 570 to 800 deaths, whereas on a global scale, these infections are estimated to cause annually an average of 684 million illnesses leading to 212,000 deaths (54). Food is an important route of transmission, and foodborne infections warrant the development of new approaches suitable for delicate fresh products that are overrepresented in foodborne NoV outbreaks. Novel technologies are needed due to the resilience of NoVs despite traditional food safety measures. HPP, ionizing radiation, and light treatments were chosen for review here because of their well-established background, industrial applications, and minor effects on nutritional value and sensory attributes (Table 1). The 47 original research articles published after 2008 were retrieved through database searches and article reference lists, and approximately half of the studies were focused on HPP techniques and the rest on light, CAP, and irradiation treatments. Because of the technical limitations associated with culture of HuNoV, only 20% of the studies were conducted with HuNoV strains and rest were conducted with the HuNoV surrogates MNV, FCV, and TV. Therefore, surrogates were also incorporated into this review to compensate for the limited number of HuNoV inactivation studies in actual food matrixes.

Common traits determining the effectiveness of the various processing methods were the food matrix and surface topography because variations in both can shield virus particles from inactivation. More research is needed to establish the role of various matrix components and their interactions with regard to food safety. Although the HuNoV surrogates share some attributes with HuNoV, none has proven to be a convincing substitute. Thus, when assessing the parameters for effective treatments, the primary choice of virus should be HuNoV strains, more precisely the circulating variants from genogroups GI and GII that have been predominantly identified in outbreaks globally (92). The pretreatments, such as binding to PGM-MBs or nucleic acid intercalators, for distinguishing infectious virus particles from noninfectious ones are important for evaluating HuNoVs but serve as only a proxy for infectivity, as indicated by the lack of infectivity in plaque assays concurrent with RT-PCR results (3, 17, 18). Nevertheless, the results of binding assays are mostly in agreement with the those of the only human and animal studies conducted with food matrixes (48, 57). In the absence of clinical human trials due to their ethical, practical, and financial challenges, the development of validated HuNoV culture methods and animal models, such as gnotobiotic piglets, is crucial for studying true HuNoV infectivity and for determining the effectiveness of nonthermal treatments against NoV in food commodities.

Of the approaches addressed in this article, HPP is the most promising because of its uniformity of pressure and its ability to reduce virus titers despite the location of the virus particles (29). Effective virus inactivation by HPP is likely to require a pressure of ≥500 MPa, a wet packaging state, and a temperature close to 0°C. This combination of conditions is expected to yield a 2- to 4-log reduction in virus titers, which was observed in naturally contaminated berry purees, whole blueberries, and oysters (9, 35). However, no universal treatment parameters can be applied to all food products because the outcomes depend on product and treatment properties. In most studies, the inoculation levels were consistently unnaturally high, which could over- or underestimate treatment outcomes. More studies with HuNoVs and food matrixes are needed to determine the inactivation potential of irradiation, light, and CAP technologies. In future studies, the use of biologically relevant inocula and the standardization in reporting of results, such as detection limits, D10-values, and exact log reductions should be encouraged. Rather than a single technology, hurdle approaches with the reviewed technologies should be assessed for possible synergistic effects. Because the amount of product in individual samples in these reviewed studies was on an experimental scale, the effects remain to be validated in pilot and industrial studies. This extension of these experiments is especially necessary for light treatments, for which the proximity of products to the light source and uneven distribution of light are likely to affect inactivation. Industrial applications require vigorous testing, and their safety should be verified with appropriate methods for each product before large-scale implementation of any inactivation technology.

## ACKNOWLEDGMENTS

This work was funded by the EU European Regional Development Fund and the Regional Council of Pohjois-Savo “Improving Shelf-Life and Ensuring Quality in Food Using New Technologies” development project.

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