Human norovirus (HuNoV), which has characteristics such as high viral load excretion, tenacious environmental sustainability, rapid propagation, and short-term immunity, causes sporadic cases of viral gastroenteritis. Owing to its high contagiousness and rapid spread around the world, it infects humans regardless of gender or age (Hartmann et al., 2015). HuNoV infection is the most common cause of acute gastroenteritis, causing approximately 680 million cases per year (CDC, 2021). Particularly, the Centers for Disease Control and Prevention report that one in five cases of acute gastroenteritis (inflammation of the intestines or stomach), which causes vomiting and diarrhea, is caused by HuNoV infections worldwide. Furthermore, in most developing countries, approximately 200 million outbreaks are recorded among children under the age of 5 years, and approximately 50,000 deaths are estimated every year.

The virus causing gastroenteritis is known to spread mainly via environmental or waterborne routes, direct transmission between people, or fecal–oral routes (Dewey-Mattia et al., 2018). Among the various transmission routes, the airborne fomite route includes aerosol droplets. Several studies demonstrate that viruses in the form of aerosol particles are a remarkable characteristic of HuNoV transmission (de Graaf et al., 2016). Bonifait et al. (2015) demonstrates that intact HuNoV particles can be aerosolized from vomit and accumulate in the upper respiratory tract during respiration and in saliva of patients. By quantifying HuNoV RNA in air samples, it was demonstrated that the HuNoV epidemic was caused by aerosol contamination of the virus in healthcare settings (Bonifait et al., 2015). In addition, Simmons et al. (2020) demonstrate that HuNoV contamination on various surfaces in airplane cabins is caused by aerosol particles via RNA quantification. Bonifait et al. (2015) emphasize that NoVs remain contagious after aerosolization in environmental fomite, where the concentrations of airborne viral particles are ample to affect humans. Aerosol droplets containing NoV particles can accumulate on certain fomites, thereby directly causing contact transmission. In such cases, viral infection occurs by touching the nose and/or mouth with hands contaminated by viral particles after touching fomites. In numerous previous studies, secondary contamination of environmental fomites by HuNoVs aerosol is proven to be a significant route of infection (Bonifait et al., 2015; Kirby et al., 2016; Simmons et al., 2020).

Recently, owing to its considerable antimicrobial effects on pathogenic microorganisms and the non-formation of disinfection by-products, slightly acidic electrolyzed water (SAEW) treatment has been considered a successful method of disinfection in healthcare settings. Particularly, among the common chemical disinfectant materials, including ethanol, chlorine and its compounds, iodine, electrolyzed water, organic compounds, hydrogen peroxide, and quaternary ammonium compounds, SAEW shows broad-spectrum virucidal efficacy against a range of viral pathogens, such as non-enveloped viruses. Several studies also suggest that SAEW disinfection has virucidal activity against pathogenic viruses, including hepatitis C and hepatitis B viruses (Morita et al., 2000; Tagawa et al., 2000; Sakurai et al., 2003). SAEW virucidal efficacy against cultivable HuNoV surrogate (e.g., murine norovirus 1; MNV-1) is verified, and sufficient virucidal efficacy against HuNoV genogroup II genotype 4 (GII.4) Sydney on stainless-steel surfaces and in suspension is reported in several studies (Montazeri et al., 2017; Moorman et al., 2017). Based on previous scientific studies, the Japanese Ministry of Health, Labor, and Welfare (June 2002) approved SAEW as a food additive to eliminate pathogenic microbial populations in foods. Furthermore, the U.S. Environmental Protection Agency authorized the use of electrolyzed water generators for inactivation of pathogens in the environmental sectors.

The use of ultraviolet C-light-emitting diode (UVC-LED) has emerged as the optimal alternative approach and is recognized as a durable UV source to replace mercury UV lamps. It is environment-friendly and relatively cost-effective (Song et al., 2016) since conventional mercury UV lamps can cause chemical pollution, have a short bulb lifetime, and have low energy efficiency (Vilhunen and Sillanpää, 2010). In addition to preventing mercury disposal, UVC-LED irradiation has numerous advantages, including lesser warmup time, better device operation, lower power consumption, higher stability, and lower operating voltage than that of mercury UV lamps (Khan, 2006). Therefore, UVC-LED treatment (ranging between 100 and 280 nm) is broadly used in various sectors to block infection transmission and ensure public health. Across the entire UVC radiation spectrum, UVC-LEDs of wavelengths 269–276 nm are highly effective for inactivating infectious viruses in water and air and on any type of environmental surface (Bhardwaj J. et al., 2021).

To date, no studies have investigated the optimal disinfection characteristics of the hurdle approach combining the sprayed SAEW treatment and UVC-LED irradiation against HuNoV on stainless-steel surfaces. The objective of the present study was to determine the optimal conditions for inactivating HuNoV droplets on stainless-steel surfaces. To verify the optimal parameters and correlation between experimental variables, including the UV dose (mJ/cm²), available chlorine concentration (ACC) of SAEW (ppm), and sprayed SAEW amount (μL), a multivariate statistical analysis based on a combination of the response surface methodology (RSM) and the Box–Behnken design (BBD) technique was applied.

HuNoV GII.4 is recognized as a major cause of acute gastroenteritis outbreaks, and sporadic illnesses caused by HuNoV GII.4 are reported (Hartmann et al., 2015). To eliminate HuNoV contamination, a potentially contaminated matrix should be disinfected properly using various methods. To this end, establishing precise qualitative and quantitative assays with improved speed and accuracy as compared with cell culture–based methods is required. In addition, there is a growing awareness of the contribution of anionic polymer as a technical tool for viral particle capture. Anionic polymer [e.g., poly (methyl vinyl ether- maleic anhydrate)] and its derivatives are copolymers with molecular and physicochemical properties that enable their application as bio-adhesives (Nunez et al., 2000). In water, such polymers dissolve as they exist in the form of ionized free acids. Polar polymer free acids are highly negatively charged in water and can be used to impart anionic charge after grafting anionic polymers to magnetic beads. They also have numerous reactive groups and can efficiently trap biomolecules, including glycine, phosphatidylcholine, oligonucleotides, enzymes, and calcium. Although the mechanism via which Poly (methyl vinyl ether-maleic anhydrate)-coated magnetic beads bind to viral particles is still unclear, components on the viral surface, such as proteins, lipids, and sugar chains, may contribute to the binding process (Sakudo et al., 2009).

There is a growing consensus that adequate and efficient disinfection, in addition to preemptive prevention of fomites contaminated with viral pathogens, are indispensable in certain environments, including hospitals, nursing homes, schools, food industries, hotels, and complex multifacilities (Koide et al., 2011). Our findings in the present study suggest that optimal disinfection conditions for HuNoV inactivation can substantially prevent exposure to the risk of viral infection. In particular, a combination of the SAEW disinfection technique demonstrated in numerous previous studies and UVC-LED irradiation technology, which has recently begun to attract attention, effectively control HuNoV GII.4, which is present on stainless-steel surfaces and is the main transmission routes. Maximum reduction effects were sufficient to eliminate the HuNoV GII.4 by combining the SAEW treatment and UVC-LED disinfection in the present study because a requirement of ≥ 3 log₁₀ reduction against infectious viral pathogens, such as HuNoV, hepatitis A virus, and rotavirus, has been considered acceptable for virucidal effects (Gulati et al., 2001). Moreover, Marks et al. (2000) demonstrate that a disinfection treatment is successful in disinfecting fomites carrying pathogenic viruses if it eliminates > 3 log₁₀ of the viral population, considering the titer of pathogenic viruses spread into the environment. Notably, statistical analysis of the main virucidal effects of the combination of two disinfection techniques confirms that the effect of SAEW treatment is more significant than that of UVC-LED irradiation.

Additionally, SAEW plays an important role in the inactivation of bacterial pathogens, and there is sufficient evidence of the inactivation of infectious viruses in contaminated fomites (Wigginton et al., 2012; Hakim et al., 2015). The potential mechanisms underlying the SAEW disinfection of pathogenic viruses could include (1) destruction of the virus envelope, (2) damage of the surface protein, (3) inactivation of viral nucleic acids encoding enzymes, and (4) destruction of viral RNA (Hanson et al., 1989; Morita et al., 2000; Kitano et al., 2003; Sakurai et al., 2003). The virucidal efficacy of SAEW has been investigated in various viruses, including enteric, human immunodeficiency, hepatitis B, and herpes simplex viruses (Morita et al., 2000; Tagawa et al., 2000; Park et al., 2007). In the cases of HuNoV GII.4 in suspension and on stainless-steel surfaces, the virucidal effect of 250 ppm neutral electrolyzed water treatment has been evaluated and its effectiveness verified (Moorman et al., 2017). In addition, SAEW’s capacity to inactivate HuNov has been evaluated and significant virucidal effects observed (Park et al., 2007). Meanwhile, in the context of the COVID-19 pandemic, there has been increasing attention on the requirement of immaculate disinfection techniques in food manufacturing facilities and large public places. However, there is a lack of evidence related to disinfection techniques for the virucidal efficacy of HuNoV; however, ULV sprayer disinfection is a notable approach for blocking surface-based viral particle propagation. The efficiency of several technical methods, particularly the virucidal efficacy of the SAEW sprayer against HuNoVs on various fomites, has not been investigated although ULV spraying techniques for disinfection have numerous potential advantages. In virucidal studies using commercial disinfectants, including hydrogen peroxide (7.5%), instead of SAEW, hydrogen peroxide droplets diffused by a fogging disinfection system achieved promising virucidal activity against feline calicivirus (FCV) by achieving 4 log₁₀ reduction criteria for an antinoroviral disinfectant (Montazeri et al., 2017).

Several mechanisms (e.g., damage to the viral genome and/or damage to viral proteins) of virus inactivation by UVC irradiation are reported, and UVC-induced virucidal mechanisms vary among different types of viruses (Sano et al., 2010; Wigginton et al., 2010; Tanaka et al., 2018). For instance, first, UVC disinfection causes oxidative damage to the viral capsid protein, which is linked with the reduction of virus infectivity in norovirus and bacteriophage MS2 (Wigginton et al., 2010) and HuNoV surrogate FCV (Sano et al., 2010; Tanaka et al., 2018). Second, UVC irradiation destroys MNV-1 viral capsid protein. The reduction of poliovirus infectivity is reported to be secondary to viral protein-genomic crosslinking by UVC irradiation (Wetz and Habermehl, 1982). UVC-induced damage to the viral genome is observed in influenza viruses (Nakaya et al., 2020). The mechanism of UVC activation against SARS-CoV-2 is reported to be induced by viral genomic damage without apparent effects on viral morphology and proteins (Gidari et al., 2021).

Surface disinfection technology using UVC-LED irradiation has recently been highlighted (Bhardwaj S. K. et al., 2021). Particularly, Kim and Kang (2021) report that conventional mercury lamps used for UV emission are currently being replaced by UVC-LED lamps, which have higher viral decontamination efficiency and emit light sources stably and efficiently in the disinfection wavelength range of 260–275 nm. UV dose uniformity with respect to time and area is the most significant variable, which must be considered before employing UVC-LED irradiation (Araud et al., 2020). Furthermore, the fomite type should be considered an important variable because irradiation efficiency differs based on whether the fomites are porous (e.g., clothes and papers) or non-porous (e.g., glass, stainless steel, plastic) Bhardwaj J. et al. (2021). Moreover, (Bhardwaj J. et al., 2021) demonstrate that non-porous fomites are the main contributors to the transmission of viral infection because the survival duration of infectious viruses on them is much longer than that on porous fomites. To investigate the survivability of HuNoV, Ha et al. (2015) report that HuNoV GII.4 exists on artificially contaminated stainless-steel surfaces for up to 4 weeks following artificial inoculation. Previous studies have already investigated the use of UVC light to disinfect noroviruses (Jean et al., 2011; Bosshard et al., 2013). Although it cannot be compared with the direct virucidal effect of UVC-LED on HuNoV, studies of human enteric virus surrogates in a water disinfection system have been conducted to determine the virucidal effects of UVC-LED. Kim et al. (2017) report that populations of human enteric virus surrogates, including MS2, upphiX174, and Qβ, of the three viruses significantly decreased as the UVC-LED dose increased. Particularly, the virucidal efficacy of UVC-LED against human enteric virus surrogates was significantly inactivated by up to 7 log₁₀ following treatment with 1 mJ/cm² for ΦX 174 and 9 mJ/cm² for Qβ and MS2 (Kim et al., 2017). For UVC-LED disinfection of SARS-CoV-2 on a rubber surface, Trivellin et al. (2021) study the design, characterization, and validation of UVC-LED disinfection and demonstrate that a 3 log₁₀ reduction could be achieved following 60 s of irradiation with a 8.31 mJ/cm² dose. Although UVC-LED disinfection can effectively inactivate infectious viruses, the shortcoming of UV irradiation is that there is no durative antimicrobial activity because the DNA damage of the microbial cells caused by UV irradiation can be repaired through photoreactivation or nucleotide excision repair. Although studies on the photoactivation properties of infectious viruses are limited., hurdle technology using chemical disinfection treatment, considering the shortcomings of UV disinfection, has recently been reported (Li et al., 2018; Nyangaresi et al., 2018; Wan et al., 2020).

To the best of our knowledge, this is the only study to investigate the virucidal efficacy of the combined technique of sprayed SAEW and UVC-LED irradiation against HuNoV strain GII.4 on stainless-steel surfaces. Our experimental results reveal the effective reduction values under optimal combination conditions (180 μL of SAEW, 30 ppm ACC of SAEW, and 2 mJ/cm² UVC-LED dose) for HuNoV inactivation by 3.21 log₁₀ genomic copies. Our experimental study included three scientific aspects: (i) Use of HuNoV to evaluate effective and promising virucidal techniques, including SAEW and UVC-LED, simultaneously; (ii) determination of the optimal parameters and correlation among experimental variables, including UV dose, SAEW concentration, and sparked SAEW amount, based on the BBD technique combined with RSM; and (iii) evaluation of the virucidal efficacy of SAEW ULV sprayer techniques on HuNoV-contaminated surfaces. However, we cannot guarantee that our results will lead to the inactivation of HuVoV under all environmental conditions owing to the lab scale. Therefore, further research is required after scaling up the SAEW ULV sprayer and UVC-LED disinfection system to a level appropriate for application in the field.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

HS and Y-MD conducted the experiments and were responsible for assessing the experimental data, and wrote the first draft of this manuscript. SH was responsible for assessing the experimental data. J-HH was responsible for data coordination, experimental design, analysis, data interpretation, writing, revision, and finalization of the manuscript. All authors have read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.