Human norovirus (HuNoV) is the leading cause of viral gastroenteritis outbreaks worldwide [1
]. Clinical symptoms of HuNoV infection include vomiting, nausea, and diarrhea within 24–48 h of infection [4
]. HuNoV has low infectious doses of ~10 viral particles and is highly infectious to susceptible individuals. In particular, young children, the elderly, and people with weak immune systems experience severe symptoms [5
]. HuNoV is transmitted mainly through the fecal-oral route and spreads easily from person to person, especially in crowded settings such as hospitals, restaurants, nursing homes, cruise ships, hotels, and schools [7
Due to the current lack of a HuNoV vaccine, preventive measures are the most effective way to reduce outbreaks of HuNoV, which is known to be resistant to environmental stresses [10
]. Therefore, current prevention methods for HuNoV include promoting personal hygiene for food handlers to avoid noroviral contamination and inactivation of infectious viral particles [12
]. Inactivation of HuNoV relies on physical methods, such as heating or radiation, and chemical methods such as sodium hypochlorite or titanium dioxide [10
]. However, the currently available methods have disadvantages for direct application to food. These include (1) increased cost of equipment and management; (2) potential acute and chronic toxic effects from chemical exposure; and (3) spoiling of food flavor or texture.
Therefore, environmentally friendly substances that might substitute for the established physical and chemical methods would be beneficial and directly applicable to food products such as oysters. Phytochemicals can be obtained from various natural plant extracts and most of them are “generally recognized as safe” (GRAS) [17
]. This has led many researchers to study natural substances with antimicrobial properties [18
]. Although previous studies have demonstrated that various phytochemicals possess antimicrobial effects, little research has been conducted to evaluate the efficacy of phytochemicals in reducing the infectiousness of norovirus [20
Cultivation methods for HuNoV using B cells [21
] or isolated enterocytes [22
] were reported recently. Although those findings are very meaningful and can be a milestone to decipher the mechanisms of norovirus infection at a molecular level, it is a bit early to apply the methods to screening various antiviral compounds. The replication of HuNoV in a B-cell culture system did not appear to be robust, as observed in murine norovirus (MNV) [23
], and the methods need to be validated with different types of HuNoV in other laboratories. Therefore, many previous studies have used viral models using MNV, feline calicivirus (FCV), and other in vitro models of HuNoV replication (e.g., HG23 cells) to evaluate the anti-noroviral activities of compounds [24
]. In this study, we used MNV and HG23 cells to investigate the ability of several phytochemicals to inactivate norovirus.
We screened the anti-noroviral effects of 18 different phytochemicals using a viral model to study norovirus biology. Dose- and time-dependent trends were evaluated for the phytochemical with the greatest efficacy. Subsequently, the results of inactivation after long-term phytochemical incubation were fitted to three different mathematical models. Inhibitory effects on norovirus replication were measured using a HuNoV replicon-bearing cell (HG23) model.
Studies on antimicrobial agents derived from phytochemicals aim to find natural products for preventing viral infection. For thousands of years, humans have consumed a variety of plant extracts as traditional medicine, natural therapies, and phytochemicals. The advantages of plant extracts in the prevention and treatment of infectious diseases include cost-effectiveness and safety, as compared to synthetic chemical antimicrobials and disinfectants. However, studies on the effectiveness of plant extracts against norovirus are limited and focus on specific phytochemical compounds such as tannins and flavonoids from berries and other fruits [28
In the present study, we investigated the anti-noroviral effect of 18 phytochemicals in terms of neutralizing viral particles and inhibiting viral replication in two models: MNV and HuNoV replicon-bearing cells (HG23). These models were used due to the difficulty of HuNoV culture. Ten different food-derived phytochemicals and eight ginsenosides with different glycosidic moieties were selected based on previous reports of potent antibacterial and/or antiviral activities. Given that the efficacy of phytochemical compounds is greatly affected by slight modification of their structure (e.g., glycosylation, methylation, acetylation, etc.), we analyzed several ginsenosides to determine whether the antiviral effects were influenced by the modified glycosyl ligands.
In the MNV neutralization assay, the most effective phytochemical was found to be CCM, which reduced the titer of MNV by 90.88% ± 10.87%. CCM is derived from the spice turmeric, and has clinical importance in improving cardiovascular and neurological health, as well as fighting breast tumors and gastric inflammation [30
]. Dietary intake of curcumin was reported up to 200 mg/day in some Asian countries where curcumin has been widely used as a common constituent in food (e.g., curry powder) and traditional medicine [33
]. In a recent clinical study, participants taking 4 g of mixed curcuminoids (2920 mg curcumin) daily for 6 days showed 363 ng/mL of curcumin in rectal tissue at day 7 [34
Several studies have reported antiviral effects of CCM. For example, it affected hepatitis C virus (HCV) envelope fluidity without changing virus integrity, resulting in inhibition of binding and fusion to the host [35
]. Other studies showed that CCM inhibited HCV using a replicon model [36
] and also reduced herpes simplex virus (HSV) [32
]. As far as we know, two groups have reported anti-noroviral effects of CCM recently [38
]. Photodynamically activated CCM was used in both reports and significantly neutralized MNV with damage to nucleic acid. The neutralized assay against virus was performed at room temperature or 37 °C. Considering norovirus is more stable at low temperature [40
], and most outbreaks were prevalent in winter [41
], we targeted 4 °C for the MNV neutralization assay.
CCM was investigated at various concentrations and anti-noroviral effects occurred in a dose-dependent manner. In our study, MNV particles were first treated with the phytochemicals, and the mixture with neutralized virus and phytochemicals was infected to host cells. There may be a possibility that the phytochemical itself affects the infection of host cells, not the neutralized viral particles. However, CCM showed a time-dependent inactivation of MNV and this suggests that the effect was based on increased neutralized virus by incubation with CCM rather than direct influence of CCM on host cells.
Short-term incubation with CCM (10, 20, 60 and 120 min) led to gradual reduction of MNV, with an 82.50% ± 1.21% reduction after 120 min. In the long-term test (1, 3, 7 and 14 days), a sharp decrease in MNV was observed within 1 day (78.87% ± 1.86% reduction). Comparing the results of 120 min and 1 day incubation, it is apparent that the major decrease occurred within the first 2 h. According to the D
-value (time in days required to inactivate 90% of the MNV) based on long-term data, 2.30 days of incubation are required to inactivate 90% of MNV. In control treatments, MNV reduction was about 80% after 7 days. The same trend has been observed in other studies. MNV titers decreased by 0.96 log after 7 days at 4 °C [42
], likely due to natural particle decay. RVT, a polyphenol abundant in grape seeds and skin, is capable of neutralizing MNV when incubated with the virus [26
]. Our results demonstrated that RVT significantly neutralized MNV (79.58% ± 6.67%) but had no inhibitory effect in the replicon model ((+) 26.62% ± 4.70%). PAC and CNA also significantly reduced MNV by approximately 60%, in agreement with previous studies showing that PAC decreased the levels of HuNoV surrogates such as FCV and MNV [18
]. In the case of CNA, this is the first report of an anti-noroviral effect. Su et al. studied the anti-noroviral effects of L-epicatechin using MNV and feline calicivirus (FCV-F9) [11
]. The exposure of 0.5 mM l
-epicatechin for 2 h at 37 °C neutralized only FCV-F9 by 1.40 log PFU/mL. In this study, EGC, a type of catechin, significantly inactivated MNV (26.83% ± 0.33%). This might be due to the differences of a chemical structure of catechin or exposure conditions.
In this study, CSC and GGR exhibited inhibitory effects in both models. CSC is a constituent of red peppers. CSC neutralized MNV (24.14% ± 2.28%) and reduced replicon RNA level (35.60% ± 8.50%). CSC has previously shown anti-microbial activity against bacterial pathogens including Helicobacter pylori
] and Staphylococcus aureus
]. CSC also reduced the mortality of HSV-infected mice [48
] and has been hypothesized to inhibit virus–neuron interaction [49
]. This study is the first report of an anti-noroviral effect of CSC. GGR is a culinary spice and is a common medicinal plant in China. GGR is an active constituent of most ginger species, and has been reported to have antimicrobial effects against Helicobacter pylori
and periodontal bacteria [50
]. In this study, we found that GGR had anti-noroviral activity in both models. GGR neutralized MNV (43.77% ± 4.50%) and inhibited the replication of norovirus (40.50% ± 3.83%) in the HG23 replicon model. This implies that GGR is a potential anti-noroviral for both in vitro inactivation of norovirus particles (high dose) and inhibition of norovirus replication in cells (low dose).
Ginsenosides are active constituents of most ginseng species. In the MNV neutralization assay, most ginsenosides showed an antiviral effect. Rh1 (80.05% ± 6.67%) and Rb1 (69.10% ± 0.00%) were particularly effective. However, five ginsenosides (F2, Rh2, Rd, Rg3, Rb1) increased, rather than repressed, the replication of norovirus in the HG23 replicon model. This result may be explained by the role of ginsenosides in the cholesterol biosynthesis pathway. It has been reported that the inhibition of cholesterol synthesis using statins (HMG-CoA reductase inhibitors) significantly increased HuNoV replicon RNA and proteins in the HG23 replicon model [52
]. Ginsenosides also suppress cholesterogenesis and lipogenesis [53
]. HMG-CoA reductase activity was significantly downregulated after treatment with ginseng powder. Our data are consistent with previous findings, in that the inhibition of cholesterol biosynthesis by ginsenosides significantly increased the level of norovirus RNA in the HG23 replicon model. Thus, it is important to consider the various stages of viral infection when evaluating the anti-noroviral effects of phytochemicals.
In summary, we evaluated 18 different types of phytochemicals and identified CCM as having the strongest anti-noroviral effects. This study indicated that phytochemicals such as CCM hold promise as natural anti-noroviral agents for the food-processing industry.