Introduction

Enteric viruses, in particular human norovirus (NoV), are the leading causes of foodborne illnesses in industrialized countries (Anonymous 2013; EFSA 2014), whereas hepatitis A virus (HAV) has recently been considered as a re-emerging foodborne public health threat in Europe (Sprenger 2014) due to the number of foodborne outbreaks associated to imported foods (Collier et al. 2014; Guzman-Herrado et al. 2014; Wenzel et al. 2014).

The transmission of enteric viruses associated with the consumption of contaminated produce is regularly reported after contamination by food handlers or by the use of polluted irrigation water. Since products considered healthy food i.e., fresh cut vegetables are prone to be contaminated, it is important to find natural antiviral additives which are safe, environment friendly and preferably inexpensive for use in the food industry.

The growing demand for the use of natural additives in food applications has generated a substantial increase in the number of studies based on natural extracts such as essential oils (EOs) or their main compounds. They are categorized as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration, and are therefore potential alternatives to chemical additives. EOs extracted from plants are rich sources of biologically active compounds, such as phenolic acids and terpenoids, and it has been long recognized that some of them show antimicrobial properties. Furthermore, the antibacterial, antifungal, insecticidal, antiparasitic and antitoxigenic activities of these compounds have extensively been reported (Burt 2004). In contrast, reports on the antiviral effects of EOs or their compounds are somewhat limited and are mainly based on NoV surrogates (Table 1).

Table 1 Inactivation rates of essential oils and their main compounds on different enteric viruses or norovirus surrogates
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In a previous study, our group reported the antiviral activity of clove, oregano and zataria EOs against norovirus surrogates (Elizaquível et al. 2013). Therefore, in this study, the effect of zataria (Zataria multiflora Boiss.) and oregano (Origanum vulgare) EOs on the infectivity of HAV has been assessed. We also studied the efficacy of thymol, one of the main components of oregano and thyme EO, on the infectivity of HAV and two norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV).

Materials and Methods

Viruses, Cells and Infections

The cytopathogenic HM-175 strain of HAV (ATCC VR-1402), the MNV-1 strain (kindly provided by Prof. H. W. Virgin, Washington University School of Medicine, USA) and the F9 strain of FCV (ATCC VR-782) were propagated and assayed in FRhK-4 (courtesy of Prof. Albert Bosch, University of Barcelona), RAW 264.7 (kindly provided by Prof. H. W. Virgin) and CRFK cell monolayers (ATCC CCL-94), respectively.

Virus stocks were obtained from the same cells by centrifugation of infected cell lysates at 660×g for 30 min at 2–3 days postinfection for MNV and FCV and 10–12 for HAV. Infectious viruses were quantified by determining the 50 % tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well (Sánchez et al. 2011; Pintó et al. 1994).

Natural Compounds

Origanum vulgare (oregano) essential oil provided by Pronarôm International (Ghislenghien, Belgium) and an in-house produced zataria essential oil (from Z. multiflora Boiss., collected in the Shiraz Province of Iran and kindly provided by Maryam Azizkhani) were used in this study. Main compounds of oregano EO are carvacrol (46.88 %), thymol (15.26 %), p-cymene (13.10 %) and γ-terpinene (11.61 %) (www.pranarom.com) and in zataria, carvacrol (71.12 %), γ-terpinene (7.34 %), α-pinene (4.26 %), eucalyptol (3.37 %) and globulol (2.32) (Elizaquível et al. 2013). Thymol (≥99 % purity) was provided by Sigma Aldrich.

Oregano and zataria EOs were diluted in 70 and 50 % ethanol, respectively, whereas thymol was diluted in 25 % ethanol and kept at 4 °C before use.

Cytotoxicity Determination of Natural Compounds on Cell Monolayers

Natural compounds at various concentrations were added to individual wells of confluent RAW 264.7, CRFK and FRhK-4 cell monolayers grown in 96-well microtiter plates (SPL Life Sciences) and incubated 2 h under 5 % CO2. Subsequently, 150 μl of DMEM supplemented with 2 % of fecal calf serum (FCS) was added per well. After incubation at 37 °C in a CO2 incubator for 2–15 days, the cell monolayers were observed for cytotoxicity effects by visual inspection under the optical microscope only.

Antiviral Effects of Natural Compounds

Natural compounds at different concentrations were added to virus suspensions (ca. 6 log TCID50/ml) in DMEM with 2 % FCS and further incubated at 37 °C in a water-bath shaker at 150 rpm for 2 h. Tenfold dilutions of treated and untreated virus suspensions were inoculated into confluent monolayers in 96-well plates. Then, infectious viruses were quantified by cell culture assays as described above. Each treatment was done in triplicate. Positive controls were virus suspensions added with ethanol in amounts corresponding to the highest quantity present. The decay of viruses was calculated as log10 (N t/N 0), where N 0 is the infectious virus titer for untreated sample and N t is the infectious virus titer for treated sample.

Statistical Analysis

The significance of differences among the mean numbers of viruses determined after the various treatments was estimated by the Student’s t test with a significance level of P < 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA).

Results

Determination of Cytotoxicity of EOs and Thymol on Cell Monolayers

Oregano and zataria EOs resulted cytotoxic for FRhk-4 cells at concentrations that exceeded 2 and 0.1 %, respectively. Thymol was found to be cytotoxic at concentrations that exceeded 2 %, for the three cell lines. Therefore, 2, 0.1 and 2 % were the maximum concentration of oregano EO, zataria EO and thymol tested to evaluate their effect on HAV, MNV and FCV.

Antiviral Efficacy of Oregano and Zataria EOs on HAV

Incubation of HAV with oregano EO at concentrations of 0.5, 1 and 2 % for 2 h at 37 °C did not decreased the HAV titers (Table 2). Zataria EO at 0.1 % reduced HAV titers by 0.42 TCID50/ml (P < 0.05). At lower zataria EO concentrations (i.e., 0.05 and 0.01 %), no differences in titer reduction were observed between HAV suspensions treated or non-treated with the tested concentrations of zataria EO (Table 2).

Table 2 Effect of oregano (O) and zataria (Z) essential oils against Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
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Effect of Thymol on the Infectivity of HAV, MNV and FCV

Incubation of FCV and MNV with thymol at concentrations from 0.5 to 2 % for 2 h at 37 °C decreased the titer of the two norovirus surrogates (Table 3). Thymol at 0.5 and 1 % reduced FCV titers to undetectable levels. On the other hand, MNV titers, the hardier NoV surrogate, were reduced by 0.50 log TCID50/ml after treatment with thymol at 0.5 %, while thymol at 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. For HAV, no differences in titer reduction were observed between HAV suspensions treated or non-treated with thymol at 0.1, 0.5, 1 or 2 % (Table 3).

Table 3 Effect of thymol against feline calicivirus (FCV), murine norovirus (MNV-1) and Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
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Discussion

The food industry is currently exploring new ways to use effective natural antimicrobials to improve food safety, and plant EOs or their compounds are potential alternative natural ingredients (reviewed by Gyawali and Ibrahim 2014). Regarding enteric viruses, oregano and zataria EO revealed efficient to reduce infectivity of MNV and FCV (Elizaquível et al. 2013). The initial aim of the present study was to evaluate them on HAV, since it has recently been considered as a re-emerging foodborne public health threat in Europe (Sprenger 2014). Oregano EO was ineffective on HAV at the maximum concentration tested, unlike our previous results where oregano EO reduced the infectivity of MNV and FCV by 1.62 and 3.75 log, respectively (Elizaquível et al. 2013). For zataria EO, although statistically significant, HAV infectivity was only reduced by 0.42 log TCID50/ml, while in our previous study, 0.1 % zataria EO reduced FCV and MNV infectivity by 4.51 and 0.25 TCID50/ml, respectively.

As an alternative, one of the active compounds, thymol, was tested. Thymol is present in oregano EO, and its isomer, carvacrol, is the main active component of zataria. It is a phenolic monoterpene that has been identified as a natural economical food preservative (Lu and Wu 2010) with potential for incorporation in food packaging (Ramos et al. 2012). Following, antiviral activity of thymol was explored on NoV surrogates and HAV. This study clearly shows that thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner, where increasing concentrations of thymol resulted in increased reduction in viral titers. Furthermore, HAV was resistant to thymol treatment at any concentration tested. Similar trends have recently been reported for carvacrol treatment; however, carvacrol was more effective than its isomer thymol against NoV surrogates and HAV. Sánchez et al. (2015) reported that carvacrol at 0.5 % completely inactivated FCV and MNV, whereas 1 % concentration was required to achieve ca. 1 log reduction of HAV. Moreover, Sánchez et al. (2015) reported the potential of carvacrol to be used as a natural disinfectant of produce.

Overall, this study showed that the evaluated natural compounds resulted in slight reductions of HAV infectivity, while they were effective in NoV surrogates with higher reduction in FCV than MNV titers. These results resemble those reported for carvacrol (Sánchez et al. 2015). However, Su and D’Souza (2011), when testing the antiviral activity of grape seed extract (GSE), reported that MNV was more resistant than HAV.

Taking together these results and previous studies (Elizaquível et al. 2013; Sánchez et al. 2015; Gilling et al. 2014a, b; Pilau et al. 2011), the use of plant extracts as antivirals in food safety should focus on the purified active compounds as they exhibited stronger activity than EO. Nevertheless, complete inactivation of foodborne viral contamination with plant extracts either EOs or active compounds cannot be foreseen since they slightly affect HAV infectivity. The application of plant antimicrobials in food sanitation should combine them with other natural compounds, e.g., GSE, to achieve a synergistic effect, and be used as part of hurdle approaches to improve food safety.