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Virucidal and antiviral effects of Thymus vulgaris essential oil on feline coronavirus

22.4.2021
Cristiana Catella, Michele Camero, Maria Stella Lucente, Giuseppe Fracchiolla, Sabina Sblano, Maria Tempesta, Vito Martella, Canio Buonavoglia, Gianvito Lanave
Original article: Virucidal and antiviral effects of Thymus vulgaris essential oil on feline coronavirus

Table of Content to hide
Basic facts
Abstract
keywords
The results
Declaration of conflict of interest
Appendix A. Additional information
References

Basic facts

  • EOs function as antibacterial, antiviral, antifungal and insecticidal agents.
  • TEO has been shown to be effective against several RNA viruses, including CoV.
  • The anti-infective activity of TEO against FCoV was assessed in CRFK cells.
  • TEO has been shown to inhibit FCoV replication in vitro.
  • TEO showed virucidal activity against FCoV up to 92,86%.

Abstract

Feline peritonitis (FIP) is a deadly systemic disease of cats caused by coronavirus (CoV) (FIPV). Despite their clinical significance and impact on cat health, therapeutic options for the treatment of FIP in cats are currently limited. The emergence of pandemic coronavirus severe respiratory syndrome type 2 (SARS-CoV-2), the etiological agent of coronavirus disease of 2019 (COVID-19), capable of infecting a wide range of animal species, including cats, has sparked interest in developing new molecules with antiviral activity to treat infections. CoV in humans and animals.

Essential oils (EOs) have attracted significant attention with their antiviral properties, which integrate and in some cases replace conventional drugs. Thymus vulgaris EO (TEO) is known to be effective against several RNA viruses, including CoV. In the present study, the antiviral activity of TEO against FIPV was evaluated in vitro.

TEO at a concentration of 27 μg / ml was able to inhibit virus replication with a significant reduction of 2 logs10 TCID50/ 50 μl. In addition, virucidal activity was tested by TEO at concentrations of 27 and 270 μg / ml, above the cytotoxic threshold, to determine a reduction in viral titer up to 3.25 log10 TCID50/ 50 μl within 1 hour of time contact. These results open up several perspectives in terms of future applications and therapeutic options for coronaviruses, as FIPV infection in cats could be a potential model for the study of antivirals against CoV.

keywords

Thyme essential oil, Cat coronavirus, Thymus vulgaris, Thyme, Thyme, Thyme, Thyme, FIP

The results

The 2019 coronavirus disease pandemic (COVID-19) caused by severe respiratory syndrome coronavirus (SARS-CoV-2) (WHO, 2020) stimulated research into treatment and immune prophylaxis using previous knowledge gathered on SARS-CoV-1 and animal CoV (Decaro et al., 2020). To date, no specific drug has been approved for the treatment of patients with COVID-19. However, remdesivir, an inhibitor of RNA-dependent RNA polymerase (RdRp), has shown promising results (Kabir et al., 2020). In addition, studies are currently underway to evaluate the efficacy of teicoplanin and monoclonal and polyclonal antibodies against SARS-CoV-2 (Kabir et al., 2020).

Drugs studied in cats for the treatment of feline infectious peritonitis (FIP) have also been tested against COVID-19 in human patients (Pedersen et al., 2018; Pedersen et al., 2019).

Coronaviruses have long been known for the existence of FIP, a deadly systemic disease in cats. FIP virus (FIPV) is a virulent pathotype of feline enteric coronavirus (FCoV) (Kummrow et al., 2005). Despite its impact on cat health, therapeutic options for the treatment of FIP in cats are limited and effective vaccines are not available. In addition, adverse effects of vaccines have been reported (Tizard, 2020). The development of new molecules with antiviral activity for the treatment of CoV infections is currently perceived as a priority in both human and animal medicine. FIPV infection in cats is considered a potential model for studying antiviral drugs against CoV (Amirian and Levy, 2020).

Herbal medicines have aroused the interest of both consumers and scientists (Hosseinzadeh et al., 2015) and essential oils (EO) extracted from aromatic and medicinal plants have increased special attention for their beneficial properties (de Sousa Barros et al., 2015). EOs have been reported to exhibit significant antiseptic, antibacterial, antiviral, antioxidant, antiparasitic, antifungal, and insecticidal effects (Chouhan et al., 2017; Ma and Yao, 2020). Recently, in catfish experimentally intoxicated with Thiamethoxam (TMX), administration of EO Thymus vulgaris (TEO) partially reduced the toxic effects of TMX (El Euony et al., 2020). EOs are also a potential reservoir of innovative therapeutic solutions that integrate and in some cases replace conventional drugs (Reichling et al., 2009). For example, EO Laurus nobilis (SARS-CoV-1) inhibited SARS CoV type 1 (SARS-CoV-1) (Loizzo et al., 2008) and the EO mixture was effective against avian coronavirus infectious bronchitis virus (IBV) (Jackwood et al. , 2010). ). EOs have also recently been shown to have antiviral activity against SARS-CoV-2 (Asif et al., 2020). TEO has been shown to be effective against several RNA viruses, including CoV (Lelešius et al., 2019; Nadi et al., 2020).

The composition of TEO (Specchiasol Bussolengo, Verona - Italy) was determined in three independent experiments by gas chromatography-mass spectrometry (GC-MS) (Rosato et al., 2020). Details on sample preparation, apparatus, and GC-MS analysis methods have already been published (Salvagno et al., 2020; Rosato et al., 2018). Data from GC / MS analyzes were expressed as % areas ± Structural Equation Modeling (SEM). In all cases, the SEM was less than 10%. Statistical analysis for SEM was performed using Microsoft Excel Office 2010 (Windows 7 Home Premium, Microsoft Corporation, USA). A total of 26 components were identified in the TEO sample, corresponding to 98.7% of the whole mixture. The detailed chemical composition of TEO is given in Additional Table 1.

TEO at a concentration of 928 mg / ml was first diluted in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri, USA) and then in Dulbecco-MEM (D-MEM).

Crandell Reese cat kidney cells (CRFK) were cultured in DMEM and strain FCoV-II 25/92 (Buonavoglia et al., 1995) with a titer of 105.25 TCID50/ 50 μl.

TEO cytotoxicity was assessed by XTT assay (Denizot and Lang, 1986) using the In Vitro Toxicology Assay Kit (Sigma - Aldrich Srl, Milan, Italy) after exposure of cells to various concentrations of compounds (7.25, 14.5, 29, 58, 116, 232 , 464, 928, 1856 μg / ml) for 72 hours. Cytotoxicity was evaluated by spectrophotometric measurement of the absorption signal (optical density, OD). In all experiments, untreated cells were used as a negative control for 0% cytotoxicity. Cells treated with equivalent dilutions of DMSO were used as a vehicle control. After logarithmic conversion of TEO concentrations, the data obtained in the cytotoxicity assays were analyzed using a non-linear curve fitting procedure. The accuracy of the match was tested by non-linear regression analysis of the dose-response curve. The maximum non-cytotoxic concentration was considered to be the concentration of compound at which the viability of treated CRFK cells decreased by no more than 20% (CC20) in view of the negative control.

CC value20 TEO was evaluated at 27 μg / ml and calculated based on the mean ± standard deviation (SD) from three experiments. In all experiments, DMSO showed no effect on cells.

Based on the results of the cytotoxicity test, the antiviral activity of TEO against the FCoV-II 25/92 strain was evaluated at 27 μg / ml and also below the cytotoxic threshold (13.5 μg / ml). Using a substance below the cytotoxic threshold allows us to reduce toxicity and achieve effective results at lower cost. Confluent CRFK cell monolayers 24 h in 24-well plates were infected with 100 μl FCoV-II containing 10,000 TCID50 with a multiplicity of infection (MOI) of 0.14. After virus adsorption for 1 hour at 37 ° C, the inocula were removed, the cell monolayers were washed once, and TEO was added. In untreated infected cells, D-MEM was used to replace the inoculum and used as a viral control. After 72 hours, aliquots of supernatants were taken for viral titration (Lanave et al., 2019) and RNA quantification (Gut et al., 1999).

The virucidal activity of TEO against FCoV-II was assessed by pre-treatment with the virus (10,000 TCID50) with TEO at a concentration of 27 μg / ml and above the cytotoxic threshold (270 μg / ml), because when used as a virucide, the molecule is not in direct contact with cells. Specifically, 100 μl of FCoV-II was treated with TEO (1 mL) at room temperature. Virus control was used for the experiments. After 10 minutes, 30 minutes, and 1 hour, aliquots of each virus-TEO and virus control mixture were subjected to virus titration (Lanave et al., 2019).

Data from antiviral and virucidal activity assays were expressed as mean ± SD and analyzed by analysis of variance (ANOVA) using Tukey's test as post hoc test (statistical significance set at 0.05).

Statistical analyzes were performed using GraphPad Prism v.8.0.0 software (GraphPad Software, San Diego, CA, USA).

Virus titers of TEO-treated CRFK cells and untreated infected cells (viral control) were expressed as log10 TCID50 / 50 μl and plotted against drug concentrations. By comparing the viral titer of untreated infected cells (4.25 log10 TCID50/ 50 μl) with infected TEO-treated cells at 13.5 and 27 μg / ml there was a decrease of 0.25 (p> 0.05) and 2.25 log10 TCID50/ 50 μl (p <0.0001), respectively (Fig. 1A). This suggests that TEO at a concentration of 27 μg / ml is able to significantly inhibit virus replication. The antiviral activity of TEO against FIPV corresponds to the results obtained with Thymus vulgaris hydrosols in vitro against swine reproductive and respiratory syndrome virus (PRRSV) (Kaewprom et al., 2017).

Figure 1. Viral titers of supernatants collected 72 hours after infection from untreated FCoV-infected CRFK cells (10 TCID50) and treated with Thymus vulgaris essential oil (TEO) at various concentrations (13.5 and 27 μg / ml). 0 = Untreated CRFK cells infected with FCoV, NA = Nucleic acid

Viral titers were evaluated by endpoint dilution, expressed as log10 TCID50/ 50 μl and plotted against TEO at various concentrations (A). Viral nucleic acids were quantified by qPCR, expressed as log10 viral NA / 10ul and plotted against TEO at various concentrations (B). Bars in the figures indicate diameters. Error bars indicate standard deviation.

Viral nucleic acids (NA) were expressed as log10 viral NA / 10μl of infected cells treated with TEO and viral control and plotted against non-cytotoxic concentrations treated. By comparing the viral load of untreated infected cells (6.53 log10 viral NA / 10μl) with infected TEO-treated cells at 13.5 and 27 μg / ml, there was a decrease of 0.61 (p = 0.0005) and 1.34 (p <0.0001) log10 NAs / 10 μl, in that order (Figure 1B).

The virucidal activity of TEO was evaluated at different concentrations and for different contact times with FCoV-II (Fig. 2). After 10 minutes of TEO at 27 and 270 μg / ml, a reduction of 1.5 (p = 0.0008) and 2.5 (p <0.0001) log10 TCID50/ 50μl compared to virus control (4.25 log10 TCID50/ 50 μl) (Fig. 2A). After 30 minutes, TEO at 27 and 270 μg / ml induced a decrease of 1.25 (p = 0.0007) and 3.375 (p <0.0001) log10 TCID50/ 50 μl compared to virus control (3.75 log10 TCID50/ 50 μl) (Fig. 2B). After 1 hour of TEO at 27 and 270 μg / ml, there was a decrease of 1.25 (p = 0.0007) and 3.25 (p <0.0001) log10 TCID50/ 50μl compared to virus control (3.50 log10 TCID50/ 50μl). Viral inactivation occurred in a dose-time manner, starting at 33,33% and reaching 92,86%, when TEO was used at the highest concentration (270 μg / ml), after 1 hour (Fig. 2C). The virucidal activity of TEO can be explained by its ability to damage the viral envelope, thereby preventing adsorption and penetration into host cells (Reichling et al., 2009), as shown by electron microscopy of the herpesvirus envelope after pretreatment with EO (Shogan et al., 2006). TEO could therefore be a valuable means of disinfecting surfaces and could be an additive in the preparation of food products.

Figure 2. Virucidal effect of TEO at various concentrations (27 and 270 μg / ml) against FCoV (10000 TCID50). The virus was incubated with TEO for 10 minutes (A), 30 minutes (B) and 60 minutes (C) at room temperature and subsequently titrated in CRFK cells. FCoV viral titers were expressed as log10 TCID50/ 50 μl and plotted against TEO at various concentrations. Bars in the figures indicate diameters. Error bars indicate standard deviation.

Thymus vulgaris is an aromatic plant of Mediterranean origin that contains EO and lipophilic substances (Nabavi et al., 2015) and its extracts are rich in thymol, carvacrol, p-cymene and γ-terpinene (Kowalczyk et al., 2020).

Thymus vulgaris has been shown to have antiviral activity against herpes simplex virus (HSV) (Nolkemper et al., 2006), influenza virus (Vimalanathan and Hudson, 2014), Newcastle disease virus (Rezatofighi et al., 2014), PRRSV (Kaewprom et al., 2017) and IBV (Lelešius et al., 2019), although the antiviral mechanism has yet to be elucidated. In contrast, inhibition of human immunodeficiency virus replication in vitro by TEO has been elucidated (Feriotto et al., 2018).

The chemical composition of TEO revealed the presence of 26 molecules, of which the main fractions were thymol, p-cymene, γ-terpinene, β-linalool, caryophyllene. To reduce the cytotoxicity of TEO, it would be interesting to identify active molecules and test them individually. As expected, the main component of TEO used in this study was thymol. The thymol fraction has been shown to be effective against HSV (Sharifi-Rad et al., 2017) and influenza virus (Alburn et al., 1972). Additional less abundant TEO fractions should be tested to evaluate their antiviral activity.

We demonstrated the in vitro antiviral and virucidal effect of TEO against FCoV in CRFK cells. These studies open up several perspectives in terms of future applications and therapeutic options for human and animal coronaviruses.

Declaration of conflict of interest

The authors declare that there is no conflict of interest.

Appendix A. Additional information

Table 1: Chemical composition of Thymus vulgaris essential oil

NumberFolderArea% + SEMSI / MSLRIAI
1propanoic acid, ethyl ester0.10+0.0986714714
2α-tricyclene0.13+0.1094915919
3α-thujene1.21+0.9897925926
4α-pinene1.81+0.1195930931
5camphene1.89+0.7096949952
61-octen-3-ol0.37+0.0183974975
7sabinene0.71+0.3293977977
8β-pinene0.56+0.0394978978
9β-myrcene1.42+0.2586985991
10α-phellandrene0.15+0.019110011003
11p-cymene19.64+2.509510241024
12lemonade0.6+0.019110331027
13eucalyptol0.89+0.079910231031
14cis-β-terpineol0.13+0.019011451147
15γ-terpinene8.83+0.879410631059
16α-terpinolene0.12+0.018110851089
17β-linalool4.07+1.559710971098
18camphor1.69+0.779811451146
19borneol1.85+0.989711661167
20terpinen-4-ol1.83+0.899611721174
21α-terpineol0.12+0.018611891190
22methyl thymol, ether0.39+0.229012351235
23isothymol methyl ether0.42+0.0294ON THE1244
24thymol47.01+1.599412901290
25caryophyllene2.18+0.999914171418
26caryophyllene oxide0.58+0.139115811592
 Totally characterized98.7   
 Others 1.3   
The legend
SEM = Structural Equation Modeling
SI / MS = Similarity Index / Mass Spectrum
LRI = Linear retention index
AI = Arithmetic Index
NA = not assessed

References

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