Green tea extract reduces viral proliferation and ROS production during Feline Herpesvirus type-1 (FHV-1) infection

Abstract

Background: Feline Herpesvirus type-1 (FHV-1) is a worldwide spread pathogen responsible for viral rhinotracheitis and conjunctivitis in cats that, in the most severe cases, can lead to death. Despite the availability of a variety of antiviral medications to treat this illness, mainly characterized by virostatic drugs that alter DNA replication, their use is often debated. Phytotherapeutic treatments are a little-explored field for FHV-1 infections and reactivations. In this scenario, natural compounds could provide several advantages, such as reduced side effects, less resistance and low toxicity. The purpose of this study was to explore the potential inhibitory effects of the green tea extract (GTE), consisting of 50% of polyphenols, on FHV-1 infection and reactive oxygen species (ROS) production.

Results: Crandell-Reese feline kidney (CRFK) cells were treated with different doses of GTE (10-400 µg/mL) during the viral adsorption and throughout the following 24 h. The MTT and TCID₅₀ assays were performed to determine the cytotoxicity and the EC₅₀ of the extract, determining the amounts of GTE used for the subsequent investigations. The western blot assay showed a drastic reduction in the expression of viral glycoproteins (i.e., gB and gI) after GTE treatment. GTE induced not only a suppression in viral proliferation but also in the phosphorylation of Akt protein, generally involved in viral entry. Moreover, the increase in cell proliferation observed in infected cells upon GTE addition was supported by enhanced expression of Bcl-2 and Bcl-xL anti-apoptotic proteins. Finally, GTE antioxidant activity was evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and total antioxidant capacity (TAC) assays. The ROS burst observed during FHV-1 infection was mitigated after GTE treatment, leading to a reduction in the oxidative imbalance.

Conclusions: Although further clinical trials are necessary, this study demonstrated that the GTE could potentially serve as natural inhibitor of FHV-1 proliferation, by reducing viral entry. Moreover, it is plausible that the extract could inhibit apoptosis by modulating the intrinsic pathway, thus affecting ROS production.

Keywords: Antiviral; Feline Herpesvirus type-1; Green tea extract; ROS.

Fig. 1

Antiviral activity of Green tea extract (GTE) against Feline Herpesvirus type-1 (FHV-1). (A) CRFK cell viability treated with increasing concentration of GTE; (B) EC₅₀ of GTE calculated by TCID₅₀ assay; (C) Real time PCR of FHV-1’s thymidine kinase gene conducted on supernatants. (*p < 0.05; ***p < 0.001; ****p < 0.0001)

Fig. 2

Green tea extract (GTE) activates the anti-apoptotic pathway during FHV-1 infection. (A) Representative western images of Bcl-2, Bcl-xL, and β-actin; (B) Densitometric analysis of Bcl-2 and Bcl-xL protein expression normalised toward β-actin. A representative actin has been inserted into the figure. Individual actins from each membrane, as well as full-size membranes, are available in the Supplementary Material. Results were expressed as means ± SD from three independent experiments (*p < 0.05; **p < 0.01; ****p < 0.0001); (C) Acridine orange (green) and Propidium iodide (red) dual stain, scale bar = 50 μm. Whole blots are available in the Supplementary file (Additional file 1)

Fig. 3

Green tea extract (GTE) reduces Feline Herpesvirus type-1 entry. (A) Representative western images of Akt 1/2, p-Akt, gB, gI, and β-actin; (B) Densitometric analysis of p-Akt, gB, and gI protein expression normalised toward β-actin. A representative actin has been inserted into the figure. Individual actins from each membrane, as well as full-size membranes, are available in the Supplementary Material. Results were expressed as means ± SD from three independent experiments (****p < 0.0001); (C) Immunofluorescence staining of FHV-1 (green) and cell nuclei (DAPI, blue), scale bar = 50 μm. Whole blots are available in the Supplementary file (Additional file 1)

Fig. 4

Green tea extract (GTE) limits ROS production during Feline Herpesvirus type-1 (FHV-1) infection. (A) DCFH-DA (2’,7’-Dichlorofluorescin diacetate) stain, scale bar = 100 μm; (B) Fluorescence intensity quantification of DCFH-DA stain; (C) Total antioxidant capacity (TAC) relative to nmol of Trolox. (**p < 0.01; *** p < 0.001)