Antiviral activity of eicosapentaenoic acid against zika virus and other enveloped viruses

Abstract

Background: Zika virus (ZIKV) is an emerging flavivirus that may cause innate microcephaly or neurological disturbances. Yet no antiviral has been approved by FDA against ZIKV infection. It was shown that some unsaturated fatty acids could inactivate enveloped viruses including SARS-CoV-2. However, studies investigating the effect of eicosapentaenoic acid (EPA) on ZIKV infection are lacking. This study aims to evaluate the antiviral effect of EPA against ZIKV and other enveloped viruses.

Methods: We first explored the toxicities of EPA in vitro and in vivo. Then we examined the antiviral effect of EPA against ZIKV via cell-based immunodetection, qRT-PCR, Western blotting, and so on. To uncover its antiviral mechanism, we performed assays for virus binding, adsorption and entry, and time-of-addition. RNase digestion and ZIKV NS2B-NS3 protease inhibition assays were also adopted. Finally, we detected its effects on dengue virus (DENV)-2, herpes simplex virus (HSV)-1 and influenza A virus via MTT, Western blotting and qRT-PCR assays.

Results: EPA was found to inhibit ZIKV infection in vitro without causing cytotoxicities. EPA exhibited antiviral activity in the early stages of the ZIKV life cycle quickly. Mechanistic experiments showed that EPA disrupted the membrane integrity of viral particles, leading to the release of viral RNA, together with the interruption of ZIKV from binding, adsorption and entry, and ultimately the inhibition of viral proliferation. Furthermore, EPA exerted antiviral effects against DENV-2, HSV-1, and influenza virus, in a dose-dependent manner.

Conclusion: These findings suggest that EPA is a promising broad-spectrum antiviral drug candidate.

Keywords: EPA; ZIKV; adsorption; antiviral activity; binding; entry.

FIGURE 1

Toxicity of EPA in vitro and in vivo. Effects of EPA on the viability of Vero (A), MDCK (B), and BHK-21 (C) cells. All experiments were repeated three times. (D) Serum ALT levels in mice were measured on days 1, 2, 3, 5, and 7 after the last administration of EPA (0–100 mg/kg) using an ALT assay kit. The error bars represent the SD of the mean. (E) Urinary CRE levels in mice were measured on days 1, 2, 3, 5, and 7 after the last administration of EPA (0–100 mg/kg) using a CRE assay kit. The error bars represent the SD of the mean. (F) Representative images of HE staining of liver and kidney tissues in each group.

FIGURE 2

Inhibition of ZIKV activity by EPA in vitro. The mRNA levels of ZIKV E (A) and NS1 (B) was evaluated in ZIKV-infected Vero cells treated with EPA via qRT−PCR. (C) The protein expression of ZIKV E was evaluated in ZIKV-infected Vero cells treated with EPA via Western blotting. (D) ZIKV activity was evaluated in ZIKV-infected Vero cells treated with EPA via cell-based immunodetection assay. (E) ZIKV activity was evaluated in ZIKV-infected Vero cells treated with EPA via indirect immunofluorescence analysis. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

FIGURE 3

EPA rapidly inhibited ZIKV replication and targeted the virus. Determination of mRNA levels of ZIKV E (A) and NS1 (B) in cells infected with EPA–ZIKV mixture pre-incubated for 0, 5, 10, 30, and 60 min. Determination of the mRNA levels of ZIKV E (C) and NS1 (D) in cells, which were divided into four groups according to the treatment of EPA and ZIKV. Cell-treatment group: EPA was incubated with Vero cells for 1 h before ZIKV infection. Virus-treatment group: ZIKV was pre-incubated with EPA for 30 min and subsequently inoculated into cells. Co-treatment group: EPA and ZIKV were simultaneously inoculated into cells. Post-treatment group: Cells were incubated with ZIKV for 1 h and washed before being treated with EPA. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

FIGURE 4

EPA disrupted the early stages of the ZIKV replication cycle. (A, B) Results of time-of-addition assay. The mRNA levels of ZIKV E (A) and NS1 (B) were evaluated via qRT-PCR. EPA was added at 0, 5, 10, 20, and 40 min after ZIKV infection or at 0, 0.5, 1, 2, and 4 h after 1 h of ZIKV infection. The effects of EPA on the binding of ZIKV to host cells (C, D), its adsorption on host cells (E–G), and its entry into host cells (H) are shown. The effects of EPA on the mRNA (C–F) and protein (G, H) expression of ZIKV E are shown. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

FIGURE 5

EPA destroyed the ZIKV envelope by binding to E protein. The mRNA levels of ZIKV E (A) or the whole viral genome (B) were evaluated after performing the micrococcal nuclease digestion assay. The three-dimensional (C) and two-dimensional (D) diagrams of the EPA–ZIKV docked complexes show interactions between EPA and amino acid residues of ZIKV E protein. (C) In the 3D diagram, the gray dotted lines represent hydrophobic bonds. (D) In the 2D diagram, the pink dotted lines represent hydrophobic bonds. (E) The effects of EPA on ZIKV NS2B-NS3 protease were assessed via FRET spectroscopy. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.

FIGURE 6

EPA exhibited broad-spectrum antiviral activities. (A) The effects of EPA on dengue virus replication were evaluated via MTT assay. (B) Semi-logarithmic coordinate graph demonstrating the inhibitory effects of EPA on dengue virus replication. (C) The effects of EPA on HSV-1 gD protein were evaluated via Western blotting. (D) The effects of EPA on HSV-1 VP16 mRNA were evaluated via qRT-PCR. (E) The effects of EPA on HSV-1 gB mRNA were evaluated via qRT-PCR. (F) The effects of EPA on influenza A virus were evaluated via MTT assay. All experiments were repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n. s = not significant.