Identification of novel antiviral of fungus-derived brefeldin A against dengue viruses
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Microbial natural products possess a wide range of biological and biochemical potential. Among them, fungal secondary metabolites are one of the most important sources for discovering new drugs or lead compounds. In the present study, we explored substances produced by the strain Penicillium sp. FKI-7127 for its antiviral activity. We identified brefeldin A as a novel antiviral agent against dengue viruses. The inhibitory effect of brefeldin A was confirmed by virus titer and immunofluorescence assay. Brefeldin A inhibited dengue viruses regardless of serotypes and other related viruses including Zika virus and Japanese encephalitis virus. Time-of-addition study showed that brefeldin A exerts its antiviral effect at an early stage of the dengue virus (DENV) life cycle. These studies demonstrate that (i) brefeldin A could be used as a lead compound for drug development of anti-DENV and other related viruses and (ii) fungal metabolites are a potential and valuable source for dengue virus drug discovery.
KeywordsDengue virus Antiviral Brefeldin A Secondary metabolite Fungus
The 50% cytotoxic concentration
Dengue hemorrhagic fever
Enzyme-linked immunosorbent assay
High-performance liquid chromatography
Half maximal inhibitory concentration
Japanese encephalitis virus
Dengue virus (DENV) is an important mosquito-borne pathogen for causing dengue fever (DF) and dengue hemorrhagic fever (DHF). DF is relatively mild, but DHF leads to the life-threatening dengue shock syndrome . It is estimated that there are 390 million dengue infections per year, of which 96 million manifest apparently . At present, no specific antiviral therapy for treatment of dengue disease is available. Thus, drug discovery research for dengue is of importance.
Natural products are valuable materials for the discovery and development of new drugs for treating many diseases since they possess a wide range of structural and functional diversity . Among varied sources of natural products, secondary metabolites produced by fungi have been recognized as an important source of lead structures for new drugs .
Some bioactive compounds that are isolated and characterized from metabolites of soil-borne and endophytic fungi have led to the development of drugs such as anticancer drug Taxol which originated from endophytic-fungal metabolites . Furthermore, a lot of antibacterial substances have been demonstrated from extracts and pure substances obtained from culture broth or fungal biomass . Some studies have identified substances that inhibit viruses [7, 8]. However, there are only few reports about DENV compounds found from fungal metabolites so far .
In this study, we examined anti-DENV activity of secondary metabolites produced by a fungal strain, Penicillium sp. FKI-7127. We isolated and identified brefeldin A (BFA) as a novel antiviral agent against DENVs. Inhibition of BFA on Japanese encephalitis virus (JEV) and Zika virus (ZIKV) was also demonstrated.
Materials and methods
Cell lines and viruses
Vero cells were maintained in a minimum essential medium supplemented with 10% fetal calf serum. The cells were grown at 37 °C with 5% CO2. The strains used for this study were patient-derived DENV1–4 from the Philippines, strains 99st, 00st-22A, SLMC50, and SLMC318, respectively; Zika virus strain 976; and Japanese encephalitis virus (JEV) Beijing strain.
Microorganisms and culture of the fungal strain
The fungal strain FKI-7127 was isolated from a soil around the root of Angelica keiskei collected in Kouzu Island, Tokyo, Japan. To observe the morphological characteristics, this strain was incubated on Miura’s medium (LcA). From the results of morphological observation, the producing strain FKI-7127 was classified as genus Penicillium. The strain Penicillium sp. FKI-7127 was maintained on an LcA slant. A loopful of spores of this strain was inoculated into a test tube, containing 10 ml of a seed medium consisting of 2% glucose, 0.5% Polypepton, 0.2% yeast extract, 0.2% KH2PO4, 0.05% MgSO4·7H2O, and 0.1% agar, and incubated for 3 days. One milliliter of the seed culture was inoculated into each of two 500-ml Erlenmeyer flasks containing 100 ml of a production medium consisting of 3% soluble starch, 1.0% glycerol, 2% soybean meal, 0.3% dry yeast, 0.3% KCl, 0.2% CaCO3, 0.05% KH2PO4, 0.05% MgSO4·7H2O, and 0.03% quercetin dihydrate, and the production culture was incubated for 6 days. Fifty percent of ethanol extract of cultured broth were prepared for antiviral test.
Antigen detection ELISA
To evaluate the antiviral activity of a sample, Vero cells were seeded in 96-well plates (1 × 104 cells/well) and infected with DENV at multiplicity of infection (MOI) of 0.5 in the presence of samples/compound or 0.1% dimethyl sulfoxide (DMSO). The cells were incubated for 3 days when infected culture fluid (ICF) was harvested and subjected for antigen detection enzyme-linked immunosorbent assay (ELISA) to determine dengue virus antigen level as described previously . The result was expressed as a percent of inhibition which determined as (OD value of DMSO-treated cells) − (OD value of compound-treated cells) × 100% divided by (OD value of DMSO-treated cells).
Cells viability assay
Vero cells in 96-well plates were treated with samples for 5 days. Cell viability was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] according to manufacturer’s instruction (Promega).
Isolation and identification of brefeldin A
Cultured broth was extracted with 200 ml of ethyl alcohol (EtOH). After the mycelia were separated by centrifugation, the extract was evaporated to remove EtOH. A part of the aqueous residue was applied to a Seppak plus ODS C18 cartridge and eluted with H2O–CH3CN system to give five fractions (pass through 100:0, 70:30, 40:60, and 0:100 each 3 ml). Brefeldin A was detected in both 40:60 fraction and 0:100 fraction by high-performance liquid chromatography (HPLC) analysis, and identification of brefeldin A was achieved by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) measurement.
Vero cells in a 24-well plate were infected with DENV-2 and added with BFA. After 48 h, infected cells were recovered, washed with PBS, and spotted onto a glass slide. Immunostaining was done as described previously .
Time-of-addition studies were performed in 96-well plate cells as follows. (i) Pre-infection assay: Vero cells were treated with 125 nM BFA or 0.1% DMSO as control for 2 h at 37 °C prior to being washed twice with PBS and infected with DENV-2 at an MOI of 10. After 1.5 h virus adsorption, the cells were washed twice with PBS and incubated in fresh media for 24 h before ICFs were harvested for virus quantification. (ii) During-infection assay: Vero cells were infected with DENV-2 in the presence of BFA. After 1.5 h virus adsorption, inoculum was removed and the cells were washed twice. The cells were then incubated with fresh media for 24 h before ICFs were harvested for virus quantification. (iii) After-infection assay: Vero cells were infected with DENV-2. After virus adsorption and washing, BFA or 0.1% DMSO as control was added at seven different time points postinfection (0, 2, 4, 6, 8, 12, and 18 h). The ICFs were harvested at 24 hpi for virus quantification.
Virus titration and focus reduction assay
Virus titers were determined using Vero cells in 96-well plates as described previously . In brief, virus stock or ICFs were diluted tenfold in the MEM and inoculated to the cells. After 60 min of virus adsorption, the MEM containing 2% FCS and 1.25% methylcellulose was overlaid on the cells. The cells were then incubated for 2 to 4 days before subjected to focus staining. For focus staining, 12D11/7E8 monoclonal antibody and HRP-conjugated goat anti-mouse IgG + M were used as a primary and secondary antibody, respectively. The infected cells were visualized with 3,3′-diaminobenzidine, tetrahydrochloride (DAB). For focus reduction assay, Vero cells in 96-well plates were infected with DENV-1, 2, 3, 4, ZIKV, or JEV at an MOI of 0.5 in the presence of BFA at different concentrations. After 48 h incubation, ICFs were harvested. A hundred microliters of diluted ICFs (100× or 1000× dilution) was infected into fresh Vero cells in 96-well plates and incubate for another 2 to 4 days. The cells were then subjected for focus staining as described above. Experiments were performed twice, duplicating each.
Results and discussion
Identification of antiviral substance in the present study was part of our drug screening study for new dengue drug discovery. We employed cell-based assay in combination with in-house antigen detection ELISA to evaluate anti-DENV activity. This technique was relatively quick and can be applied for high-throughput screening.
Inhibitory effect and cytotoxicity of fraction #17 were further evaluated in increasing dilution (Fig. 1b). Fraction #17 inhibited DENV-2 in a dose-dependent manner. The 50% cytotoxic concentration (CC50) of the fraction after 5 days of simultaneous incubation was > 16×, the lowest dilution tested.
We next performed the identification of active components in fraction #17. Using HR-ESI-MS and NMR methods, brefeldin A was revealed as an active compound (Fig. 1c).
Antiviral activity of BFA against DENV-1 to 4, ZIKV, and JEV in Vero cell
61.3 ± 13.5
54.6 ± 0.9
57.9 ± 0.1
65.7 ± 6.3
54.8 ± 0.4
58.4 ± 0.3
BFA is a fungal secondary metabolite which was first isolated from Penicillium decumbens . BFA has various biological actions including antitumor and antibacterial activities . BFA has also been reported to have antiviral activities against some viruses including poliovirus  and Rotavirus . Recently, Zhou et al. demonstrated the inhibitory effect of BFA on Japanese encephalitis virus (JEV) in BHK-21 cells . To our knowledge, this is the first report to demonstrate antiviral activity of BFA on DENV.
The time-of-addition study results are in line with an already known mode of action of BFA. BFA has been known to inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus indirectly by interfering with the function of the Golgi apparatus . BFA disturbs maturation and egress of herpes simplex virus particles during infection . BFA has also been reported to interfere processing and secretion of the envelope of glycoproteins of HIV-1 in T-lymphoblast cells leading to inhibition of viral particle formation . In case of poliovirus, BFA inhibits viral RNA synthesis by preventing the formation of secretory vesicles . We speculate that, like other enveloped viruses, BFA inhibits the maturation of the DENV, ZIKV, and JEV by directly blocking the trafficking of glycoprotein from the ER to Golgi apparatus leading to the prevention of formation and release of the viruses from infected cells. In addition in our study, no inhibitory effect of BFA on DENV was shown in C6/36 cells (data not shown) indicating that BFA could not block intracellular protein transport in mosquito cell line.
Despite the fact that BFA possesses antiviral activity as demonstrated in this study and other previous studies, its toxicity would become a crucial issue in order to develop it as antiviral agents. Toxicity of BFA indeed is not unexpected because it targets Golgi apparatus which eventually causes cell death. However, BFA could be served as a lead compound. In the future, it can be structurally and phenotypically optimized by reducing its toxicity. Furthermore, its antiviral activity can also be improved through derivative analyses or another approaches.
Aside from its antiviral property, mechanism of disruption of the proper vesicular transport between ER and Golgi by BFA which is a critical step for the viral replication and release could provide a new tool to characterize the lifecycle of the virus. It is also possible to help researchers develop novel inhibitors of DENV and other viruses.
In conclusion, here we isolated, identified, and characterized an anti-DENV agent of fungus-derived BFA which is potentially used as a lead compound for drug development of anti-DENV and other related viruses. Fungal secondary metabolites are a potential and valuable source in drug screening for the development of antiviral agents.
This research was supported by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) of Japan Agency for Medical Research and Development (AMED).
Availability of data and materials
KM and MR conceived and designed the study. MR, MM, KN, MA, and AM performed the experiments. MR, MM, KS, AM, and KM analyzed the data. MR, MM, KN, KS, and KM wrote the paper. All authors read and approved the final manuscript.
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