Antiviral activities and putative identification of compounds in microbial extracts from the Hawaiian coastal waters

Abstract

Marine environments are a rich source of significant bioactive compounds. The Hawaiian archipelago, located in the middle of the Pacific Ocean, hosts diverse microorganisms, including many endemic species. Thirty-eight microbial extracts from Hawaiian coastal waters were evaluated for their antiviral activity against four mammalian viruses including herpes simplex virus type one (HSV-1), vesicular stomatitis virus (VSV), vaccinia virus and poliovirus type one (poliovirus-1) using in vitro cell culture assay. Nine of the 38 microbial crude extracts showed antiviral potencies and three of these nine microbial extracts exhibited significant activity against the enveloped viruses. A secosteroid, 5α(H),17α(H),(20R)-beta-acetoxyergost-8(14)-ene was putatively identified and confirmed to be the active compound in these marine microbial extracts. These results warrant future in-depth tests on the isolation of these active elements in order to explore and validate their antiviral potential as important therapeutic remedies.

Keywords: marine extract; antiviral activity; antiviral drug; enveloped virus; secosteroids.

Figure 1

Cytotoxic sensitivities of Vero cells to selected marine microbial extracts. (^* Samples with significant differences among different concentrations in ANOVA tests. Vero cell at their exponential growth phase were seeded in 96-well plates and then exposed to different concentrations of selected marine extracts (4 wells per concentration). Following a 2-day incubation time at 37 °C, cell viability was determined using MTT assay by measuring absorbance reading at 492 nm. Results shown represent mean values of cell viability from two independent experiments and error bars denote the standard deviation.)

Figure 2

Marine microbial extracts mediated anti-viral attachment and entry into Vero cells. (HSV-1 (Left), VSV (middle), and Vaccinia virus (right) were pre-incubated with selected marine extracts at subtoxic concentration, incubated in room temperature for 2 h, and then used to infect Vero cells prepared in 24-well plates. Following 1 h viral adsorption, removed the viruses, then overlay medium was added and plates were transferred to 37 °C incubator for 36–48 h to allow viral plaque development. Plates were fixed and stained with crystal violet staining solution and photomicrographs were taken. Viral induced plaques were visually counted and marine extracts mediated inhibitory effect was determined by comparing the plaques produced in the control cultures.)

Figure 3

Inhibitory effect of marine microbial extracts on viral replication. (Vero cells were seeded into T-12.5 cm² flasks and then infected with VSV (A) or poliovirus-1 (B) at an MOI of 0.001. Following 1 h viral adsorption, culture fluid was completely removed and infected cells were washed three times and then incubated with the medium containing selected marine extracts at subtoxic concentrations. Photomicrographs were taken to show the progression of viral-induced CPE at selected post-infection times. In addition, delayed progression of CPE in the presence of marine extracts 298M(1) was documented compared to control cells under the same experimental condition except with no marine extract.)

Figure 4

GC/ITMS chromatograms of marine microbial crude extracts including blank control, standard, 298M(1), 457P(1), 474M(1), 476M(1) and 495M(1). (A) Mass peak of target compound in marine microbial crude extracts; (B) Mass peak of standard in database of GC-ITMS workstation. Arrow point denotes target compound.