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Investigation of Stilbenoids as Potential Therapeutic Agents for Rotavirus Gastroenteritis.

Ball JM, Medina-Bolivar F, Defrates K, Hambleton E, Hurlburt ME, Fang L, Yang T, Nopo-Olazabal L, Atwill RL, Ghai P, Parr RD - Adv Virol (2015)

Bottom Line: Cell viability counts showed no cytotoxic effects on HT29.f8 cells.Two stilbenoids, trans-arachidin-1 and trans-arachidin-3, show a significant decrease in RV infectivity titers.Western blot analyses performed on the infected cell lysates complemented the infectivity titrations and indicated a significant decrease in viral replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Rotavirus (RV) infections cause severe diarrhea in infants and young children worldwide. Vaccines are available but cost prohibitive for many countries and only reduce severe symptoms. Vaccinated infants continue to shed infectious particles, and studies show decreased efficacy of the RV vaccines in tropical and subtropical countries where they are needed most. Continuing surveillance for new RV strains, assessment of vaccine efficacy, and development of cost effective antiviral drugs remain an important aspect of RV studies. This study was to determine the efficacy of antioxidant and anti-inflammatory stilbenoids to inhibit RV replication. Peanut (A. hypogaea) hairy root cultures were induced to produce stilbenoids, which were purified by high performance countercurrent chromatography (HPCCC) and analyzed by HPLC. HT29.f8 cells were infected with RV in the presence stilbenoids. Cell viability counts showed no cytotoxic effects on HT29.f8 cells. Viral infectivity titers were calculated and comparatively assessed to determine the effects of stilbenoid treatments. Two stilbenoids, trans-arachidin-1 and trans-arachidin-3, show a significant decrease in RV infectivity titers. Western blot analyses performed on the infected cell lysates complemented the infectivity titrations and indicated a significant decrease in viral replication. These studies show the therapeutic potential of the stilbenoids against RV replication.

No MeSH data available.


Related in: MedlinePlus

HT29.f8 Cell viability at 24 hpi with stilbenoids. (a) Resveratrol (t-Res). (b) Piceatannol (t-Pa). (c) Arachidin-1 (t-A1). (d) Arachidin-3 (t-A3).
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fig2: HT29.f8 Cell viability at 24 hpi with stilbenoids. (a) Resveratrol (t-Res). (b) Piceatannol (t-Pa). (c) Arachidin-1 (t-A1). (d) Arachidin-3 (t-A3).

Mentions: The objective of the study was to test the effect(s) of the four stilbenoids on RV replication in HT29.F8 cells with variable concentrations of the stilbenoids and different collection times. The dose was based on a previous study that assessed the effects of 20 μM resveratrol and 0.02% DMSO on two cell lines infected with polyomaviruses [13, 14]. Another study utilized different concentrations of DMSO and higher concentrations of resveratrol (50, 100, and 200 μM) on influenza A-infected cell line. Fifty and 100 μM concentrations of resveratrol were not cytotoxic to the cells [13, 14]. Based on these results, we choose to test 10 μM and 20 μM concentrations of each stilbenoid solubilized in DMEM with 0.02% DMSO. A total of five experimental sets were performed per stilbenoid. In the first experimental set, cells were infected with SA114F RV at a multiplicity of infection (MOI) of 2 as previously reported [22]. In the second experimental set, 0.02% DMSO was added to the RV infection to prove that 0.02% DMSO used to solubilize the stilbenoids had no effect on cell viability or production of RV. In the third and fourth experimental sets, 10 μM or 20 μM concentrations of the stilbenoids, respectively, were solubilized in 0.02% DMSO in DMEM, added to the RV inoculum, and used to infect the cells. The fifth set was uninfected HT29.f8 cells treated with the stilbenoids. The sixth set was uninfected HT29.f8 cells treated with 0.02% DMSO, and the seventh set was uninfected HT29.f8 cells alone (Figure 2). Each experimental set was tested in four wells of a 24-well tissue culture (TC) plate. The media from the four wells were pooled and centrifuged and the supernatants were stored at −80°C and used to determine viral infectivity titers. The cells were collected in PBS, frozen, thawed 3 times, and centrifuged. The supernatants were collected as the cell lysates and stored at −80°C until used in western blot assays. Viral infectivity titers were performed in triplicate using two assays, the focus forming units (FFU) and the plaque forming units (PFU) assays. Equal amounts of the cell lysates were used in western blot assays to resolve the viral proteins and probe for RV NSP4.


Investigation of Stilbenoids as Potential Therapeutic Agents for Rotavirus Gastroenteritis.

Ball JM, Medina-Bolivar F, Defrates K, Hambleton E, Hurlburt ME, Fang L, Yang T, Nopo-Olazabal L, Atwill RL, Ghai P, Parr RD - Adv Virol (2015)

HT29.f8 Cell viability at 24 hpi with stilbenoids. (a) Resveratrol (t-Res). (b) Piceatannol (t-Pa). (c) Arachidin-1 (t-A1). (d) Arachidin-3 (t-A3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563088&req=5

fig2: HT29.f8 Cell viability at 24 hpi with stilbenoids. (a) Resveratrol (t-Res). (b) Piceatannol (t-Pa). (c) Arachidin-1 (t-A1). (d) Arachidin-3 (t-A3).
Mentions: The objective of the study was to test the effect(s) of the four stilbenoids on RV replication in HT29.F8 cells with variable concentrations of the stilbenoids and different collection times. The dose was based on a previous study that assessed the effects of 20 μM resveratrol and 0.02% DMSO on two cell lines infected with polyomaviruses [13, 14]. Another study utilized different concentrations of DMSO and higher concentrations of resveratrol (50, 100, and 200 μM) on influenza A-infected cell line. Fifty and 100 μM concentrations of resveratrol were not cytotoxic to the cells [13, 14]. Based on these results, we choose to test 10 μM and 20 μM concentrations of each stilbenoid solubilized in DMEM with 0.02% DMSO. A total of five experimental sets were performed per stilbenoid. In the first experimental set, cells were infected with SA114F RV at a multiplicity of infection (MOI) of 2 as previously reported [22]. In the second experimental set, 0.02% DMSO was added to the RV infection to prove that 0.02% DMSO used to solubilize the stilbenoids had no effect on cell viability or production of RV. In the third and fourth experimental sets, 10 μM or 20 μM concentrations of the stilbenoids, respectively, were solubilized in 0.02% DMSO in DMEM, added to the RV inoculum, and used to infect the cells. The fifth set was uninfected HT29.f8 cells treated with the stilbenoids. The sixth set was uninfected HT29.f8 cells treated with 0.02% DMSO, and the seventh set was uninfected HT29.f8 cells alone (Figure 2). Each experimental set was tested in four wells of a 24-well tissue culture (TC) plate. The media from the four wells were pooled and centrifuged and the supernatants were stored at −80°C and used to determine viral infectivity titers. The cells were collected in PBS, frozen, thawed 3 times, and centrifuged. The supernatants were collected as the cell lysates and stored at −80°C until used in western blot assays. Viral infectivity titers were performed in triplicate using two assays, the focus forming units (FFU) and the plaque forming units (PFU) assays. Equal amounts of the cell lysates were used in western blot assays to resolve the viral proteins and probe for RV NSP4.

Bottom Line: Cell viability counts showed no cytotoxic effects on HT29.f8 cells.Two stilbenoids, trans-arachidin-1 and trans-arachidin-3, show a significant decrease in RV infectivity titers.Western blot analyses performed on the infected cell lysates complemented the infectivity titrations and indicated a significant decrease in viral replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Rotavirus (RV) infections cause severe diarrhea in infants and young children worldwide. Vaccines are available but cost prohibitive for many countries and only reduce severe symptoms. Vaccinated infants continue to shed infectious particles, and studies show decreased efficacy of the RV vaccines in tropical and subtropical countries where they are needed most. Continuing surveillance for new RV strains, assessment of vaccine efficacy, and development of cost effective antiviral drugs remain an important aspect of RV studies. This study was to determine the efficacy of antioxidant and anti-inflammatory stilbenoids to inhibit RV replication. Peanut (A. hypogaea) hairy root cultures were induced to produce stilbenoids, which were purified by high performance countercurrent chromatography (HPCCC) and analyzed by HPLC. HT29.f8 cells were infected with RV in the presence stilbenoids. Cell viability counts showed no cytotoxic effects on HT29.f8 cells. Viral infectivity titers were calculated and comparatively assessed to determine the effects of stilbenoid treatments. Two stilbenoids, trans-arachidin-1 and trans-arachidin-3, show a significant decrease in RV infectivity titers. Western blot analyses performed on the infected cell lysates complemented the infectivity titrations and indicated a significant decrease in viral replication. These studies show the therapeutic potential of the stilbenoids against RV replication.

No MeSH data available.


Related in: MedlinePlus