Limits...
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

Western blot analysis of HT29.f8 cell lysates. Five micrograms of HT29.f8 cell lysates was separated on a 12.5% SDS-PAGE, electroblotted onto nitrocellulose membranes, probed with rabbit anti-NSP4150–175 peptide-specific and goat anti-rabbit HRP-conjugated IgG, and visualized with Super Signal West Pico chemiluminescent substrate (Pierce) followed by exposure to Kodak X-OMAT film. (Lane 1) RV-infected HT29.f8 cells and (Lane 2) RV-infected HT29.f8 cells with 0.02% DMSO, respectively, show cleavage fragments and unglycosylated, mono-, diglycosylated, and multimeric forms of NSP4. (Lane 3) RV-infected HT29.f8 cells with 20 μM t-A1 and (Lane 4) RV-infected HT29.f8 cells with 20 μM t-A3 only show the diglycosylated form of NSP4. (Lane 5) HT29.f8 with no virus showed NSP4 banding pattern.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4563088&req=5

fig6: Western blot analysis of HT29.f8 cell lysates. Five micrograms of HT29.f8 cell lysates was separated on a 12.5% SDS-PAGE, electroblotted onto nitrocellulose membranes, probed with rabbit anti-NSP4150–175 peptide-specific and goat anti-rabbit HRP-conjugated IgG, and visualized with Super Signal West Pico chemiluminescent substrate (Pierce) followed by exposure to Kodak X-OMAT film. (Lane 1) RV-infected HT29.f8 cells and (Lane 2) RV-infected HT29.f8 cells with 0.02% DMSO, respectively, show cleavage fragments and unglycosylated, mono-, diglycosylated, and multimeric forms of NSP4. (Lane 3) RV-infected HT29.f8 cells with 20 μM t-A1 and (Lane 4) RV-infected HT29.f8 cells with 20 μM t-A3 only show the diglycosylated form of NSP4. (Lane 5) HT29.f8 with no virus showed NSP4 banding pattern.

Mentions: The percentage of live/dead cells was calculated using the trypan blue exclusion dye assay (Figures 2(a)–2(d)). At 24 hpi, the cell viability between all groups tested (HT29.f8 cells with RV, HT29.f8 cells with RV and 0.02% DMSO, HT29.f8 cells with RV and 10 μM stilbenoids, HT29.f8 cells with RV and 20 μM stilbenoids, HT29.f8 cells with 20 μM stilbenoids only, HT29.f8 cells with 0.02% DMSO, and HT29.f8 cells only) was not statistically significantly different (p < 0.05). These data revealed that the addition of RV increases cell death, but not significantly in the time frame examined. Also, the addition of 20 μM concentrations of the stilbenoids decreased cell viability but not significantly, while the addition of 0.02% DMSO to the culture system did not adversely affect the viability of the HT29.f8 cells in culture or diminish viral replication (Figures 2(a)–2(d) and 6). These data demonstrate that HT29.f8 cells were not adversely affected by RV, 0.02% DMSO, or concentrations up to 20 μM of the four stilbenoids tested (t-Res, t-PA, t-A1, or t-A3).


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)

Western blot analysis of HT29.f8 cell lysates. Five micrograms of HT29.f8 cell lysates was separated on a 12.5% SDS-PAGE, electroblotted onto nitrocellulose membranes, probed with rabbit anti-NSP4150–175 peptide-specific and goat anti-rabbit HRP-conjugated IgG, and visualized with Super Signal West Pico chemiluminescent substrate (Pierce) followed by exposure to Kodak X-OMAT film. (Lane 1) RV-infected HT29.f8 cells and (Lane 2) RV-infected HT29.f8 cells with 0.02% DMSO, respectively, show cleavage fragments and unglycosylated, mono-, diglycosylated, and multimeric forms of NSP4. (Lane 3) RV-infected HT29.f8 cells with 20 μM t-A1 and (Lane 4) RV-infected HT29.f8 cells with 20 μM t-A3 only show the diglycosylated form of NSP4. (Lane 5) HT29.f8 with no virus showed NSP4 banding pattern.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Western blot analysis of HT29.f8 cell lysates. Five micrograms of HT29.f8 cell lysates was separated on a 12.5% SDS-PAGE, electroblotted onto nitrocellulose membranes, probed with rabbit anti-NSP4150–175 peptide-specific and goat anti-rabbit HRP-conjugated IgG, and visualized with Super Signal West Pico chemiluminescent substrate (Pierce) followed by exposure to Kodak X-OMAT film. (Lane 1) RV-infected HT29.f8 cells and (Lane 2) RV-infected HT29.f8 cells with 0.02% DMSO, respectively, show cleavage fragments and unglycosylated, mono-, diglycosylated, and multimeric forms of NSP4. (Lane 3) RV-infected HT29.f8 cells with 20 μM t-A1 and (Lane 4) RV-infected HT29.f8 cells with 20 μM t-A3 only show the diglycosylated form of NSP4. (Lane 5) HT29.f8 with no virus showed NSP4 banding pattern.
Mentions: The percentage of live/dead cells was calculated using the trypan blue exclusion dye assay (Figures 2(a)–2(d)). At 24 hpi, the cell viability between all groups tested (HT29.f8 cells with RV, HT29.f8 cells with RV and 0.02% DMSO, HT29.f8 cells with RV and 10 μM stilbenoids, HT29.f8 cells with RV and 20 μM stilbenoids, HT29.f8 cells with 20 μM stilbenoids only, HT29.f8 cells with 0.02% DMSO, and HT29.f8 cells only) was not statistically significantly different (p < 0.05). These data revealed that the addition of RV increases cell death, but not significantly in the time frame examined. Also, the addition of 20 μM concentrations of the stilbenoids decreased cell viability but not significantly, while the addition of 0.02% DMSO to the culture system did not adversely affect the viability of the HT29.f8 cells in culture or diminish viral replication (Figures 2(a)–2(d) and 6). These data demonstrate that HT29.f8 cells were not adversely affected by RV, 0.02% DMSO, or concentrations up to 20 μM of the four stilbenoids tested (t-Res, t-PA, t-A1, or t-A3).

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