Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: Evaluation of cytotoxicity and antiviral activity of plant materials used for HFMD in China*.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: Antiviral activity evaluation of HCT and MHB extracts against EV71 and CVA16. (a, b) Pretreatment of Vero cells with HCT or MHB blocks the cytopathic effect by virus infection. Vero cells were pretreated with or without a herb extract at concentrations as indicated for 2 hr, and the cells were then infected with EV71 (a, Fuyang strain) or CVA16 (b) at an MOI of 0.2 TCID 50 /cell or PFU/cell. After incubation for 72 hr, the cells were fixed with 3% formaldehyde, stained with 0.5% crystal violet. The cells were photographed. To quantitatively measure the staining, crystal violet was extracted by DMSO, and the absorbance at 570 nm was measured colorimetrically. An increase in reading compared with that of an infected control indicates an inhibitory effect of the extract against virus infection. Data are presented as mean ± SE of duplicate samples. (c, d) HCT and MHB treatments reduce infectious virion production of EV71 and CVA16, respectively. Vero cells were infected with EV71 or CVA16 (MOI = 0.2) in the presence or absence of a herbal extract at indicated concentrations. The cells and culture supernatants were harvested at 48 hr PI and determined for virus titration by TCID 50 assay for EV71 (c) and by plaque forming assay for CVA16 (d). Data are presented as mean ± SE of triplicate samples. The results are representative of two independent experiments.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Activity Assay, Infection, Incubation, Staining, Titration

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: HCT directly inactivates EV71 virion, whereas MHB blocks CVA16 infection by targeting both the virion and cellular factors. (a) Time course study of HCT and MHB antiviral activity. HCT at 40 μ g/mL and MHB at 200 μ g/mL were added at 2 hr prior to (−2 hr), during (0 hr), or postvirus inoculation at times as indicated (2 hr, 4 hr, 8 hr, 10 hr, 12 hr, and 24 hr PI). Virus infection was determined at 72 hr PI by measuring cell viability colorimetrically. Data are presented as an inhibition rate which is calculated as described in Materials and Methods. (b, c) Coincubation of EV71 with HCT abolishes EV71 infectivity, and MHB inhibits CVA16 infection likely through viral and cellular factors. EV71 (b) or CVA16 (c) in 20 μ L culture medium was mock-treated or treated with HCT at a concentration of 40 μ g/mL or MHB at 200 μ g/mL in a 37°C water bath, respectively, for 2 hr (virus). The treated samples were then added to 1.0 mL fresh culture medium and used to infect Vero cells (final MOIs at 0.2 and final concentrations of HCT and MHB were at 0.8 μ g/mL and 4 μ g/mL, resp.). In parallel experiments, Vero cells were pretreated with HCT at 40 μ g/mL or with MHB at 200 μ g/mL at 37°C for 2 hr. The drugs were replaced with fresh culture medium (cell) or left in the culture medium (plus drug). The cells were then infected with EV71 or with CVA16 at equal MOIs. Virus infection was assayed by secondary infection assays at 48 hr PI. Pretreatment of EV71 but not Vero cells with HCT abolished the infectivity of EV71 virus. Treatment of CVA16 or Vero cells with MHB blocked CVA16 infection. Data are presented as mean ± SE of triplicate samples. The results are representative of two independent experiments.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Infection, Activity Assay, Inhibition, Concentration Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: Treatment with flavonoids or chlorogenic acid of HCT does not block EV71 infection. (a) HPLC profiling of water extract of HCT. The identity of the water extract of HCT was characterized by HPLC profiling on a Hypersil GOLD column (4.6 mm × 250 mm, 5 μ m) and acetonitrile with increasing concentration of 0.1% trifluoroacetic acid as a mobile phase. Hyperoside (peak 1), isoquercitrin (2), quercitrin (3), and quercetin (4) were used as standards. (b) Flavonoids or chlorogenic acid in HCT has no antiviral effect against EV71 infection. Vero cells were pretreated with or without quercetin, quercitrin, isoquercitrin (Isoq), hyperoside (Hepe), or chlorogenic acid (Chlo) at indicated concentrations. The cells were then infected with EV71 at an MOI of 0.2 TCID 50 /cell for 72 hr, and cell viability was measured colorimetrically. Data are presented as mean ± SE of triplicate samples. The results are representative of two independent experiments.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Blocking Assay, Infection, Concentration Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: Inhibition of infection-induced I κ B α degradation by HCT and MHB. (a, d) Determination of the time courses of I κ B α degradation during EV71 and CVA16 infections. Vero cells were infected with EV71 (a) or CVA16 (d) at an MOI of 3 for indicated times. The cytoplasmic levels of I κ B α proteins were determined by immunoblotting assay. At 10 hr PI, I κ B α was significantly degraded implying the NF- κ B activation. (b, c) HCT treatment blocks I κ B α degradation induced by EV71 infection and TNF α treatment. Vero cells were infected with EV71 (b, MOI = 3) for 10 hr or treated with 1 ng/mL TNF α (c) for 20 min in presence or absence of HCT at indicated concentrations. I κ B α degradation was detected by immunoblotting assay. (e, f) MHB treatment blocks I κ B α degradation induced by CVA16 infection and TNF α treatment. Vero cells were infected with CVA16 (e, MOI = 3) for 10 hr or treated with 1 ng/mL TNF α (f) for 20 min in presence or absence of MHB at indicated concentrations. I κ B α degradation was detected by immunoblotting assay. GAPDH was used as a loading control. The results are representative of two independent experiments. TNF α (+) at 3 ng/mL was used as a positive control for I κ B α degradation.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Inhibition, Infection, Western Blot, Activation Assay, Positive Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: The effect of herb extracts on virus-induced I κ B α degradation. Vero cells were pretreated with extracts (1: EFT, 2: IIFL, 3: GUF, 4: GJE, 5: MHB, 6: HCT, 7: IIFS, 8: LJT, 9: FSV, 10: ALL, 11: SBG, 12: AAB) for 2 hr and then infected with EV71 (a) or CVA16 (b) at an MOI of 3 for 10 hr. I κ B α degradation was detected by immunoblotting assay.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Infection, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Laboratory Evaluation of Medicinal Herbs Used in China for the Treatment of Hand, Foot, and Mouth Disease

doi: 10.1155/2013/504563

Figure Lengend Snippet: The herb extracts inhibit EV71 or CVA16 infection induced proinflammatory response. Vero cells were infected with EV71 (a) or CVA16 (b) at an MOI of 3 in the presence or absence of HCT or MHB at indicated concentrations. The total RNA was isolated by the TRIzol reagent at 16 hr PI. Five hundred nanograms of total RNA was reverse transcribed and amplified by PCR (upper panels) or by quantitative real-time PCR (lower panels) using primers specific for IL-6 and GAPDH. PCR products were run on a 2.0% agarose gel and visualized with ethidium bromide staining. Data are presented as mean ± SE of triplicate samples. The results are representative of two independent experiments.

Article Snippet: Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) BrCr strain were obtained from China Center for Type Culture Collection (CCTCC) at Wuhan University.

Techniques: Infection, Isolation, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: COPI subunits (A) αCOP and (B) ζ1COP silencing with two individual siRNA duplexes blocked replication of EV71 ( P <0.05). COPII subunits (C) Sec13p and (D) Sec23p depletion by siRNA was dispensable to EV71 replication ( P >0.05). Values were obtained from triplicate wells and are mean ± SD.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: (A) αCOP, (B) ζ1COP silencing with two individual siRNA duplexes blocked EV71 viral protein VP1 expression by Western blotting with the indicated antibodies ( P <0.05). Quantification analysis of EV71 VP1 synthesis was performed by Image J. Standard deviations of three independent experiments are shown. (C) αCOP and ζ1COP depletion by siRNA inhibited EV71 virions production ( P <0.05). Values were obtained from triplicate wells and are mean ± SD.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: RD cells were transfected with αCOP and ζ1COP siRNA and infected with EV71. At 24 hpi, cells were fixed with 4% paraformaldehyde and permeabilized. EV71 infection was determined by immunofluorescence staining on VP1. RD cells were observed by DAPI.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques: Transfection, Infection, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: RD cells were infected with EV71 at 0.1 MOI with various concentrations of BFA. (A) Concentrations of 20 ng/ml BFA or higher significantly inhibited EV71 replication ( P <0.05). Standard deviations of three independent experiments are shown. (B) Viral VP1 protein and internal cell tubulin protein was detected by in-cell Western blot. Concentrations of 20 ng/ml or higher BFA inhibited VP1 expression. (C) Viral titre was suppressed by 20 ng/ml or higher BFA ( P <0.05). Standard deviations of three independent experiments are shown. (D) 50 ng/ml GCA inhibited EV71 replication in RD cells ( P <0.05). Standard deviations of three independent experiments are shown.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques: Infection, In-Cell ELISA, Expressing

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: ( A ) Cells were pretreated with siRNA, infected at 4°C to allow surface binding, followed by 3 hr at 25°C to resume endocytosis. Viral entry was measured by determining viral genome copies, which was almost unchanged between COPI knocked down group and control group ( P >0.05). ( B ) BFA and GCA inhibited EV71 replication ( P <0.05), when added up to 6 hpi. RD cells were infected with at 0.1 MOI and harvested at 24 hpi. The experiment was performed in triplicate, and the bars represent means ± SD.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques: Infection, Binding Assay, Control

Journal: PLoS ONE

Article Title: COPI Is Required for Enterovirus 71 Replication

doi: 10.1371/journal.pone.0038035

Figure Lengend Snippet: Quantitative real-time PCR primers.

Article Snippet: Cells were washed twice with PBS and incubated with anti-EV71 VP1 monoclonal antibody (eENZYME, Maryland, USA) and mouse anti-tubulin (Beyotime, Suzhou, China) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction