Journal: Virology Journal

Article Title: Antiviral activity of micafungin against enterovirus 71

doi: 10.1186/s12985-016-0557-8

Figure Lengend Snippet: Identification of micafungin as an anti-EV71 inhibitor from a screen of the FDA-approved drug library. a Schematic diagram of DNA encoding the EV71 replicon. b Vero cells were transfected with in vitro-transcribed EV71-replicon RNAs, immediately treated with 968 FDA-approved drugs (10 μM) for 8 h, and then assayed for firefly luciferase activity. Rupintrivir (10 μM) was used as a positive control. The luciferase activities from cells treated with 21 primary hits including micafungin were presented in graph. The luciferase activity from DMSO-treated cells was considered to be 100 %. c The antiviral activities of the primary hits were further evaluated in EV71-infected LLC-MK2 Derivative cells. The LLC-MK2 Derivative cells were infected with EV71 (100 CCID 50 ), simultaneously treated with the 21 primary hits (2 and 10 μM) for 96 h, and then cell viabilities were analyzed by using MTT assay. Rupintrivir (2 and 10 μM) was used as a positive control. The viability of DMSO-treated cells was considered to be 0 %, and that of uninfected cells was considered to be 100 %. d The chemical structure of micafungin

Article Snippet: EV71 (strain BrCr) (ATCC VR-1775), EV71 (strain H) (ATCC VR-1432) were purchased from ATCC, and EV71 (strain 1095) was kindly provided by Yorihiro Nishimura [ ].

Techniques: Drug discovery, Transfection, In Vitro, Luciferase, Activity Assay, Positive Control, Infection, MTT Assay

Journal: Virology Journal

Article Title: Antiviral activity of micafungin against enterovirus 71

doi: 10.1186/s12985-016-0557-8

Figure Lengend Snippet: Micafungin potently inhibits the replication of the EV71 replicon. a Vero cells were transfected with in vitro-transcribed EV71-replicon RNAs, simultaneously treated with the indicated concentrations of micafungin for 8 h, and then assayed for firefly luciferase activity. The luciferase activity of DMSO-treated cells was considered to be 100 %. b In the same condition, another set of EV71 replicon-transfected cells was assayed for cell viability using CellTiter-Glo reagent. The activity of DMSO-treated cells was considered to be 100 %

Article Snippet: EV71 (strain BrCr) (ATCC VR-1775), EV71 (strain H) (ATCC VR-1432) were purchased from ATCC, and EV71 (strain 1095) was kindly provided by Yorihiro Nishimura [ ].

Techniques: Transfection, In Vitro, Luciferase, Activity Assay

Journal: Virology Journal

Article Title: Antiviral activity of micafungin against enterovirus 71

doi: 10.1186/s12985-016-0557-8

Figure Lengend Snippet: Micafungin potently inhibits EV71 proliferation in LLC-MK2 Derivative cells. a LLC-MK2 Derivative cells were infected with EV71 (100 CCID 50 ) and immediately treated with increasing concentrations of micafungin. Four days after treatment, antiviral activity was determined by the reduction of the cytopathic effect in an MTT assay. Cell viability of DMSO-treated cells was set to 0 % and that of uninfected cells was set 100 %. b Same cells treated with indicated concentrations of micafungin without EV71 infection were also analyzed for cell viability by using MTT assay. c-e LLC-MK2 Derivative cells were infected with EV71 (1 MOI) and simultaneously treated with increasing concentrations of micafungin. c Twenty hours post-infection, total cell extracts were prepared from cells and subjected to Western blot analysis with anti-3C antibody. β-actin was also analyzed as a loading control. d Total RNAs were prepared from cells in ( c ) and then subjected to RT-PCR for 3BC region of EV71 viral RNA. β-actin mRNAs were also analyzed as a negative control. e Twenty hours post-infection, dsRNAs were stained by using specific antibody and visualized by FITC-conjugated secondary antibody (green). Nuclear DNA was also visualized by DAPI staining (blue)

Article Snippet: EV71 (strain BrCr) (ATCC VR-1775), EV71 (strain H) (ATCC VR-1432) were purchased from ATCC, and EV71 (strain 1095) was kindly provided by Yorihiro Nishimura [ ].

Techniques: Infection, Activity Assay, MTT Assay, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Staining

Journal: Virology Journal

Article Title: Antiviral activity of micafungin against enterovirus 71

doi: 10.1186/s12985-016-0557-8

Figure Lengend Snippet: Antiviral activity of micafungin depending on the time of addition. LLC-MK2 Derivative cells were infected with EV71 at 1 MOI and treated with 20 μM of micafungin or 4 μM rupintrivir at the indicated times prior to or after virus infection. Twenty hours post-infection, virus-infected cells were visualized by staining with anti-dsRNA antibody and percentage of infected cells out of total cells were calculated

Article Snippet: EV71 (strain BrCr) (ATCC VR-1775), EV71 (strain H) (ATCC VR-1432) were purchased from ATCC, and EV71 (strain 1095) was kindly provided by Yorihiro Nishimura [ ].

Techniques: Activity Assay, Infection, Virus, Staining

Journal: Virology Journal

Article Title: Antiviral activity of micafungin against enterovirus 71

doi: 10.1186/s12985-016-0557-8

Figure Lengend Snippet: Anti-EV71 effect of micafungin is not related with IRES-dependent translation, polyprotein processing, and 2C and 3A. a 293 T cells were transfected with dual luciferase reporter DNA measuring EV71 IRES-dependent translation and then treated with indicated concentrations of micafungin. Twenty-four hours after compound treatment cells were assayed for firefly and renilla luciferases. Luciferase activities from DMSO-treated cells were set to 100 %. b 293 T cells were transfected with plasmid expressing flag-EV71(3CD) and then treated with 10 μM of micafungin. Nine hours after compound treatment total cell extracts were prepared and subjected to Western blot analysis with anti-flag antibody. Rupintrivir (10 μM) was included as a positive control. c Vero cells were transfected with in vitro transcribed CVB3-wt, CVB3-2C mt, or CVB3-3A mt replicon RNAs and simultaneously treated with indicated concentrations of micafungin. Eight hours after compound treatment cells were assayed for luciferase activity. Luciferase activities from DMSO-treated cells were set to 100 % for each replicons

Article Snippet: EV71 (strain BrCr) (ATCC VR-1775), EV71 (strain H) (ATCC VR-1432) were purchased from ATCC, and EV71 (strain 1095) was kindly provided by Yorihiro Nishimura [ ].

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Positive Control, In Vitro, 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: 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: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: Unique changes in the metabolism of EV71-infected cells. Vero cells were infected without or with EV71 at MOIs of 0.3125, 0.625, or 1.25 for 24 h, and extracted for metabolomic analysis with UPLC-Q-TOF-MS operated in electrospray positive (ESI+) and negative (ESI-) modes (n = 9). The datasets were analyzed by OPLS-DA, and the score plots for the features obtained in ESI+ and ESI– modes are shown ( A ). Some of the identified metabolites with VIP scores >1.5 are shown ( B ). The summary plot for metabolite set enrichment analysis for EV71-infected cells ( C ).

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Infection

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: EV71 infection is associated with variations in amino acid pools. Vero cells were infected and extracted for metabolomic analysis, as described in . Features corresponding to specific amino acids were extracted. The levels of metabolites are expressed relative to those of mock-infected cells. Data are mean ± SD (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.005, significant difference from mock-infected cells.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Infection

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: EV71-induced changes in metabolism of epithelial cells. Vero cells were infected and extracted for metabolomic analysis as described in . The features of interest were extracted, and the validated metabolites of interest were mapped onto biochemical pathways. The levels of metabolites are expressed relative to those of mock-infected cells. The arrow indicates the metabolic pathway, and the dashed one indicates the transport of biomolecules. Data are mean ± SD (n = 9). * p < 0.05, ** p < 0.01, *** p < 0.005, significant difference from mock-infected cells.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Infection

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: Glutamine deprivation rescues cells from viability loss and cytopathic effect. Vero cells were cultured in medium containing the indicated concentrations of glutamine (Gln) ( A , B ), or in glutamine-free medium supplemented with the indicated concentrations of DM-αKG ( C , D ). They were mock- (solid bar) or infected (empty bar) with EV71 at MOI of 1.25 for 24 ( A , C ) and 48 h ( B , D ), and the viability was assayed by neutral red assay. The data are expressed as percentage relative to those of mock-infected cells and presented as mean ± SD of six separate experiments. * p < 0.05, significant difference from mock-infected cells at a specified concentration. ( E ) Cells were cultured in glutamine-free medium (no treatment), medium containing 2 mM Gln, or glutamine-free medium containing 3.5 mM DM-αKG, and were mock- (Con) or infected ( EV71 ) with EV71 for 24 h. They were stained with Hoechst 33342 and examined under a fluorescence microscope. The white arrows indicate cells with CPE. The results shown are representative of six experiments (original magnification: × 200). The cells with CPE are those characterized by chromatin condensation and formation of crescent-shaped nuclei. ( F ) The percentage of such cells, taken as a measure of CPE, was determined by IN Cell Analyzer 1000. Data are means ± SD of three experiments. * p < 0.01, significant difference from mock-infected cells for the indicated treatment; § p < 0.05, significant difference from the no-treatment group.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Cell Culture, Infection, Neutral Red Assay, Concentration Assay, Staining, Fluorescence, Microscopy

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: Glutamine and DM-αKG are essential to viral replication. ( A ) Vero cells were infected with EV71 in the medium containing the indicated concentrations of glutamine for 1 h, and were overlaid with 0.3% agarose in MEM/2% FCS, supplemented with the indicated concentrations of glutamine. The infected cells were processed for crystal violet staining as described in Materials and Methods. Representative plates of three experiments are shown here (upper panel). The number of plaques was counted, and is expressed as percentage relative to that formed in the medium supplemented with 2 mM glutamine. Data are mean ± SD. * p < 0.01, significant difference from that of 2 mM glutamine group. ( B , C ) Vero cells were cultured in medium containing the 0 (solid bar) and 2 (empty bar) mM glutamine ( B ), or in glutamine-free medium supplemented without (solid bar) or with 3.5 (empty bar) mM DM-αKG ( C ). They were infected with EV71 at MOI of 1.25. The total RNA was extracted from cells at 0 and 24 h p.i., and qRT-PCR was performed to determine the level of viral genomic RNA. The result is expressed as fold change relative to that at 0 h p.i. Data are mean ± SD of six experiments. * p < 0.01, significant difference from that at 0 h. § p < 0.05, significant difference from the no-treatment group.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Infection, Staining, Cell Culture, Quantitative RT-PCR

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: Increased expression of GDH and CAD proteins during viral infection. ( A ) Vero cells were mock- (Mock) or infected with virus ( EV71 ) at MOI of 1.25, and were harvested at the indicated time-points p.i. for western blotting with antibodies to GLS, GDH, CAD and actin. A representative result of three experiments is shown. ( B ) The expression levels were quantified by densitometry and Image J software and normalized to that of actin, and are expressed as fold change relative to that of mock-infected cells at 0 h p.i. * p < 0.05, significant difference from cells at 0 h; § p < 0.05, significant difference from mock-infected cells at the same time-points.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Expressing, Infection, Virus, Western Blot, Software

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: GLS, GDH, and CAD are essential to viral replication. ( A ) Vero cells were transfected with NC siRNA, siGDH, siGLS and siCAD, and 48 h after transfection, the cells were analyzed for expression of GDH, GLS and CAD by western blotting. An equal amount of the lysate of each transfectant was analyzed for expression of actin by western blotting. A representative result of three experiments is shown. ( B – D ) Vero cells were transfected with NC siRNA, siGDH, siGLS or siCAD for 48 h, and the transfected cells were mock- (solid bar) or infected (empty bar) with virus at MOI of 1.25. After 48 h, the infected cells were analyzed for cell viability ( B ) and the percentage of CPE ( C ). The cell viability data are expressed as percentage relative to those of mock-infected cells. Data are mean ± SD of six experiments. ( D ) Total RNA was extracted from mock- (Mock) or infected ( EV71 ) cells for determination of viral genomic RNA. The result is expressed as fold change relative to that at 0 h p.i. Data are mean ± SD of six experiments. § p < 0.05, significant difference from mock-infected cells; * p < 0.05, significant difference from NC siRNA-transfected cells.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Transfection, Expressing, Western Blot, Infection, Virus

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: Interaction between VP1 and CAD. ( A ) Vero cells were transfected with the plasmid encoding GFP-VP1-Myc and that coding for N-FLAG-CAD, C-FLAG-CAD, or C34V. Forty-eight hours later, the cell lysate was prepared for immunoprecipitation with anti-c-Myc antibody (αMyc; c-Myc monoclonal antibody 9E10), and the subsequent western blotting with HRP-conjugated anti-FLAG antibody (αFLAG-HRP; MA1-91878) for detection of the co-immunoprecipitated N- or C-FLAG-CAD, or with HRP-conjugated anti-c-Myc antibody (αMyc-HRP; R951-25) for detection of GFP-VP1-Myc. ( B ) Reciprocal immunoprecipitation with anti-FLAG antibody (αFLAG; FLAG M2 monoclonal antibody) was followed by western blotting with αMyc-HRP for detection of the co-immunoprecipitated GFP-VP1-Myc, or with αFLAG for detection of N-FLAG-CAD. The cell lysate was immunoblotted with αFLAG-HRP, αMyc-HRP or anti-actin (αActin) antibodies. A representative result of three experiments is shown. ( C , D ) Vero cells were transfected with expression vector of GFP-VP1-Myc ( C ), GFP-VP1(FY)-Myc ( D ) or GFP-Myc (negative control) for 48 h, and the cell lysate was subjected to immunoprecipitation with αMyc and western blotting with anti-CAD antibody (αCAD) or with αMyc-HRP. The cell lysate was immunoblotted with αCAD, αMyc-HRP and αActin. A representative result of three experiments is shown. ( E ) Vero cells were transfected with the plasmid encoding N-FLAG-CAD for 48 h, and the transfected cells were infected with EV71 at MOI of 1.25 for 24 h. The cell lysate was subjected to immunoprecipitation with αFLAG and western blotting with anti-VP1 antibody (αVP1) and αFLAG. The cell lysate was immunoblotted with αVP1, αFLAG-HRP and αActin. A representative result of three experiments is shown.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Negative Control, Infection

Journal: Cells

Article Title: Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

doi: 10.3390/cells9020473

Figure Lengend Snippet: VP1 expression promotes pyrimidine biosynthesis that is essential to viral replication. ( A ) A simplified diagram shows the de novo pyrimidine biosynthesis pathway. It is noted that BRQ selectively inhibits DHODH. ( B ) (Upper panel) Vero cells were transfected with 0, 1, or 2 μg of VP1 expression vector (plus empty expression vector to maintain the total amount of transfected DNA at 2 μg). Twenty-four hours later, cells were harvested for western blotting with anti-VP1 antibody (αVP1) or anti-actin (αActin) antibodies. A representative result of three experiments is shown. (Lower panel) Vero cells were transfected in a similar manner. Twenty hours later, cells were labeled in NaH 13 CO 3 -containing medium and treated with BRQ as described in Materials and Methods. The cell extracts were subjected to mass spectrometric analysis. The abundance of [ 13 C]-dihydroorotate ( m / z 158.0283) was quantified from the corresponding extracted ion chromatogram and divided by the labeling time, and the flux is expressed as fold change relative to that of mock-transfected cells. Data are mean ± SD of six replicates. # p < 0.05, significant difference from the mock-transfected cells. ( C ) Vero cells were infected with EV71 at MOIs of 1, 2 and 5, labeled in NaH 13 CO 3 -containing medium, and treated with BRQ, as described in Materials and Methods. The abundance of [ 13 C]-dihydroorotate was quantified and divided by the labeling time, and the flux is expressed as fold change relative to that of mock-infected cells. Data are mean ± SD of six replicates. # p < 0.05, § p < 0.01, significant difference from the mock-infected cells. ( D ) Vero cells were mock- or infected with EV71 at the indicated MOIs for 1 h, and treated with 25 or 50 nM BRQ till 48 h p.i. The cell viability was assayed, as described above. The data are expressed as percentage relative to those of the untreated mock-infected cells, and presented as mean ± SD of six separate experiments. # p < 0.05, § p < 0.01, significant difference from the untreated cells (Con). ( E ) Vero cells were mock- or infected with EV71 at MOI of 0.3125 for 1 h, and treated with 25 or 50 nM BRQ till 30 h p.i. Total RNA was extracted for quantification of genomic RNA. The result is expressed as fold change relative to that at 0 h p. i. The data are presented as mean ± SD of six separate experiments. # p < 0.05, § p < 0.01, significant difference from the untreated cells. ( F ) Vero cells were infected with EV71 for 1 h, and were overlaid with 0.3% agarose in MEM/2% FCS, which was supplemented with 25 or 50 nM BRQ. The infected cells were processed for crystal violet staining as described in Materials and Methods. The number of plaques was counted, and is expressed as percentage relative to that formed in the absence of BRQ. Data are mean ± SD of six experiments. # p < 0.05, § p < 0.01, significant difference from that formed in the control plates.

Article Snippet: EV71 prototypic strain BrCr (ATCC VR784) was cultivated in Vero cells, as previously described [ ].

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Labeling, Infection, Staining, Control