mouse anti-ev-a71 vp2  (Merck KGaA)

Journal: PLoS Pathogens

Article Title: Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan

doi: 10.1371/journal.ppat.1008857

Figure Lengend Snippet: Phylogenetic analyses were based on viral protein VP1, VP2 and VP3 nucleotide sequences of the Taiwan EV-A71 isolates identified between 2018 and 2019 (black circles) and a representative set of EV-A71 and enterovirus isolates (891 bases for VP1, 762 bases for VP2, 726 bases for VP3). The 2018–2019 EV-A71 that were isolated from patients with severe neurological manifestations were labeled with open triangles (refer to ). Trees were constructed by using the neighbor-joining method with 1,000 replicates through MEGA 7.0.25 ( http://www.megasoftware.net/ ). Coxsackievirus A16 strain Tainan/5079/98 (AF177911.1) was used as the outgroup. The distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Genotype assignment, country, and year of isolation are provided in the virus names. TW, Taiwan.

Article Snippet: Mock-infected RD cells were used as an antigen control, and the EV-A71 convalescent serum, an anti-EV-A71 VP2 mouse monoclonal antibody MAB979 (EMD Millipore, USA) and PBS were used as antibody controls for each experiment.

Techniques: Isolation, Labeling, Construct, Virus

Journal: PLoS Pathogens

Article Title: Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan

doi: 10.1371/journal.ppat.1008857

Figure Lengend Snippet: ( A ) Canyon epitopes, including VP1 D110, VP1 T232, VP1 K162, VP1 D164 and VP3 F186, recognized by human anti-EV-A71 MAbs 16-2-9D, 16-2-12D, 16-3-3C and 16-2-2D, were colored in cyan . The surface residue 232 of VP3, located at the canyon region of capsid, was distinct in genotype C1 EV-A71 and interacted with VP3 D234 and L236 canyon residues (colored in orange). ( B ) Epitope mapping on the viral capsid by genotype C1 EV-A71-neutralizing MAbs. Escape variants were selected using MAbs 16-2-11B, 16-3-4D and 17-1-12A and revealed single amino acid substitutions at capsid residues VP1 S241 (colored in red), VP3 E81 (colored in blue) and VP2 D84 (colored in green), respectively. VP2 E88V and VP2 K149E were previously identified in the escape variant of genotype B5 and C4 EV-A71 with MAb 17-1-12A . The surface view of pentamer shown with the 5-fold vertex at the center are created using the software program PyMOL (PDB 3VBS). The capsid VP1 protein is colored in black, VP2 colored in grey and VP3 colored in white. Abbreviations: 3, 3-fold axis; 2, 2-fold axis.

Article Snippet: Mock-infected RD cells were used as an antigen control, and the EV-A71 convalescent serum, an anti-EV-A71 VP2 mouse monoclonal antibody MAB979 (EMD Millipore, USA) and PBS were used as antibody controls for each experiment.

Techniques: Residue, Variant Assay, Software

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSP70 family chaperones are required for the EV-A71 replication cycle. RD cells were treated with DMSO or the HSP70 inhibitor JG40 at the indicated concentrations 1 h prior to EV-A71 infection (MOI = 1). The drug was retained in the culture medium throughout the experiment. Twelve hours postinfection, (A) viral RNA was quantified by RT-qPCR using a primer pair targeting the 3D region. Cell viability, shown by the number below each bar, was determined by using alamarBlue methods following the manufacturer’s instructions; (B) the levels of the viral proteins VP1 and 3D were analyzed by immunoblot analysis using mouse antibodies. The relative expression levels are indicated by the numbers shown below the blot; and (C) total virus was prepared from the cultured supernatant and the cell lysates of the EV-A71-infected cells, and viral titers were measured by TCID 50 assays. (D) Two independent shRNA clones targeting each of the HSPA1 , HSPA5 , HSPA8 , or HSPA9 genes were used separately to knock down the expression of individual HSP70s in RD cells. The knockdown efficiencies of all the shRNAs were measured by RT-qPCR using primers specific to the individual HSP70. The dotted line indicates 50% knockdown efficiency. (E) EV-A71 viral RNA replication levels in each of the HSP70- knockdown cells were measured by RT-qPCR 6 h.p.i. The dotted line indicates the vRNA levels in the control knockdown cells. (F) EV-A71 viral protein levels in the knockdown cells were analyzed 6 h.p.i. by immunoblotting using antibodies against VP1 and 3D. (G) Total viral titers were determined 12 h.p.i. by TCID 50 assays. All the quantitative results are presented as the means ± standard deviation (SD) ( n = 3), with the DMSO control (A) or the shCtrl-treated cells (D,E,G) set at 100%. One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare the differences between the DMSO group and the JG40-treated group (A,C) or between the shCtrl group and the knockdown groups (D,E,G) . *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Virus, Cell Culture, shRNA, Clone Assay, Knockdown, Control, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSP70 family chaperones are involved in various stages ofthe EV-A71 life cycle. (A) Schematic diagrams show the timecourse of the time-of-addition experiments. RD cells were infected with EV-A71 (MOI = 1) at time 0 and harvested 12 h.p.i. for analysis. JG40 (5 μM) was either absent (DMSO Ctrl) or present throughout the experiment (full-time) or was present during a specific time period, e.g., –1∼1 h.p.i., 2∼8 h.p.i., or 8∼12 h.p.i., which roughly represented the times of viral entry, translation/replication and assembly/release stages, respectively. (B) Viral RNA levels were analyzed by RT-qPCR analysis based on the DMSO control and JG40-treated cells. (C) Total viral titers and (D) extracellular viral titers were determined by TCID 50 assays, with the DMSO control set at 100%. (E) The ratio of extracellular viral titer/total viral titer, representing virion release efficiency, was determined with that of the DMSO-treated cells set at 100%. (F) The EV-A71 RNA replicon, R1 (shown in the schematic), was transcribed in vitro , and 1 μg of the RNA was introduced into RD cells by a transfection method in which the viral entry step is bypassed. RD cells were treated with DMSO or JG40 (2.5 or 5 μM) 1 h prior to RNA transfection and throughout the experiment. RNA was analyzed by RT-qPCR 3 h post transfection (h.p.t.) and (G) luciferase activity was analyzed 9 h.p.t. All the quantitative results are presented as the means ± SD ( n = 3). One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare the differences between the DMSO group and the JG40-treated group. (E) Two-tailed Student’s t -test was performed to compare the differences between the ratios of the groups treated with DMSO or with JG40 at the assembly/release stage. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Infection, Quantitative RT-PCR, Control, In Vitro, Transfection, Luciferase, Activity Assay, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: Multiple HSP70 chaperones are involved in EV-A71 entry into cells. (A) RD cells were treated with DMSO or JG40 (5 μM) together with CHX (100 μg/ml) 1 h prior to EV-A71 infection (MOI = 300). CHX was used to prevent viral RNA translation. After 1 h of infection, the cells were fixed and permeabilized. EV-A71 viral particles were detected with mouse anti-VP0/VP2 antibody, followed by Alexa Fluor 488-labeled secondary antibody. Cells were imaged by confocal fluorescence microscopy. (left) DIC image of the field; (middle) of stained viral VP0/VP2; and (right) the two images merged. One representative confocal slice is shown. Bar, 10 μm. (B) Quantitative comparison of the fluorescence intensity in the DMSO and JG40 groups is shown. The average integrated fluorescence intensity was calculated per cell based on the images taken of 10–15 fields (20–30 cells/field). The results are presented as the means ± SD of the integrated intensity of one representative experiment. Similar results were obtained from at least two independent experiments. (C,D) RD cells depleted of HSPA1, HSPA8 or HSPA9 and control knockdown cells were used for viral internalization experiments, and the integrated intensity was quantified as described in the legends to (A,B) , respectively. (B) Two-tailed Student’s t -test of the differences between the DMSO group and the JG40-treated group was performed. (D) One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare the differences in the shCtrl group and the knockdown groups. **P < 0.01, and ***P < 0.001.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Infection, Labeling, Fluorescence, Microscopy, Staining, Comparison, Control, Knockdown, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: Multiple HSP70 chaperones are required for EV-A71 IRES translational activity. (A) Schematic diagrams show the monocistronic IRES-Luc reporter RNA and the R1 3D D330A mutant replicon that had a D330A mutation in the 3D polymerase region of the R1 replicon. RD cells treated with DMSO or JG40 (2.5 or 5 μM) 1 h prior to RNA transfection and throughout the experiment were transfected with IRES-Luc RNA (B) or with the R1 3D D330A replicon RNA (C) . IRES translational activity was determined by luciferase activity assays 9 h.p.t. To determine the IRES activity in the HSP70- knockdown cells, RD cells were first infected with lentiviruses expressing control shRNA (shCtrl) or shRNA targeting HSPA1 , HSPA8 , or HSPA9 . The cells were then transfected with IRES-Luc RNA (D) or with R1 3D D330A replicon RNA (E) . The knockdown efficiency of each shRNA in each transfection experiment is indicated at the bottom. IRES activity was determined by luciferase activity assays 9 h.p.t. One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare the differences between the DMSO group and the JG40-treated group (B,C) or between the shCtrl cells and the HSP70 -knockdown cells (D,E) . *P < 0.05 and ***P < 0.001.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Activity Assay, Mutagenesis, Transfection, Luciferase, Knockdown, Infection, Expressing, Control, shRNA

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSP70 inhibition decreases the stability of the EV-A71 2C, 3C, and 3D proteins in the EV-A71-infected cells. (A) Schematic diagram shows the time course of the CHX chase experiment during EV-A71 infection. RD cells were infected with EV-A71 (MOI = 5) at time 0. DMSO or JG40 (5 μM) was added to the cell culture 2 h.p.i., and CHX (100 μg/ml) was added 9 h.p.i. to prevent further protein synthesis. Viral protein levels remaining at 1, 2, 4, and 6 h after the CHX treatment were examined by immunoblot analysis. The intensities of the protein bands were quantified by ImageJ software, with the values 1 h after CHX treatment arbitrarily set at 100%. The stability levels of the EV-A71 (B) 2C, (C) 3C, and (D) 3D proteins in the DMSO-treated and JG40-treated cells were compared by the regression curves (GraphPad Prism), shown on the right. (E) EV-A71-infected RD cells (MOI = 5) were treated with bafilomycin A1 (100 nM) or JG40 (5 μM) alone or cotreated with JG40 and bafilomycin A1 2 h.p.i., and the levels of the EV-A71 2C, 3C, and 3D proteins were determined by immunoblotting 5 h.p.i. using specific antibodies. The relative intensities of the protein bands are shown at the bottom of each lane.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Inhibition, Infection, Cell Culture, Western Blot, Software

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSPA8 and HSPA9 stabilize viral proteins to facilitate replication complex formation. (A) RD cells treated with shCtrl or with HSPA1, HSPA8, or HSPA9 depleted were infected with EV-A71 (MOI = 5) at time 0. CHX (100 μg/ml) was added to the cell culture 9 h.p.i., and the viral protein levels at 1, 2, 4, and 6 h after CHX treatment were examined by immunoblot analysis. The stability levels of the viral proteins 2C, 3C, and 3D in the HSPA1 -, HSPA8- and HSPA9 -knockdown cells were compared to those in the shCtrl cells, as shown in the regression curves, and the immunoblot data for these regression curves are presented in . To perform the coimmunoprecipitation experiments, RD cells were infected with EV-A71 (MOI = 30), and JG40 was added to the cell culture 2 h.p.i. Cell lysates were harvested 5 h.p.i. and immunoprecipitated with the indicated antibodies or control IgG, and then, an immunoblot analysis was performed using the antibodies indicated. (B) The antibody against 2C coimmunoprecipitated HSPA9 with 2C, an outcome attenuated by JG40 treatment. (C) The antibody against 3D coimmunoprecipitated HSPA8 with 3D, an outcome also greatly reduced by JG40 treatment. (D) The antibody against 2C coimmunoprecipitated viral 3CD and 3D with 2C, a finding indicative of the formation of the replication complex. The replication complex formation was greatly disrupted by JG40 treatment.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Infection, Cell Culture, Western Blot, Knockdown, Immunoprecipitation, Control

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSPA8 and HSPA9 are involved in EV-A71 viral particle assembly. (A) A coimmunoprecipitation experiment was performed to examine the effects of JG40 on viral capsid formation. RD cells were infected with EV-A71 (MOI = 1), and JG40 was added to the cell culture 8∼12 h.p.i. Cell lysates were harvested 12 h.p.i. and immunoprecipitated with anti-VP0 antibody or control IgG, followed by immunoblot analysis using the antibodies indicated. (B–D) RD cells depleted of HSPA8 or HSPA9 were infected with EV-A71 (MOI = 1). Twelve hours after infection, (B) the total viral titer in each group was determined by TCID 50 assay, and (C) the level of total vRNA, prepared by collecting cultured supernatant and cell lysates, was determined by RT-qPCR analysis. The relative levels of virus or vRNA among groups are indicated by the number shown at the bottom, with that of the control knockdown cells set at 100%. (D) The relative ratios of total viral levels/total vRNA levels, representing the assembly efficiency, are shown. One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare the differences between the shCtrl cells and the HSP70 knockdown cells. *P < 0.05 and **P < 0.01.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Infection, Cell Culture, Immunoprecipitation, Control, Western Blot, Quantitative RT-PCR, Virus, Knockdown

Journal: Frontiers in Microbiology

Article Title: The Heat Shock Protein 70 Family of Chaperones Regulates All Phases of the Enterovirus A71 Life Cycle

doi: 10.3389/fmicb.2020.01656

Figure Lengend Snippet: HSP70 inhibition suppresses autophagy- and apoptosis-mediated viral release. RD cells were infected with EV-A71 (MOI = 10), and JG40 was added to the cell culture 2 h.p.i. (A) Cells were harvested for immunoblotting and detection of LC3 at 6, 8, 10 and 12 h.p.i. The ratios of LC3-II/LC3-I, indicative of autophagy activation, are shown by the numbers below the blot. (B) Cells were collected for immunoblotting and assessed for cleaved PARP, indicative of apoptosis, 10, 12, 14, and 16 h.p.i. The relative intensities of the protein bands are shown by the numbers below the blot, based on the absence of JG40 at 12 h.p.i. arbitrarily set at 1. Staurosporine (0.1 μM) incubated with cells for 16 h was used as a positive control for the apoptosis assay. (C–E) RD cells depleted of HSPA1, HSPA8, or HSPA9 were infected with EV-A71 (MOI = 1). Twelve hours postinfection, the levels of extracellular virion (C) and total virion (D) were determined by TCID 50 assays. The relative levels of viral titers among groups are indicated by the number shown at the bottom, with that of the control knockdown cells set at 100%. (E) The relative ratios of extracellular viral titer/total viral titer, representing the release efficiency, were calculated with that of the control knockdown cells set at 1. One-way ANOVA followed by Dunnett’s post hoc analysis was performed to compare differences between the shCtrl cells and the HSP70 -knockdown cells. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The antibodies used in this study included mouse anti-EV-A71 VP1 from Abnova, mouse anti-EV-A71 VP0/VP2 (MAB979, which cross-reacts with the CVA16 capsid protein), mouse anti-CVB1 (MAB9410, which cross-reacts with CVB3) and mouse anti-Echo 11 (MAB9670) from Millipore, rabbit anti-EV-A71 VP3 and anti-HSPA9 from GeneTex, mouse anti-HSPA8 from Abcam, mouse anti-β-actin from Sigma, mouse anti-cleaved poly (ADP-ribose) polymerase (PARP) and anti-light chain 3B (LC3B) from Cell Signaling, mouse control IgG from BD Pharmingen, goat anti-mouse (H+L) conjugated to Alexa Fluor Plus 488 from Invitrogen, sheep anti-mouse IgG conjugated to horse radish peroxidase (HRP) and donkey anti-rabbit IgG conjugated to HRP from GE Healthcare.

Techniques: Inhibition, Infection, Cell Culture, Western Blot, Activation Assay, Incubation, Positive Control, Apoptosis Assay, Control, Knockdown

Journal: Scientific Reports

Article Title: Drug repurposing of pyrimidine analogs as potent antiviral compounds against human enterovirus A71 infection with potential clinical applications

doi: 10.1038/s41598-020-65152-4

Figure Lengend Snippet: Inhibition of viral genomic RNA and protein synthesis by gemcitabine. ( a ) For time-of-drug-addition study, EV-A71-infected RD cells were treated with 1 μM gemcitabine at different time points. For time-of-drug-removal study, EV-A71-infected RD cells were treated with 1 μM gemcitabine and were removed at different time points. The EV-A71 inhibition curves crossed at 4 hpi showing early gemcitabine inhibition. ( b ) RD cells were infected with EV-A71 at MOI. 1 and treated with gemcitabine at three concentrations (0.5, 1 or 10 μM). The relative viral genome RNA fold change showed a significant reduction in all three-gemcitabine concentrations at 6 hpi. Gemcitabine is represented by Gem. ( c ) EV-A71-infected RD cells and treated with gemcitabine were assessed for viral protein synthesis by Western blotting. Gemcitabine was observed to inhibit non-structural (3CD and 3D) and structural protein (VP0 and VP2) synthesis from ≥0.5 μM gemcitabine. ( d ) FLuc: RLuc ratio was determined in RD cells transfected with EV-A71 bicistronic plasmid. Results showed a similar amount of FLuc: Rluc ratio for all gemcitabine concentration tested indicating no inhibition of IRES-dependent translation. ( e,f ) Luminescence readings from cells transfected with either replication-deficient replicon (3Ddel) or replication-competent construct. RLU showed a gradual decrease from 1 to 10 μM gemcitabine treated transfected cells specifically in the replication-competent construct. Average from three independent experiments are shown for each treatment with error bars representing standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) followed by student’s T-test comparing against vehicle-control (0.1% DMSO) for each compound treatment and *indicates p < 0.05.

Article Snippet: EV-A71-infected RD cells were fixed and permeabilized with warm 4% paraformaldehyde containing 0.01% Triton-X for 10 min at room temperature, washed three times with PBS and stained with primary antibody mouse anti-EV-A71 VP2 protein (1:1000 dilution, Millipore, #MAB979) for 1 h at 37 ° C. The primary and secondary antibodies used were and followed by.

Techniques: Inhibition, Infection, Western Blot, Transfection, Plasmid Preparation, Concentration Assay, Construct, Standard Deviation, Control