Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: (A) RD cells were cultured in 96-well plates for 24 h and infected with the EV71 strain for varying MOI. Cell viability was monitored by using a MTT assay kit at 48 hpi. (B) RD cell monolayers infected with the EV71 strain at an MOI of 1 were harvested at different time points, and then were stained with PI and Annexin V and examined by Flow Cytometry. (C) Apoptotic rate of RD cells at different points post infection. Data are expressed as means ± SEM. * P <0.05, vs 0 hpi (n=3); *** P <0.001, vs 0 MOI (n=3).

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Cell Culture, Infection, MTT Assay, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: The RD cell monolayers were infected with the EV71 strain of 1 MOI for different periods. The changes of cell morphology (A) were observed and captured using a light microscope with amplification (100×) and the levels of SOD activity (B) , iNOS (C) , MDA (D) and NO (E) were measured with corresponding assay kit at varying points of infection. Data are expressed as means ± SEM. * P <0.05, vs 0 hpi (n=3); ** P <0.01, vs 0 hpi (n=3).

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Infection, Light Microscopy, Amplification, Activity Assay

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: RD cells (5×10 5 ) cultured in confocal dish for 24 h were infected with EV71 and harvested at different time points. (A) JC-1 probe was used to investigate ΔΨm after EV71 infection in RD cells under a confocal microscopy (bar=50 μm). (B) The ultramicro variation of organellae in infected RD cells under TEM (bar=1 μm). N and M represents nucleus and mitochondria, respectively. (C) The location of EV71 particles in infected RD cells (bar=0.25 μm). (D) The red/green rate representing ΔΨm was evaluated by Image-Pro Plus 6.0 software. (E) Mitochondrial dysfunction (%) in RD cells after EV71 infection at different time points. Data are expressed as means ± SEM. * P <0.05, vs 0 hpi (n=3); ** P <0.01, vs 0 hpi (n=3).

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Cell Culture, Infection, Confocal Microscopy, Software

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: (A) Mitochondrial dysfunction in brain and skeletal muscle from mice with EV71 infection at 7 dpi was observed under TEM (bar=1 μm). N, M and ER represent nucleus, mitochondria and endoplasmic reticulum, respectively. Mitochondrial dysfunction (%) in brain cells (B) and skeletal muscle cells (C) was calculated by the number of damaged mitochondria in the total number of mitochondria under three different versions. Data are expressed as means ± SEM. * P <0.05, vs 0 hpi (n=4); ** P <0.01, vs Control (n=4).

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Infection, Control

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: Three days old BALB/c mice were intraperitoneally incubated with EV71 (2×10 6 PFU) and were sacrificed at 1 dpi, 3 dpi, 5 dpi, 7 dpi. Slices of brain, spinal cord, skeletal muscle, lung and heart of mice were stained with anti-EV71 VP1 antibody. The red triangle indicates the positive staining (brown). Bar=50 μm.

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: Tissues dissected out from EV71-infected 3-day-old mice at different time points post infection were sliced and stained with H&E. Brain tissues exhibited perivascular cuffing and neuronal degeneration (indicated by red solid arrows). Hyperchromatic and pyknotic nerve cells, neuronal loss and glial nodules (indicated by black solid arrows) were found in spinal cord. Cardiac muscle and skeletal muscle appeared necrotizing myositis with muscle fibers rupture and inflammatory cell infiltration (indicated by black hollow arrows and yellow arrows, respectively) at day 5 and 7 post infection. Lung comprised swollen alveolus pulmonis at 5 dpi and erythrocyte-filled fluid in the alveolar spaces (indicated by red hollow arrows) at 7 dpi. Bar= 50 μm.

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Infection, Staining

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: (A) iNOS expression level in target organs from mice with EV71 infection was evaluated by immunofluorescence under confocal microscopy (bar=50 μm). White arrows showed positive staining (red). Positive areas (%) of iNOS expression in the cytoplasm of brain (B) , spinal cord (C) , skeletal muscle (D) , lung (E) and heart (F) were estimated by Image-Pro Plus 6.0 software. Data are expressed as means ± SEM. * P <0.05, vs 0 hpi (n=4); ** P <0.01, vs Control (n=4).

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Expressing, Infection, Immunofluorescence, Confocal Microscopy, Staining, Software, Control

Journal: Oncotarget

Article Title: Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection

doi: 10.18632/oncotarget.21250

Figure Lengend Snippet: Characteristics of HFMD patients and controls

Article Snippet: Briefly, the paraffin section slides were incubated with 0.5% H 2 O 2 for 10 min and washed three times with phosphate buffer saline (PBS) after microwaved, and then blocked in a 5% bovine serum albumin (BSA) solution for 30 min. Rabbit anti-EV71 VP1 polyclonal antibodies (GeneTex, Inc, San Antonio, USA 1:500 dilution) were used to incubate infected sections at 4°C overnight.

Techniques: Control

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

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: Synthetic RGDS peptides significantly inhibit EV71 replication. RD cells were pretreated with RGDS and RGES peptides at a concentration of 5 mg/ml for 9 h. (A and B) Cells were infected with EV71 (MOI = 1) and harvested for RNA analysis (A) and Western blotting (B). (C) Cells were measured for cell viability by MTT assay, and the unit of the y axis is the readout optical density (OD) value. N.S, not significant. The data are representative of the means and standard deviations (error bars) for three samples per group (**, P < 0.01). All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Concentration Assay, Infection, Western Blot, MTT Assay

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: RGDS impairs MA-EV71 infection in mice. (A) One-day-old BALB/c mice were mock injected (n = 7) or i.p. injected with PBS containing RGES (5 mg/kg) (n = 8) or RGDS (n = 7) (5 mg/kg). Body weight changes were monitored every other day, and the average weights at the time point of injection were set as 1. (B, C, D) One-day-old BALB/c mice were mock infected (n = 14) or infected via i.p. injection with 1 × 107 PFU mouse-adapted EV71 along with RGES (n = 23) or RGDS (n = 21) (5 mg/kg). Body weight changes (B) and clinical symptoms (C) of the mice were monitored every day. The clinical scores were defined as follows: 0, healthy; 1, ruffled hair and hunchbacked; 2, limb weakness; 3, paralysis in one limb; 4, paralysis in both limbs; and 5, death. (D) Survival was monitored for the indicated periods. Kaplan-Meier curves were plotted using Origin 9, and the difference between the RGES-treated infected mice and the RGDS-treated infected mice was statistically significant as determined by Gehan-Breslow-Wilcoxon test analysis. (E, F) One-day-old BALB/c mice were mock infected (n = 9) or infected via i.p. injection with 1 × 107 PFU mouse-adapted EV71 along with RGES (n = 9) (5 mg/kg) or RGDS (n = 10) (5 mg/kg). The mice were euthanized at 8 dpi. Viral loads in the brainstem and skeletal muscle were quantified by qRT-PCR. N.D., not detectable (E). The viral antigen VP1 in the brainstem and skeletal muscle was examined by IHC; magnification, ×200 (F). The error bars indicate the means ± standard errors of the means (SEM). Data are representative of two independent experiments. (**, P < 0.01; *, P < 0.05). All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Infection, Injection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: FN expression upregulates EV71 proliferation. RD cells were transfected with empty vector or FN expression plasmid for 24 h and then infected with EV71 (MOI = 1). (A) Viral titers in the supernatant were measured at 0 and 12 hpi. (B) The infection of RD cells was quantified by qRT-PCR at 0 and 9 hpi. (C) EV71 VP1 and FN expression was determined by immunoblot analysis at 9 hpi. (D) CPE induced by EV71 in RD cells at 0, 6, and 9 hpi, as well as the ratio of cells with CPE, are shown. (E) RD cells transfected with or without the indicated plasmids were measured for cell viability by MTT assay, and the unit of the y axis is the readout OD value. N.D., not detectable. N.S., not significant; Vec, vector. The data are representative of the means and standard deviations (error bars) of three samples per group (*, P < 0.05). All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, MTT Assay

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: Knockout of FN decreases the yield of EV71. WT or FN-KO RD cells were infected with EV71 (MOI = 2). (A) Viral titers in the supernatant were measured at 0 and 12 hpi. (B) The infection of RD cells was quantified by qRT-PCR at 0 and 9 hpi. (C) EV71 VP1 and FN expression was determined by immunoblot analysis at 9 hpi. (D) CPE were induced by EV71 in RD cells at 0, 6, and 9 hpi, and the ratio of cells with CPE are shown. (E) Flow cytometry studies of the WT or FN-KO RD cells infected with EV71-GFP virus (MOI = 5) for 24 h. The frames indicate the virus-infected cells, and the ratio of infected cells is indicated in the inset. The data below the frames are representative of the means and standard deviations (error bars) for three samples per group. SS, side scatter. (F) WT or FN-KO RD cells were preincubated with BSA or FN protein for 6 h and then infected with EV71 (MOI = 2). The infected RD cells were collected and quantified by qRT-PCR at 9 hpi. (G) Cell viability of WT or FN-KO RD cells was determined by MTT assay, and the unit of the y axis is the readout OD value. N.D., not detectable. N.S., not significant. The data are representative of the means and standard deviations (error bars) of three samples per group (**, P < 0.01; *, P < 0.05). All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Virus, MTT Assay

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: FN increases the efficiency of EV71 binding to host cells, but not intracellular viral replication. (A) The EV71 infectious replicon SDLY107RV was transcribed in vitro and transfected into WT or FN-KO RD cells for 24 h. EV71 VP1 expression was then determined by qRT-PCR or immunoblot analysis in cell lysates of WT or FN-KO RD cells. N.S., not significant. (B) Binding assay of EV71 virions (MOI = 20) with WT or FN-KO RD cells. Bound virus was measured by qRT-PCR, and the value of the control group was set as 1. (C) Binding assay of EV71-GFP virions (MOI = 20) with WT or FN-KO RD cells. The ratio of GFP-positive cells was measured by flow cytometry. (D) RD cells were preincubated with anti-SCARB2 (5 μg/ml) and/or anti-FN (5 μg/ml) antibody, or IgG (5 μg/ml) for 6 h, followed by EV71 binding, and then bound virus was measured by qRT-PCR, and the value of the control group was set as 1. (E) Binding assay of EV71 virions (MOI = 20) with WT or FN-KO RD cells pretreated with BSA, FN protein (at the indicated concentrations), RGES, or RGDS (5 μg/ml) for 9 h. Bound virus was measured by qRT-PCR, and the value of the control group was set as 1. (F, G) Effects of adding oligopeptide RGES (control, 5 μg/ml, −6 h) or RGDS (treatment group, 5 μg/ml) at various times during the EV71 replication cycle. RD cells were infected with EV71, and the RGDS was then added at −9, −6, −3, 0, 3, and 6 hpi. Infection of RD cells was quantified by qRT-PCR at 9 hpi (G). Viral titers in the supernatant were measured at 12 hpi (H). The data are representative of the means and standard deviations (error bars) for three samples per group (**, P < 0.01; *, P < 0.05). All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Binding Assay, In Vitro, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Virus, Control, Flow Cytometry, Infection

Journal: Journal of Virology

Article Title: Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry

doi: 10.1128/JVI.02251-17

Figure Lengend Snippet: FN interacts with EV71 VP1. (A) RD and RD-FN-KO cell lysates were subjected to Western blotting (left) with anti-FN antibodies (left) and VOPBA with anti-EV71 VP1 antibodies (right). The arrows indicate bands observed on the membrane. (B) FN protein (1.5 μg), EV71 virions (5 × 106 viral genome copies), and BSA (20 μg) were incubated at 4°C overnight with constant agitation and immunoprecipitated with anti-FN antibody/isotype IgG and protein G-agarose; after being washed five times with washing buffer, the samples were subjected to immunoblotting with anti-FN and anti-EV71 VP1 antibodies. (C) The lysates of 293T cells transfected with Flag-VP1 or Flag-VP3 along with the FN plasmid were harvested at 24 h posttransfection and immunoprecipitated with anti-FN antibodies prior to immunoblotting with anti-Flag antibodies. (D) The lysates of EV71-infected RD cells (MOI = 2) were harvested at 12 hpi and immunoprecipitated with anti-FN antibodies prior to immunoblotting with anti-VP1. (E) Schematic map of the domains and truncated mutations of FN and VP1. (F) The lysates of 293T cells transfected with empty vector, Flag-VP1, and truncated VP1 mutations along with FN were harvested at 24 h and immunoprecipitated with anti-Flag antibodies prior to immunoblotting with anti-FN antibodies. (G) The lysates of 293T cells transfected with empty vector, FN, and truncated FN mutations along with VP1 were harvested at 24 h and immunoprecipitated with anti-HA antibodies prior to immunoblotting with anti-Flag antibodies. All experiments were repeated at least three times with consistent results.

Article Snippet: Rabbit anti-EV71 polyclonal antibodies against VP1 (catalog number PAB7631-D01P; Abnova) were used to detect viruses in all experiments.

Techniques: Western Blot, Membrane, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Infection

Journal: Nature Communications

Article Title: A second open reading frame in human enterovirus determines viral replication in intestinal epithelial cells

doi: 10.1038/s41467-019-12040-9

Figure Lengend Snippet: A second ORF of EV-A71 is efficiently translated in challenged cells. a Schematic diagram of the EV-A71 structure. The EV-A71 genome contains a long ORF flanked by a 5′UTR and 3′UTR. The ORF encodes a 250-kDa polyprotein that is processed into the P1, P2 and P3 regions, which are further cleaved into mature proteins (VP1 to VP4, 2 A to 2 C, and 3 A to 3D). Red box, the second ORF, which begins at the 3’ border of the IRES in the 5’UTR, encodes a 64-76-amino acid polypeptide in diverse sub-genotypes of EV-A71. b Amino acid and nucleic acid sequences of ORF2p from EV-A71 strain AH08/06. c HT-29 cells and isolated primary IECs were infected with EV-A71 or EV-A71ΔORF2p at an MOI of 0.1. Cells were harvested and prepared for immunoblotting at 48 hpi and 72 hpi using antibodies against EV-A71 ORF2p, VP1 and α-tubulin. Source data are provided as a Source Data file

Article Snippet: An anti-Enterovirus 71 VP1 antibody (dilution 1:1000; GTX132338) and anti-Enterovirus D68 VP1 antibody (dilution 1:1000; GTX132313) were purchased from GeneTex (San Antonio, USA).

Techniques: Isolation, Infection, Western Blot

Journal: Nature Communications

Article Title: A second open reading frame in human enterovirus determines viral replication in intestinal epithelial cells

doi: 10.1038/s41467-019-12040-9

Figure Lengend Snippet: ORF2p facilitates the release of enterovirus particles from isolated IECs and HT-29 cells. a – c Isolated primary IECs were transfected with 1 µg in vitro -synthesized RNA transcripts of EV-A71 or EV-A71ΔORF2p ( n =3 biologically independent experiments). Error bars denote SEM. Viral RNA in the culture supernatant a and cell lysates b was detected by an RT-PCR assay at the indicated times. c Virus titres of the supernatant or frozen-thawed lysates were determined at 24, 36, 48 h post-transfection. d , e Isolated primary IECs were challenged with EV-A71 or EV-A71ΔORF2p at an MOI of 10. d Virus titres of EV-A71 or EV-A71ΔORF2p in the supernatant or cell lysates were determined at the indicated times ( n =3 biologically independent experiments). Error bars denote SEM. e The supernatant and cells were harvested at indicated time, followed by immunoblot analysis, as described above. f HT-29 cells stably expressing EV-A71 ORF2p or control HT-29-CDH cells were challenged with EV-A71ΔORF2p at an MOI of 0.1. The supernatants and cells were harvested for immunoblotting using antibodies against EV-A71 VP1 and α-tubulin. CDH, control HT-29-CDH cells. g Detection of the EV-D68-induced cytopathic effect (CPE). HT-29-ORF2p or control HT-29-CDH cells were infected with equal amounts of EV-D68, and CPE was imaged via light microscopy. Scale bars equal 50 μm. h , i HT-29-ORF2p or control HT-29-CDH cells were infected with equal amounts of EV-D68. h The supernatant was harvested at 24 and 48 hpi for RT-PCR assays ( n =3 biologically independent experiments). Error bars denote SEM. i Cells were harvested at 8, 24, and 48 hpi, followed by immunoblot analysis. Source data are provided as a Source Data file

Article Snippet: An anti-Enterovirus 71 VP1 antibody (dilution 1:1000; GTX132338) and anti-Enterovirus D68 VP1 antibody (dilution 1:1000; GTX132313) were purchased from GeneTex (San Antonio, USA).

Techniques: Isolation, Transfection, In Vitro, Synthesized, Reverse Transcription Polymerase Chain Reaction, Virus, Western Blot, Stable Transfection, Expressing, Control, Infection, Light Microscopy

Journal: PLOS ONE

Article Title: USP26 suppresses type I interferon signaling by targeting TRAF3 for deubiquitination

doi: 10.1371/journal.pone.0307776

Figure Lengend Snippet: Primer pairs for real-time PCR.

Article Snippet: Antibodies for USP26 (13126-1-AP), anti-GAPDH and anti-hemagglutinin (HA)-HRP (561–7) were obtained from the Protein TECH Group (Chicago, USA) and MBL (Japan) respectively; EV71/VP1 (169442), K63 linkage-specific ubiquitin (179434) and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG secondary antibodies (97023)were from Abcam (UK); antibodies ubiquitin (SC-8017) was from Santa Cruz Biotechnology (USA); anti-Flag (M2)-horseradish peroxidase (A8592) was from Sigma (USA); anti-rabbit-IgG (sc-52336) was from Santa Cruz (USA).

Techniques: Sequencing

Journal: Virologica Sinica

Article Title: The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10

doi: 10.1007/s12250-020-00198-2

Figure Lengend Snippet: Construction of an infectious clone for prototype CVA10. A The cartoon shows the structure of the CVA10 infectious clone which is led by a T7 promotor. P1, P2 and P3 represent VP4-VP2-VP3-VP1, 2A-2B-2C and 3A-3B-3C-3D polyproteins, respectively. B The linearized PL451-EVA71 and PL451-CVA10 were transcribed by T7 polymerase. The purified mRNAs for EVA71 and CVA10 were analyzed by electrophoresis on agarose gel. Lane M, ssRNA ladder marker. C RD cells were transfected with 0.5 μg CVA10 mRNA per well. The cytopathic effect (CPE) was detected 24 h after transfection. D The electron microscopic characterizations of the CVA10 particles. Scale bar, 100 nm. E The rescued CVA10 from initial mRNA transfection was passaged 3 times and harvested as the stock. The viral titer of the CVA10 stock was determined by plaque assay. F RD cells were infected by EVA71 and CVA10 at an MOI of 1. At 6 h post-infection, cells were fixed and stained with anti-dsRNA J2 antibody, followed by Alexa Flour 594 conjugated secondary antibody. DAPI was used to visualize the nuclei. (Red, dsRNA; Blue, nuclei).

Article Snippet: Mouse monoclonal antibody against EV71 capsid protein VP1 was from Abcam (Cambridge, UK).

Techniques: Purification, Electrophoresis, Agarose Gel Electrophoresis, Marker, Transfection, Plaque Assay, Infection, Staining

Journal: Virologica Sinica

Article Title: The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10

doi: 10.1007/s12250-020-00198-2

Figure Lengend Snippet: Characterization of the rescued CVA10. A The growth curves of the two CVA10 strains on different cell lines. The pictures above showed the CPE and the charts below depicted the viral growth pattern by quantitative RNA levels. B, C RD SCARB2 WT and KO cells were infected with EVA71 or CVA10 at an MOI of 1. The infectivity was monitored by VP1 (EVA71) or 3A (CVA10) staining at 6 h post-infection (B). CPE was detected at 24 h post-infection (C). *P < 0.05; **P < 0.01.

Article Snippet: Mouse monoclonal antibody against EV71 capsid protein VP1 was from Abcam (Cambridge, UK).

Techniques: Infection, Staining

Journal: American Journal of Translational Research

Article Title: Brequinar inhibits enterovirus replication by targeting biosynthesis pathway of pyrimidines

doi:

Figure Lengend Snippet: Brequinar potently inhibits EV71 infection without significant cytotoxicity. RD cells were infected with EV71 (MOI=0.5) for 1 h and were incubated with the indicated concentrations of Brequinar for an additional 24 h: (A) The viral RNA copy number was quantified by RT-qPCR, the β-actin was used as housekeeping gene; (D) The EV71 VP1 protein expression in cells was determined by western blot, the β-actin was determined as loading control; (E) The EV71 infected cells were visualized by indirect fluorescence microscopy, nuclei were labeled with DAPI (blue). (B) Cell viability after cultured with indicated concentrations of Brequinar was assessed by CCK8 assay at 48 h later (mean ± SD, n=6). (C) The IC50 of Brequinar against EV71 on RD cells was calculated with GraphPad. (F) The culture supernatant of each sample in (A) was collected and infected RD cells for 24 h. The viral RNA copy number indicating the virus yield in (A) was quantified by RT-qPCR (mean ± SD, n=3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Rabbit Enterovirus 71 VP1 antibody was purchased from GeneTex (GTX132338, Hsinchu, China). β-actin monoclonal antibody (mAb) was purchased from ProteinTech (66009-1-Ig, Wuhan, China).

Techniques: Infection, Incubation, Quantitative RT-PCR, Expressing, Western Blot, Fluorescence, Microscopy, Labeling, Cell Culture, CCK-8 Assay

Journal: American Journal of Translational Research

Article Title: Brequinar inhibits enterovirus replication by targeting biosynthesis pathway of pyrimidines

doi:

Figure Lengend Snippet: Brequinar inhibits EV71 replication post virus entry. (A) Schematic representation of the different treatment strategy. The RD cells in 12-well plates were incubated with serially diluted Brequinar and EV71 (MOI=0.5) at pre-treatment (B), attachment (C), entry (D), replication (E), and post-infection (F) stages, the EV71 viral RNA copy number was quantified by RT-qPCR (mean ± SD, n=3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Rabbit Enterovirus 71 VP1 antibody was purchased from GeneTex (GTX132338, Hsinchu, China). β-actin monoclonal antibody (mAb) was purchased from ProteinTech (66009-1-Ig, Wuhan, China).

Techniques: Incubation, Infection, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: Brequinar inhibits enterovirus replication by targeting biosynthesis pathway of pyrimidines

doi:

Figure Lengend Snippet: Supplement of exogenous uridine reversed the anti-EV71 effect of Brequinar. A. EV71 VP1 protein level in RD cells was measured by western blot after 24 h of treatment with 1 μM Brequinar by adding serially diluted exogenous uridine, β-actin was set as the loading control. B. EV71 RNA in RD cells was quantified by RT-qPCR after 24 h of treatment with 1 μM Brequinar by adding serially diluted exogenous uridine, data were normalized to the untreated control (set as 100%). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit Enterovirus 71 VP1 antibody was purchased from GeneTex (GTX132338, Hsinchu, China). β-actin monoclonal antibody (mAb) was purchased from ProteinTech (66009-1-Ig, Wuhan, China).

Techniques: Western Blot, Quantitative RT-PCR