Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 1. Host range of EV70-Rmk14 and EV70-Dne in cultured cells. One-step growth analysis of viruses was performed in human (HeLa), monkey (LLC-MK2), and murine (L, L-hDAF) cell lines with EV70-Rmk14 (top) or EV70-Dne (bottom) virus at an MOI of 5. Infections were halted at different times postinfection, and virus titers were determined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Cell Culture, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 2. Host range in cell lines derived from human eye and brain. One-step growth analysis of viruses was performed using EV70-Rmk14 (top) or EV70-Dne (bottom) virus to infect (MOI 5) the following eye and brain derived cell lines: HeLa, T98, LLCMK2, U373MG, 15C4, SY5Y, or HCE. Infections were halted at different times postin- fection, and virus titers were determined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Derivative Assay, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 3. Effect on viral replication of enzyme or antibody treatment of cultured cells. HeLa cells were incubated with one of the following as indicated along the x axis: phosphate-buffered saline (PBS [mock treated]), neuraminidase, PI-PLC, or antibodies specific for hapten or the SCR1 or SCR2 domains of the human DAF molecule. Cells were washed and infected with EV70-Rmk14 or EV70-Dne at an MOI of 3. At 24 h postinfection, the total RNA was isolated from infected cells and transferred onto a nitrocellulose membrane for slot blot analysis. Positive-strand viral replication was assessed by hybridization with a radiolabeled negative-strand EV70 RNA probe. The amount of hy- bridized probe was determined with a PhosphorImager and Image- Quant software and reported as the optical density. The data were normalized to the mock (PBS)-treated sample (PBS treatment 100% replication).

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Cell Culture, Incubation, Saline, Infection, Isolation, Membrane, Dot Blot, Hybridization, Software

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 4. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in replication in HeLa cells. One-step growth anal- ysis was performed with mutants of EV70-Dne harboring single amino acid substitutions to the EV70-Rmk14 sequence. HeLa cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were determined by plaque assay. (A) Growth analysis of viruses DDDDD (EV70-Dne encoding amino acids K14, M238, L133, P178, and D226), DRRRR (K14K, I238, F133, R178, N226), RDRRR (E14, M238, F133, R178, and N226), RRDRR (E14, I238, L133, R178, and N226), RRRDR (R14, I238, F133, P178, and N226), and RRRRR (EV70-Rmk, E14, I283, F133, R178, and N226). (B) Growth analysis of viruses DDDDD (see above), RDDDD (E14, M238, L133, P178, and D226), DRDDD (K14, I238, L133, P178, and D226), DDRDD (K14, M238, F133, P178, and D226), DDDRD (K14, M238, L133, R178, and D226), and DDDDR (K14, M238, L133, P178, and N226).

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Sequencing, Infection, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 5. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in hDAF usage. One-step growth analysis was per- formed using mutants of EV70-Dne virus (described in the legend to Fig. 4). L-hDAF cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were deter- mined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Virus, Infection, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 6. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in cell killing. HeLa cells were infected at an MOI of 3, using EV70-Dne (DDDDD), EV70-Rmk14 (RRRRR), or mu- tant viruses described in the legend to Fig. 4. Infections were halted at different times postinfection, and cells were pelleted by low-speed centrifugation, resuspended in PBS with trypan blue dye, and exam- ined by light microscopy. The percent viability was determined by dividing the number of cells that excluded dye by the number of cells examined.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Infection, Centrifugation, Light Microscopy

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 7. Predicted location in the viral capsid of EV70 amino acids that influence host range and cell killing. The known crystallographic structure of BEV-1 was used to predict the locations in the viral capsid of amino acid residues that differ between EV70-Rmk14 and EV70-Dne. Capsid protomer proteins are color coded, with VP1 blue, VP2 yellow, and VP3 red. VP4 is omitted for clarity. Amino acid changes are enumerated in white. (A) Exterior view of a pentamer, comprising five copies each of VP1, VP2, VP3, and VP4 (not shown), revealing the locations of four of the five strain-specific amino acid changes: in the VP1 DE loop, amino acid 133 of EV70 VP1 (F1133L, BEV 1128 in figure), and three in the canyon, including 238 of EV70 VP3 (I3238M, BEV 3240), 178 of EV70 VP1 (R1178P, BEV 1151), and 226 of EV70 VP1 (N1226D, BEV 1219). Residues on only one protomer are labeled. (B) Closer exterior view of canyon and fivefold axis of symmetry. Amino acid changes in all five protomers are labeled.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Labeling

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: Profile of Vero cell growth and EV71 amplification at different MOIs.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Amplification

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: (A) Purification of EV71 antigens from different MOI cultures by sucrose density gradient ultracentrifugation (SDG) and identification of the viral particle-enriched fractions using SDS-PAGE analysis with silver staining. (a)-(d). EV71 cultures at different MOIs as indicated above. The lines labeled with FP and EP represent the distributed fractions of the infectious full particles and defective empty particles, respectively. The molecular weights of VP0, VP1, VP2, and VP3 are indicated. The molecular weight marker is shown on the right. (B) The ELISA yield, total protein, and specific activity of EV71 vaccines purified from different MOI cultures. After SDG purification, the EV71 particle-enriched fractions (fractions 6–13) were subsequently pooled and inactivated for productivity and quality evaluation. (C) Viral neutralization titers against the B4(E59) and C4(E36) subgenotypes elicited by EV71 vaccines from different MOI preparations. Significant differences between vaccine groups are indicated with the following symbols: *, p <0.05; **, p <0.01; and ***, p <0.001.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Purification, SDS Page, Silver Staining, Labeling, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: (A) Pooled viral particles from the vaccines prepared from (a) MOI 10 −1 , (b) MOI 10 −2 , (c) MOI 10 −4 , and (d) MOI 10 −6 cultures were observed by TEM. The morphologies of the FPs and EPs are indicated by arrows. The total numbers of viral particles in the different MOI samples were statistically counted, and the average sums of the FPs (B) and EPs (C) per field were plotted. (D) Percent normalization of FP and EP content to the total particles from each EV71 vaccine prepared from cultures with different MOIs. The total particle number for each EV71 vaccine produced using different MOIs was set as 100%. Significant differences between groups are indicated with the following symbols: *, p <0.05; **, p <0.01; and ***, p <0.001. ns: no significant difference was present between the groups.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Produced

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: Quality confirmation of purified FPs and EPs based on TEM observations (A) and SDS-PAGE analysis with silver staining (B). The molecular weights of VP0, VP1, VP2, and VP3 are indicated. (C) Measurement of neutralizing efficacy against the B4(E59) or C4(E36) subgenotype EV71 provided by anti-FP and anti-EP. The neutralizing titers raised from FPs were set as 100% normalized neutralization. The percent reduction in neutralization conferred by anti-EP was calculated. Evaluation of antigenic competition between EV71 vaccines composed of different FP and EP ratios. The neutralizing titer raised from 1 μg of FP was set as 100% normalized neutralization. The B4(E59) (D) or C4(E36) (E) neutralization conferred by antisera from manually prepared vaccines was calculated. Significant differences between groups are indicated by the following symbols: *, p <0.05; **, p <0.01; and ***, p <0.001.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Purification, SDS Page, Silver Staining, Neutralization

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: (A) The binding specificities of anti-FP and anti-EP to the target virus B4(E59) were examined by adding virus antigens from B4(E59) or C4(E36) as competitors in a competition ELISA. The binding efficiencies of anti-FP (B) and anti-EP (C) to FP or EP from the B4(E59) virus were evaluated by competitive ELISA assay. The antisera, competitors, and binding targets are indicated. Inhibition of viral neutralization by anti-FP or anti-EP against the B4(E59) (D) or C4(E36) (E) virus was examined by FP- or EP-adsorption experiments. BSA was used as the non-EV71 competitor/antigen, as a negative control in the competitive ELISAs and viral neutralization inhibition assays. The substantial neutralizing titers (≥1:128) conferred by BSA-pre-adsorbed anti-FP or anti-EP against the viruses were set as 100% normalized neutralization. The increase in the neutralization inhibition percentage was measured by pretreating anti-FP and anti-EP with FP or EP adsorption. Significant differences between groups are indicated by the following symbols: *, p <0.05; **, p <0.01; and ***, p <0.001. ns: no significant difference was present between the groups.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA, Inhibition, Neutralization, Adsorption, Negative Control

Journal: PLoS ONE

Article Title: The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus

doi: 10.1371/journal.pone.0210553

Figure Lengend Snippet: (A) The purity of maltose-binding protein (MBP) fusions with the VP0, VP1, VP2, and VP3 EV71 capsid proteins was confirmed by SDS-PAGE analysis with Coomassie blue staining. (B) The neutralization activities of protein-preabsorbed antisera or antibodies against the B4(E59) or C4(E36) virus were measured. The tested antisera are labeled. MAB979 is a monoclonal VP2-specific antibody that neutralizes the B4(E59) virus and was used as a positive control for MBP-VP2 adsorption. The anti-VP1/C4 monoclonal antibody confers neutralization activity against C4(E36) and was used as a positive control for MBP-VP1 adsorption. (C) Measurement of EV71 subgenotype neutralization inhibition by preabsorbed anti-FP. The neutralizing titer provided by anti-FP pre-adsorbed with MBP against individual viruses was set as 100% normalized neutralization. The increase in the percentage of neutralization inhibition conferred by anti-FP pre-adsorbed with MBP-VP1 or MBP-VP2 was calculated and normalized to the MBP-adsorption groups. The EV71 virus strains used for this experiment are labeled. Significant differences between groups are indicated by the following symbols: *, p <0.05; **, p <0.01; and ***, p <0.001. ns: no significant difference was present between the groups.

Article Snippet: The commercial Enterovirus 71 VP1 neutralizing antibody against EV71 genotype C4 (VP1/C4; 40013-H136, Sino biological Inc.) and VP2-specific MAB979/Anti-Enterovirus 71 antibody (MAB979; Merck Millipore) were treated with the same process to deplete the anti-VP1 and anti-VP2 antibodies.

Techniques: Binding Assay, SDS Page, Staining, Neutralization, Labeling, Positive Control, Adsorption, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Validation of the easyscreen flavivirus dengue alphavirus detection kit based on 3base amplification technology and its application to the 2016/17 Vanuatu dengue outbreak

doi: 10.1371/journal.pone.0227550

Figure Lengend Snippet: Materials used in sensitivity/specificity studies.

Article Snippet: DENV-4 , Vircell , MBC058 , enterovirus 71 , Vircell , MBC019.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition

doi: 10.3389/fmicb.2020.01105

Figure Lengend Snippet: Remdesivir potently inhibits EV71 infection without significant cytotoxicity. (A) HeLa cells were infected with EV71 (MOI = 1) and treated with varying concentrations of remdesivir at 1 hpi. The viral RNA copy number was quantified by qRT-PCR at 24 hpi (mean ± SD, n = 3). (B) Cell viability after culture with different doses of remdesivir at 24 hpi assessed using a CCK8 assay (mean ± SD, n = 6). (C) Western blot of EV71 VP2 protein expression in HeLa cells. HeLa cells were infected with EV71 (MOI = 0.5). After adsorption, the cells were treated with the indicated concentrations of remdesivir and collected at 24 hpi. Then, the proteins were resolved by SDS-PAGE and western blotting. (D) Fluorescence microscopy examination of EV71-infected HeLa cells treated with different amounts of remdesivir (24 hpi). The dsRNA-specific antibody J2 (red) was used for staining viral dsRNA replication intermediates, and nuclei were labeled with Hoechst 33258 (blue). Bar = 100 μm. (E) Cell culture supernatants collected from (A) were used to infect HeLa cells, and after 1 h of adsorption at 37°C, fresh medium was added. At 24 hpi, cells were collected and subjected to viral RNA copy number quantification by qRT-PCR (mean ± SD, n = 3). NS, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Rabbit Enterovirus 71 VP2 antibody was purchased from GeneTex (GTX132340, Hsinchu, China).

Techniques: Infection, Quantitative RT-PCR, CCK-8 Assay, Western Blot, Expressing, Adsorption, SDS Page, Fluorescence, Microscopy, Staining, Labeling, Cell Culture