Figure 3. Neutralizing mAbs block MAYV infection at postattachment steps. (A and B) Attachment inhibition assay. MAYV-CH was incubated with soluble heparin (0.5 to 2 mg/ml; A), BSA (0.5–2 mg/ml; A), or anti-MAYV mAbs (10 µg/ml; B) for 1 h before addition to Vero cells at 4°C. After unbound virus was removed by extensive rinsing, cell-adsorbed viral RNA was quantified by qRT-PCR, standardized to GAPDH levels, and plotted relative to an untreated (A) or isotype mAb-treated (B) control. Data are the mean and SD of three experiments performed in triplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype control mAb). (C) Pre/postattachment neutralization assays. Serial dilutions of anti-MAYV mAbs were incubated with 102 FFU of MAYV and added to Vero cells. Infection proceeded for 18 h before foci were stained, counted, and plotted relative to a no-antibody control. Data are the mean and SD of two experiments performed in triplicate. (D) Postattachment neutralization assay. 102 FFU of MAYV was adsorbed to Vero cells at 4°C. Unbound virus was removed by extensive washing, and serial dilutions of anti-MAYV mAbs were added. Infection proceeded for 18 h at 37°C. Viral foci were stained, counted, and plotted relative to a no-mAb control. (E and F) FFWO assay. Cells were incubated with virus at 4°C. After removing unbound virus by rinsing, cells were treated with 1 µg/ml of the indicated mAbs and then pulsed for 2 min with medium at pH 7.6 (E) or pH 5.5 (F) at 37°C. After pH neutralization, cells were cultured in medium supplemented with 20 mM NH4Cl to prevent viral fusion via canonical endosomal pathways. Fusion inhibition was measured by flow cytometry by staining cells for MAYV E2 antigen 18 h later. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). (G and H) Egress inhibition assay. RNA isolated from MAYV-infected cells was transfected into Vero cells. Medium was added with the indicated concentrations of anti-MAYV mAbs. RNase A–resistant encapsidated viral RNA in the supernatant was quantified by qRT-PCR at 1 h (G) or 6 h (H) after transfection. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 3.

Neutralizing mAbs block MAYV infection at postattachment steps. (A and B) Attachment inhibition assay. MAYV-CH was incubated with soluble heparin (0.5 to 2 mg/ml; A), BSA (0.5–2 mg/ml; A), or anti-MAYV mAbs (10 µg/ml; B) for 1 h before addition to Vero cells at 4°C. After unbound virus was removed by extensive rinsing, cell-adsorbed viral RNA was quantified by qRT-PCR, standardized to GAPDH levels, and plotted relative to an untreated (A) or isotype mAb-treated (B) control. Data are the mean and SD of three experiments performed in triplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype control mAb). (C) Pre/postattachment neutralization assays. Serial dilutions of anti-MAYV mAbs were incubated with 102 FFU of MAYV and added to Vero cells. Infection proceeded for 18 h before foci were stained, counted, and plotted relative to a no-antibody control. Data are the mean and SD of two experiments performed in triplicate. (D) Postattachment neutralization assay. 102 FFU of MAYV was adsorbed to Vero cells at 4°C. Unbound virus was removed by extensive washing, and serial dilutions of anti-MAYV mAbs were added. Infection proceeded for 18 h at 37°C. Viral foci were stained, counted, and plotted relative to a no-mAb control. (E and F) FFWO assay. Cells were incubated with virus at 4°C. After removing unbound virus by rinsing, cells were treated with 1 µg/ml of the indicated mAbs and then pulsed for 2 min with medium at pH 7.6 (E) or pH 5.5 (F) at 37°C. After pH neutralization, cells were cultured in medium supplemented with 20 mM NH4Cl to prevent viral fusion via canonical endosomal pathways. Fusion inhibition was measured by flow cytometry by staining cells for MAYV E2 antigen 18 h later. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). (G and H) Egress inhibition assay. RNA isolated from MAYV-infected cells was transfected into Vero cells. Medium was added with the indicated concentrations of anti-MAYV mAbs. RNase A–resistant encapsidated viral RNA in the supernatant was quantified by qRT-PCR at 1 h (G) or 6 h (H) after transfection. Data are the mean and SD of three experiments performed in duplicate (one-way ANOVA with Dunnett’s post-test compared with the isotype mAb control). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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