Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

Delvecchio, Rodrigo; Higa, Luiza M.; Pezzuto, Paula; Valadão, Ana Luiza; Garcez, Patrícia P.; Monteiro, Fábio L.; Loiola, Erick C.; Dias, André A.; Silva, Fábio J. M.; Aliota, Matthew T.; Caine, Elizabeth A.; Osorio, Jorge E.; Bellio, Maria; O’Connor, David H.; Rehen, Stevens; de Aguiar, Renato Santana; Savarino, Andrea; Campanati, Loraine; Tanuri, Amilcar

Vero, hBMEC, and NSC were seeded on black 96-well plates with clear bottoms and infected with ZIKV MR 766 or ZIKV BR strain at an MOI of 2 for 1 h. Neurospheres were seeded on coverslips and infected with ZIKV MR766 with 2.5 × 10 5 PFU After infection, the viral inoculum was removed and cells were incubated with medium containing chloroquine (1.56 to 50 µM) for 4 to 5 days, depending on cell type. Cells and neurospheres were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fixative was removed and the samples were washed three times with PBS. Blocking of unspecific binding of the antibody and permeabilization were performed with PBS supplemented with 3% bovine serum albumin (BSA, Sigma Aldrich) and 0.1% Triton X-100 for 40 min at room temperature. Incubation with anti-Map2 antibody was performed following the manufacturer's instructions (ABCAM, Cambridge, Cambridgeshire, UK, #32454; 1:1000) and the 4G2 antibody was diluted 1:4 in PBS with 3% BSA and incubated with cells for 1 h. After washing three times with PBS, cells were incubated with secondary antibodies coupled to Alexa fluorochromes (Thermo Fisher Scientific) for 40 min and washed five times. Coverslips with neurospheres were mounted with Prolong Gold mounting medium (Thermo Fisher Scientific). Samples were imaged using either Leica SP5 or Leica SPE (Leica Biosystems, Wetzlar, Hesse, DE) confocal microscopes and a Nikon TE300 (Tokyo, Japan) inverted microscope coupled to a Leica DFC310FX camera (Leica Biosystems, Wetzlar, Germany).