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 cells (ATCC, Manassas, VA, USA) are derived from the kidney of African green monkey and were grown in DMEM High Glucose (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS) (GIBCO). Human brain microvascular endothelial cells (hBMEC) were a kind gift from Dr. Julio Scharfstein (Federal University of Rio de Janeiro, Rio de Janeiro, Brazil), and hBMEC isolation was performed as previously described [25] . These cells were cultured in DMEM High Glucose supplemented with 20% FBS. The C6/36 cell line is derived from Aedes albopictus. C6/36 cells (ATCC, Manassas, VA, USA) were grown in Leibovitz L-15 medium (GIBCO) supplemented with 2.95 g/L tryptose phosphate broth (Sigma Aldrich, Boston, MA, USA), 2 mM glutamine (GIBCO), 0.075% sodium bicarbonate (GIBCO), 1X non-essential amino acids (GIBCO) and 5% FBS. Neural stem cells (NSCs) were derived from human induced pluripotent stem cells (iPSCs). iPSCs were provided by the Biobank of iPSCs of the Brazilian Ministry of Health (CONEP B-027 # 25000.111598/2014-04). According to the supplier, fibroblasts were reprogrammed using the protocol developed by Paulsen et al. [26] , and transduced with the CytoTune-iPS Sendai kit (Thermo Fisher Scientific, Waltham, MA, USA). iPSCs presented a normal karyotype and the expression of pluripotency markers. These cells were cultured with E8 culture media (GIBCO) on a Matrigel (BD Biosciences, San Jose, CA, USA) coated surface. iPSC colonies were manually passaged every 5-7 days until they reached 70%-80% confluence and were maintained at 37 • C in humidified air with 5% CO 2 . To produce NSCs, human iPSCs were exposed to the serum-free neural induction medium (GIBCO), containing Neurobasal medium (GIBCO) and the pluripotent stem cell neural induction supplement (GIBCO), according to the manufacturer's protocol [27] . Briefly, the medium was changed every other day until day 7, when initial NSCs are split and grown on neural expansion medium (Advanced DMEM/F12 and neurobasal medium (1:1) with neural induction supplement; GIBCO). NSCs were used after four passages in neural expansion media. Mouse central nervous system (CNS) cells were harvested from Swiss mouse embryos at embryo day 14 (E14) and grown for 72 h as free floating neurospheres in neurobasal culture media (GIBCO) supplemented with 1X B27 (GIBCO).