C6 36

Vero (WHO stock), C6/36 (ATCC # CRL-1660) and JEG-3 (ATCC # HTB-36) cells were maintained in Minimum Essential Medium Eagle (MEM) (Gibco-BRL, Gaithersburg, MD) supplemented with fetal bovine serum (FBS) (Hyclone, Logan, UT; respectively at 5, 5 and 10%), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (Gibco BRL) at 37 °C (Vero and JEG-3 cells) or at 30 °C (C6/36) in a humidified atmosphere containing 5% CO₂.
Virus isolation was performed by infecting Vero, C6/36 and JEG-3 cells in 6-well plates at 80% confluence with 20 µL/cm² of plasma diluted 1:2 in MEM containing 2% FBS for 1 h with gentle rocking every 15 min, followed by addition of fresh medium and incubation under the conditions described above. The cells were observed daily for cytopathic effect (CPE) as an indicator of infectivity. Supernatants were harvested when extensive CPE was observed, or at Day 6 post infection, if no CPE was observed. Two additional passages (P2 and P3) of all samples were performed in 6-well plates using 20 µL/cm² of supernatant from the first culture (P1). ZIKV-RNA titers in the supernatants from passage 3 (P3) were determined by TaqMan reverse-transcriptase PCR (RT-PCR), and infectious particle titers were determined by Focus Forming Assay (FFA).

The human lung cell line A549 (ATCC Cat No. CCL-185), the human microglial cell line CHME-5 [49 (link)] the human hepatoma cell line Hep3B (ATCC Cat No. HB-8064) and the human embryonic kidney cell line HEK293T/17 (ATCC Cat No. CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units of penicillin and 100 mg streptomycin/mL at 37 °C with 5% CO₂. The monkey kidney cell line Vero (ATCC Cat. No. CCL-81) was cultured under the same conditions but supplemented with 5% FBS. The baby hamster kidney cell line BHK-21 (ATCC Cat No. CCL-10) was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) at 37 °C with 5% CO₂. The Ae. albopictus cell line C6/36 (ATCC CRL-1660) was cultured at 28 °C in MEM (Gibco, Invitrogen) supplemented with 10% FBS and 100 units of penicillin and 100 mg streptomycin/mL. Cell lines were selected based upon our previous studies [50 (link),51 (link),52 (link)].
DENV 2 (strain 16681) and ZIKV (strain SV0010/15) virus stocks were propagated in C6/36 cells previously described [53 (link)], and virus titers were determined by standard plaque assay as described previously [52 (link),54 (link)] using Vero cells for ZIKV and LLC-MK₂ cells for DENV.

The Aedes albopictus cell line C6/36 (ATCC CRL-1660) was used for amplification of all virus stocks and testing of mosquito saliva samples. C6/36 cells were maintained at 28 °C under atmospheric CO₂ in Leibovitz’s L-15 medium (Gibco Thermo Fisher Scientific) with 10% fetal bovine serum (FBS), 2% tryptose phosphate broth (Gibco Thermo Fisher Scientific), 1× nonessential amino acids (Gibco Thermo Fisher Scientific), 10 U/ml of penicillin (Gibco Thermo Fisher Scientific) and 10 μg/ml of streptomycin (Gibco Thermo Fisher Scientific)^{57 (link),93 (link)}. The Cercopithecus aethiops cell line Vero (ATCC CCL-81) was used for titration of virus stocks by FFA. The C. aethiops cell line Vero E6 (ATCC CRL-1586) was used for titration of virus stocks by plaque assay. Vero cells were maintained at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (Gibco Thermo Fisher Scientific) with 10% FBS, 10 U/ml of penicillin, and 10 μg/ml of streptomycin^{57 (link),93 (link)}.