Bhk 21 cells

The hTSCs used in this study included the hTSCs originally established by Wang lab^{25 (link)}, the hTSC strain BT1 gifted by Arima lab^{23 (link)}, and hESC-derived trophoblast-like cells (H1-TS) gifted by Pan lab⁶⁹ . The hTSCs, BT1 and H1-TS were cultured following the previously published protocol^{25 (link)}. In brief, the hTSCs were cultured in 6-well plates pre-coated with 5 μg/ml collagen-IV at 37 °C in 5% CO₂. The hTSCs culture medium (hTSM) was prepared as follows: DMEM/F12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.2% fetal bovine serum (FBS), 0.5% Penicillin-Streptomycin, 0.3% BSA, 1% ITS-X supplement, 0.5 μM A83-01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mM VPA. The hTSCs were dissociated with TrypLE Express Enzyme (Gibco) and passaged every 4-5 days at a 1:6 split ratio.
BHK-21 cells and C6/36 cells were purchased from American Type Culture Col lection (ATCC). BHK-21 cells (ATCC, #CCL-10) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin.
C6/36 cells (ATCC, #CRL-1660) were cultured in RPMI-1640 with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin.

CHIKV 181/25 was obtained from Dr. Robert B. Tesh (University of Texas Medical Branch). The Mayaro Guyane virus (NR-49911) was obtained through BEI Resources, NIAID, NIH, as part of the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) program. CHIKV 181/25 and MAYV Guyane viruses were propagated and titered on BHK-21 cells (ATCC). CHIKV LR2006_OPY1 and AF15561 (United States Army Medical Research Institute of Infectious Diseases) were used as previously described [28 (link), 29 (link)].

The ZIKV^PR strain, PRVABC59, was obtained from BEI resources and propagated in C6/36 mosquito cells in serum-free neuro-glial differentiation media (62 (link)). This protocol was adapted from standard techniques (63 (link)) for infections in neural models and produces high titer virus. Virus-containing cell supernatants were harvested after the appearance of cytopathic effect in the C6/36 cells and clarified by centrifugation. Zika-conditioned media was collected from uninfected C6-36 cells grown in parallel. Viral titer was determined by focus-forming assay to calculate BHK-21 focus-forming units: BHK-21 cells (ATCC) were infected with dilutions of viral stock and overlaid with 3.2% carboxymethylcellulose solution mixed 1:1 with Dulbeccos modified Eagle medium (DMEM) containing 2% Fetal Bovine Serum (FBS) (63 (link)). At 4 dpi, cells were fixed and stained with 5 ug/mL anti-ZIKV NS1 protein monoclonal antibody #110 (64 (link)), followed by secondary staining with Horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (Promega) and detection using the Vector VIP HRP substrate kit (Vector Laboratories).

TBE and YF NTs were performed as described previously.
¹² (link),
³⁰ (link) Briefly, serially diluted serum samples were mixed with TBEV strain Neudoerfl
³³ (link),
³⁴ (link) or YFV strain 17D.
³⁵ (link) BHK‐21 cells (ATCC) were added and incubated for 3 (TBEV) or 4 days (YFV). YF NT titers were expressed as the reciprocal of the serum dilution that prevented the development of a virus‐induced cytopathic effect (CPE). NT titers ≥ 20 were considered positive. Since TBEV strain Neudoerfl does not produce a CPE of BHK cells, inhibition of virus replication was determined by measuring the presence of the virus in the cell culture supernatants by ELISA.
³⁶ (link),
³⁷ (link) The NT titer was defined as the reciprocal of the serum dilution that gave a 90% reduction in the absorbance readout in the assay compared with the control without antibodies. NT titers ≥ 10 were considered positive.

C6/36 cells (ATCC: CRL-1660) were cultured in Leibovitz’s L-15 medium (Gibco, 41300039) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, 10270) and 10% Tryptose Phosphate Broth (TPB) solution (Sigma, T9157). The C6/36 cells were grown at 25°C without CO₂. BHK-21 cells (ATCC: CCL-10) were used for virus titration. BHK-21 cells were cultured in Dulbecco’s modified Eagles Medium (DMEM, Gibco, 11995065) supplemented with 10% FBS (Gibco, 10270) at 37°C and 5% CO₂. In this study, DENV serotype 1 (DENV-1) of Hawaiian strain, and ZIKV of strain H/PF/2013 were used. Both viruses were propagated in C6/36 cells. Virus titer of DENV and ZIKV was determined using tissue culture infectious dose 50 (TCID50) assay. DENV-1 (Hawaiian strain) was a gift from David Perera, University Malaysia Sarawak, and ZIKV (H/PF/2013 strain) was a gift from Shee Mei Lok, Duke-NUS Medical School, Singapore.

Mammalian cells: A549 cells (human lung adenocarcinoma epithelial cell line, ATCC, CCL-185) and Huh7 cells (human hepatoma cell line, JCRB Cell Bank #0403) were maintained in Dulbecco’s Modified Eagle’s Medium/ F-12 (Ham’s), HEK 293 cells (human embryonic kidney cell lines, ATCC CRL-1573) were maintained in Dulbecco’s Modified Eagle’s Medium, HeLa cells (human epithelioid cervix carcinoma cell line, ATCC, CCL2) ‎ were maintained in RPMI-1640 Medium, BHK-21 cells (baby hamster kidney cell line, ATCC, CCL-10) were maintained in Minimum Essential Medium Alpha. All the media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Mosquito cells: C6/36HT (Aedes albopictus cells ATCC, CRL-1660 were adapted to grow at 33°C) were maintained in Leibovitz's L-15 Medium, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.3% tryptose phosphate, 0.02% glutamine, and 1% MEM non-essential amino acids solution.

Lentivirus particles were produced in HEK293T cells (ATCC CRL-3216) maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin. HEK293T cells were co-transfected with the desired pXLG3 vector and second-generation lentivirus packaging plasmids. The HEK293T media containing lentiviral particles was harvested 48 h after transfection. Cells were incubated with lentivirus for 5 days prior to experiments. FLAG-MEF2A was cloned into pSinREP5 for production of Sindbis particles in BHK-21 cells (ATCC CCL-10), which were maintained in Minimum Essential Medium Alpha supplemented with 5% foetal bovine serum and 1% penicillin/streptomycin. Briefly, the pSinREP5 vector and a defective helper vector were linearised and purified then transcribed in vitro using mMessage mMachine SP6 transcription kit (Thermo Fisher). RNA was introduced into BHK-21 cells by electroporation using a MicroPulser electroporator. The BHK-21 media containing Sindbis viral particles was harvested 36 h after electroporation. Cells were incubated with Sindbis virus for 24 h prior to experiments.