C6 36

Human embryonic kidney HEK293T/17 (ATCC No. CRL-11268) and human hepatocellular HepG2 (ATCC No. HB-8065) cells were cultured in complete medium, composed of Dulbecco’s modified Eagle medium (DMEM; Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C with 5% CO₂. Dengue virus serotype 2 (DENV 2) strain 16681, DENV 1 (strain 16007), DENV 3 (strain 16562), DENV 4 (strain 1036), ZIKV (strain SV0010/15) and JEV (strain BJ1) were all propagated in the Aedes albopictus cell line C6/36 (ATCC No. CRL-1660). Viral progenies in the supernatant were collected and centrifuged at 1,000g to remove cell debris. The virus stocks were kept at − 80 °C until used. Virus titers were determined by standard plaque assay on LLC-MK2 cells (ATCC No. CCL-7).

Cell lines used for virus isolation were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). As BTV and EHDV were assumed to be the main agents of hemorrhagic disease in Florida’s farmed deer, virus isolation was attempted in two cell lines that are usually susceptible to and permissive for the viruses: C6/36 (Aedes albopictus (mosquito), ATCC CRL1660) and Vero E6 (Cercopithecus aethiops (African green monkey) kidney, ATCC CRL 1586). Both cell lines were grown as monolayers in a humidified atmosphere containing 5% CO₂, the C6/36 cells at 28 °C and Vero E6 cells at 37 °C as described by Ahasan et al. [12 (link)].

(i) Cell lines. Human lung carcinoma A549 cells and baby hamster kidney (BHK-21) cells were obtained from ATCC (Manassas, VA, USA) and grown in minimum essential medium (MEM; Thermo Fisher Scientific [TFS], Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine (TFS), and 0.075% sodium-bicarbonate (TFS). The mosquito cell line C6/36 (isolated from Aedes albopictus) was obtained from ATCC and cultured in Leibovitz’s L-15 medium (TFS) supplemented with 10% FBS, 0.01 M HEPES (TFS), and penicillin/streptomycin (TFS). Cell cultures were maintained at 37°C in a humidified environment with 5% CO₂, except for C6/36 cells, which were cultured at 28°C in the absence of CO₂. The cells were passaged every 3 to 4 days.
(ii) Viruses. ZIKV prototype strain MR766 (isolated from sentinel Rhesus monkey, Uganda, 1947) was obtained from ATCC. ZIKV was propagated in C6/36 cell cultures from which supernatant containing the virus was harvested 5 to 9 days after infection and stored at −80°C. Viral titers were determined using plaque assays in BHK-21 cells, as described below. All viruses were obtained and used as approved according to the rules of a Belgian institutional review board (Departement Leefmilieu, Natuur en Energie, protocol SBB 219 2011/0011n) and the Biosafety Committee at the Katholieke Universiteit Leuven.

C6/36 mosquito cell cultures (ATCC CRL-1660) were maintained in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (FBS), 1% (1×) non-essential amino acids (NEAA), 10 mM HEPES buffer, 100 units/mL penicillin, and 100 µg/mL streptomycin (Penicillin-Steptomycin) at 28 °C without CO₂. Vero E6 cell cultures (Vero C1008; ATCC CRL-1586) were maintained in MEM Rega-3 medium supplemented with 10% FBS, 2 mM L-glutamine, and 0.075% sodium bicarbonate. For cell culture assays that involved virus or virus infected material, the concentration of FBS in the medium was reduced to 2% (2% medium). All tissue culture media and supplements were obtained from Gibco, Thermo Fisher Scientific (Merelbeke, Belgium).
ZIKV (SL1602, Suriname isolate) was obtained from Prof. Martijn van Hemert, Leiden University Medical Center, Leiden, The Netherlands. Lyophilized virus was reconstituted in 2% MEM Rega-3 medium and virus stocks were generated on C6/36 mosquito cell cultures as described before [20 (link)]. The aforementioned cell types tested negative for mycoplasma.
7-deaza-2′-C-methyl-d-adenosine (7DMA) was purchased from Carbosynth (Berkshire, UK).

LLC/MK2 (ATCC^®, Manassas, VA, USA CCL-7), HEK-293 (ATCC^® CRL-1573), HepG2 (ATCC^® HB-8065), THP-1 (ATCC^® TIB-202), and C6/36 (ATCC^® CRL-1660) cell lines were propagated and maintained as previously described ([9 (link),11 (link),37 (link)]. Huh-7 cells were maintained in DMEM (Gibco^®, Langley, OK, USA) supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. Reference DENV2 strains New Guinea C strain (NGC) and 16,681 were propagated in C6/36 cells, as described [9 (link),11 (link)].

African Green Monkey kidney cells [Vero; American Type Culture Collection (ATCC) #CCL-81] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM l-glutamine, sodium bicarbonate (1.5 g/liter), penicillin (100 U/ml), and streptomycin (100 μg/ml) and incubated at 37°C in 5% CO₂. Aedes albopictus mosquito cells (C6/36; ATCC #CRL-1660) were maintained in DMEM supplemented with 10% FBS (HyClone, Logan, UT), 2 mM l-glutamine, sodium bicarbonate (1.5 g/liter), penicillin (100 U/ml), and streptomycin (100 μg/ml) and incubated at 28°C in 5% CO₂. HEK293 cells (ATCC #CRL-1573) were maintained in DMEM supplemented with DMEM supplemented with 10% FBS, 2 mM l-glutamine, sodium bicarbonate (1.5 g/liter), penicillin (100 U/ml), and streptomycin (100 μg/ml) and incubated at 37°C in 5% CO₂. The cell lines were obtained from ATCC, were not further authenticated, and were tested and confirmed negative for mycoplasma.