Leibovitz medium

The human cerebral micro-vascular endothelial cells (hCMEC/D3 - shortly D3) were grown on rat tail collagen-coated dishes in EBM-2 medium (Lonza) supplemented with EGM-2 Bullet Kit (Lonza) and 2.5% Fetal Bovine Serum (FBS) from Sigma [19] (link), [20] (link). MDCK (Madin-Darby canine kidney) cells were maintained in DMEM/F-12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) (Lonza) supplemented with 5% FBS.
Cells were cultured at 37 °C, in 5% CO₂ atmosphere, seeded at 1.5*10⁴ cell/cm² in Falcon Petri dish (lid) with 3.5 cm diameter. MDCK monolayers were fed with fresh medium first after 24 h (post-seeding) than every second day until they reached confluence (3rd day).
All measurements were performed in serum free Leibovitz medium (Sigma) at 37 °C within 3 h after taking the cells out from the incubator. The temperature was kept constant using a home made heating stage at accuracy of 0.1 degree. According to our observations and to literature, in Leibovitz medium cells preserve their viability within this time period [21] (link).

For each DENV-2 lineage, 5 BALB/c mice were infected and euthanized at 72 hpi. Five noninfected mice were used as a negative control. Viral RNA was extracted from liver samples of infected and noninfected mice. Organ samples were macerated with 500 µl of Leibovitz medium (Sigma). RNA was extracted from 140 µl of supernatant of macerated liver samples by using a QIAmp Viral RNA mini kit (Qiagen) as described by the manufacturer’s protocol. Viral RNA quantitation was carried out using the primers and probes DEN2-R (5-ACCATAGGAACGACACATTTCC-3) and DEN2-F (5-CAACGCATTGTCATTGAAGGA-3) and (FAM-5-AGGGCCTTGATTTTCATCTTACTGACAGC-3-TAMRA). TaqMan Fast Virus One-step Master Mix (Applied Biosystems) was used for the amplification reaction. Five microliters of extracted RNA and a mix containing 12.5 µl of reaction mix, 1 µl of DEN2-F and DEN2-R primers (Sigma), 0.75 µl of DEN2-P probe, 3.65 µl of nuclease-free water (Gibco), 1 µl of MgSO₄ and 0.5 µl of SuperScript III Platinum One-Step Quantitative RT-PCR (Invitrogen) were applied to a 96-well microplate. The assay was performed using a 7500 FAST platform (Applied Biosystems). Thermal cycling parameters were as follows: reverse transcription at 50 °C for 15 min (min), 1 cycle of enzyme activation at 95 °C for 2 min, 1 cycle of denaturation at 95 °C for 15 s, 40 cycles of annealing/elongation at 60 °C for 1 min (Table 3).

Virus propagation and titration were done in cultured secondary duck embryonic fibroblast (DEF) cells which were derived from SPF Khaki-Campbell ducks. Cells were cultured in medium composed of equal parts Leibovitz medium (L4386–10 L, SIGMA, St. Louis, MO, USA) and McCoy’s 5A medium (M4892–10 L, SIGMA), supplemented with 10% premium grade heat-inactivated FBS (Atlanta Biologicals, Flowery Branch, GA, USA) except after viral inoculation for titration, when 2% FBS was used.