Leibovitz 15 medium

Dissected liver tissues were incubated for 30 min at 37°C in dissociation medium composed of Leibovitz-15 medium (Sigma-Aldrich), 45% glucose, 1 g/L DNAse I (Applichem) and collagenase A (Roche, Basel, Switzerland). Digested liver fragments were passed through a nylon cell strainer, and cells free in suspension were collected in 50 ml wash medium/liver and stored at 4°C. Cells were centrifuged at 30 × g for 15 min at 4°C to pellet parenchymal cells, i.e., hepatocytes. Parenchymal cells were washed 3 times with FACS buffer and analyzed by flow cytometry.
Non-parenchymal cells (NPCs) remaining in suspension were collected and centrifuged at 300 × g for 10 min at 4°C. Pelleted cells were resuspended in ice-cold-HBSS, mixed with freshly prepared 30% Histodenz (Sigma-Aldrich), and centrifuged at 1,500 × g for 20 min at 4°C. The NPCs at the Histodenz interface were collected, washed, and suspended in FACS buffer for analysis. The NPCs were phenotyped using dye-conjugated monoclonal antibodies specific for CD45 (clone 30-F11), F4/80 (clone BM8), and CD31 (clone 390) (BD Biosciences, Franklin Lake, NJ, United States). Stained samples were acquired on a multichannel cytometer BD LSR II equipped with FACS Diva software (BD Biosciences; Franklin Lake, NJ, United States) and analyzed using FlowJo 7.6.5 (Becton, Dickinson Company; Ashland, OR, United States).

Dengue virus, DENV-1 (Hawaii strain, GenBank: KM204119), DENV-2 (New Guinea C strain, GenBank: AF038403), DENV-3 (H87 strain, GenBank: M93130) and DENV-4 (H241 strain, Genbank: AY947539) are used as reference strains in this study and produced under BSL2 safety conditions. Briefly, 8×10⁶Aedes albopictus C6/36 cells were seeded in a 75 cm² flask and grown overnight at 28℃, and infected with virus at an MOI of 0.1 and cultured for 5–7 days at 28 °C in Leibovitz 15 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% L glutamine (Gibco), 10% tryptose-phosphate (Gibco) and 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco). At 5–7 days post infection, the virus culture supernatants were harvested and concentrated using 40% polyethylene glycol (PEG) 8000 solution (Sigma Aldrich) as described before^{39 (link)}. The virus concentrate was resuspended in FBS and stored at − 80 °C.

VeroE6 cells were used for the detection of neutralizing antibodies via the foci reduction neutralization test (FRNT). These cells were cultivated in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, Steinheim, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 100 U/ml penicillin-streptomycin (Gibco) at 37 °C in a 5% CO₂ atmosphere. CHIKV isolates were grown in C6/36 Ae. albopictus cells and harvested from the supernatant. The mosquito cells were cultured in Leibovitz-15 medium (Sigma-Aldrich) supplemented with 10% FBS, 1% l-glutamine (Gibco), 10% tryptose-phosphate (Gibco), and 100 U/ml penicillin-streptomycin at 28 °C.