25 cm2 tissue culture flask

To obtain primary human monocyte-derived MΦs, CD14⁺ monocytes were seeded and cultured at a concentration of 1 × 10⁶ cells per mL in 25 cm² tissue culture flasks (Corning Inc., Corning, NY, USA) with RPMI 1640 media containing l-glutamine (Lonza, Morristown, NJ, USA), supplemented with sodium pyruvate (1 mM; Lonza, Morristown, NJ, USA), 1X non-essential amino acids (NEAAs; Gibco, ThermoFisher Scientific, Waltham, MA, USA), 10% (v/v) fetal bovine serum (FBS; ATCC, Manassas, VA, USA), and 50 ng/mL recombinant human M-CSF (PeproTech Inc., Cranbury, NJ, USA) for 9 days at 37 °C, 5% CO₂. Media and cytokines were replenished every 3 days. Following differentiation, cell surface marker characterization of primary human monocyte-derived MΦs was performed using flow cytometry (Figure S2).

UC-MSC were harvested and resuspended in a final concentration of 1 × 10⁶ cells/mL. Cells were transferred to 1000 μL precooled medium, containing DMEM and 10% dimethyl sulfoxide (DMSO) supplemented with either 30% of hPL (n = 22) or 30% FBS (n = 22). For each group, cells were then cryopreserved by either using a CryoMed controlled-rate freezer under a freezing rate of 1°C per minute or placing cryovials in precooled isopropanol racks (Mr. Frosty, Nalgene®) and transferring them to −80°C freezer for 24 h prior to final liquid nitrogen vapor storage. Cell samples were stored for one month until thawing. For viability and recovery assessment, UC-MSC were thawed and washed. Viable and dead cells were counted with a Neubauer chamber after staining with trypan blue (0.4%, Life Technologies). Cells were further seeded in 25cm² tissue culture flasks (Corning, USA) in a density of 4.6 × 10³ cells/cm² and seeded until confluency in order to obtain PDL, CPD, and PDT postthawing values. For long-term expansions, four MSC donors were kept in culture for up to 23 successive passages and PDL, CPD, and cumulative cell numbers were determined.

Dami, HEL and Meg-01 cells were purchased from ATCC (ATCC number: CRL-9792, TIB-180 and CRL-2021, respectively) and grown in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% fetal calf serum (FCS) (Zhejiang Tianhang Biological Technology Co., Ltd., Hangzhou, Zhejiang, China) for Dami cells or fetal bovine serum (FBS) (Hyclone, Logan, UT) for HEL and Meg-01 cells. CMK cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany) and cultured in RPMI 1640 medium with 10% FCS. The cultures were maintained in a humidified atmosphere of 5% CO₂ at 37°C. To determine the growth curve, the cells were seeded at 2×10⁵/ml in 10 ml of complete media and cultured in 25-cm² tissue culture flasks (Costar, Corning Incorporated, Corning, NY). Cell counts were performed using the trypan blue dye exclusion method.

The entire brain from CyHV-2 latently infected fish was collected and washed twice in Medium 199 containing streptomycin (100 µg/mL) and penicillin (100 U/mL). The washed brain was minced with scissors into small pieces and digested into a single cell suspension with 0.25% trypsin-versene solution (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 15 min. The digested cells were then placed in M199 medium containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and filtered with a 70-µm steel mesh. The filtered cell suspensions were centrifuged at 200× g for 5 min and resuspended in M199 medium supplemented with 20% FBS containing streptomycin and penicillin antibiotics and cultured in 25 cm² tissue culture flasks (Corning, NY, USA) at 28 °C with 5% CO₂. Approximately 50% of the medium was replaced with fresh cell culture medium every 3 days. The confluent monolayer was split at a ratio of 1:2 every 6–8 days. After 15 subcultures, the cells were cultured in M199 medium with 10% FBS.

A. albopictus cells (A.T.C.C.: CRL-1660) were maintained at 28°C in Leibovitz's L-15 medium (HyClone), supplemented with 10% heat inactivated FBS (Gibco) and 100 unit/ml of penicillin/streptomycin (PAA Laboratories GmbH). The cell was maintained in 25 cm² tissue culture flasks (Corning) and subcultured by detaching cells with 0.25% trypsin/EDTA solution. For the treatment experiments, 1 ml of 1:5 cell dilution was seeded on to 15 mm round cover slips (Menzel-Glaszer) in 12-well plates (Nunc) or 8-well culture slide cover glass (LabTek II). The cells were allowed to adhere on to cover slips at 28°C for 48 h with approximated cell density of 80%.

Cells were seeded onto six-well tissue culture plates (30 x10⁶ cells/well; Corning, NY, USA) pre-coated with type I rat tail collagen (BD Biosciences, Bedford, MA, USA) and incubated at 37°C with 5% CO₂ in a humidity-saturated environment. After 24 h of culture, no adherent cells or debris were aspirated and complete EGM-2 medium was added to each well. Medium was changed daily until colonies of ECs (ECCs) appeared. ECCs were counted by visual inspection using an inverted microscope (Olympus CKX41, Tokyo, Japan) under 40x magnification. ECCs were released with TrypLE Express (Invitrogen) from tissue culture plates, suspended in 2 ml of complete EGM-2 media, and plated onto 25-cm² tissue culture flasks (Corning) coated with type 1 rat tail collagen for further passage.

The HMEC-1 cells were grown overnight in a 25 cm² tissue culture flasks (Corning, New York, USA). Then, cells were mock-infected or infected with DENV-2 at 5 MOIs for 48 h. Further, the samples were fixed with 2.5% of glutaraldehyde in 0.1 M Sodium cacodylate buffer (pH 7.2) for 1 h at room temperature (RT), and post-fixed with 1% osmium tetroxide for 1 h at RT. The samples were dehydrated through an ethanol gradient and propylene oxide, and then were embedded in Polybed epoxy resins and polymerized at 60°C for 24 h. Finally, 70 nm thin sections were stained with uranyl acetate and citrate and then the preparations were analyzed by using a Jeol JEM-1011 transmission electron microscope (JeolLtd., Tokyo, Japan).