Dulbecco s modified eagle medium

FMDV reference strains were propagated in fetal porcine kidney cell line (LFBK‐α_Vβ₆) cultures, as described in a previous study (LaRocco et al., 2013 (link)). Viral isolation from clinical samples of FMD outbreaks was attempted using the cell line. Tissue homogenates (20%) were prepared in Dulbecco's modified eagle medium (Corning, NY, USA) and inoculated into LFBK‐α_Vβ₆ cell lines prepared one day before inoculation. The cells and culture media were harvested and stored at −80°C until use when a 90% cytopathic effect (CPE) was observed.
Seneca valley virus (SVV) and swine vesicular disease virus (SVDV) were also included in the evaluation to differentiate FMDV from other viruses causing FMD‐like clinical signs. SVV and SVDV strains were grown in pig kidney cell (IBRS)‐2 cultures as previously described in the World Organization for Animal Health (OIE) manual (OIE, 2019 ).
Cells were cultured in T‐75 cell culture flasks (Corning∖), using Dulbecco's‐modified eagle medium (Corning) for LFBK‐α_Vβ₆ cells and minimum essential medium (Corning) for IBRS‐2 cells, both supplemented with 2% antibiotic‐antimycotic (Gibco, NYC, USA) and 5% fetal bovine serum (Corning). Each cell line was counted using a TC20 automated cell counter (Bio‐Rad, CA, USA), and the concentration normalized to a seeding density of 1.3 × 10⁷ cells/mL was incubated at 37°C and 5% CO₂ incubator.

Human hepatocellular carcinoma cell lines HepG2 and HuH7 were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). The HBV stable expression cell lines HepG2.2.15 38 and HepAD38 39 were maintained in Dulbecco's modified Eagle medium (CellGro, Herndon, VA) supplemented with 10% fetal bovine serum (Gibco) and 400 μg/mL G418 (Merck, Kenilworth, NJ). HepAD38 cells were also supplemented with 2 μg/mL doxycycline (Merck) to block HBV replication, and doxycycline was removed for at least 1 week to produce HBV. Primary human hepatocytes (PHHs) were kindly provided by Professor Hongkui Deng from the Stem Cell Research Center and the Department of Cell Biology, Peking University. PHH cells were maintained in primary hepatocyte maintenance medium (PMM). HepG2 and HuH7 cells were seeded in a 12‐well plate at 4 × 10⁵ and 2 × 10⁵ cells/well, and in a 35 mm dish at 8 × 10⁵ and 4 × 10⁵ cells/well, respectively. Following the manufacturer's protocol, the cells were transfected with plasmids using lipofectamine 2000 (Life Technologies, Carlsbad, CA).

Parasite cultures were maintained in primary human foreskin fibroblasts (HFF) as previously described (47 (link)). All of the genetically modified strains generated here (for the full list of parent and transgenic strains, see Data Set S3) were grown in confluent monolayers of HFF cells in Dulbecco’s modified Eagle medium with 4.5 g/liter glucose, l-glutamine, and sodium pyruvate (Corning Cellgro); 5% fetal bovine serum (Sigma); and 1% penicillin-streptomycin-amphotericin B (HyClone) at 37°C with 5% CO₂.