Streptomycin sulfate

HB cell line HUH6 was obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)-glutaMAX (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin sulfate (Gibco, Waltham, MA, United States). HB cell line HB-243 from patient-derived xenograft (PDX) was provided by XenTech (Evry, France) (Kats et al., 2019 (link)). HB-243 cells were cultured in Advanced DMEM/F12 (Gibco, Waltham, MA, United States) supplemented with 8% fetal bovine serum (FBS) (Gibco), 2 mM glutaMAX (Gibco), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin sulfate (Gibco) and 20 μM rock kinase inhibitor Y-27632 (S1049; SelleckChem, Houston, TX, United States). Absence of mycoplasma was regularly confirmed with PCR-based method (PromoCell, Heidelberg, Germany).

PK-15 and MDBK cell lines were stored in our laboratory and cultured in MEM or DMEM (Corning, Tewksbury, MA, USA) supplemented with 100 μg/ml penicillin, 100 IU/ml streptomycin sulfate (Gibco, Grand Island, NY, USA), and 10% fetal bovine serum (FBS) (Corning, USA) at 37°C and 5% CO₂. Insect cell lines Sf9 and High Five (Life Sciences) used for the expression of CSFV E2 and E^rns proteins were cultured at 27°C in Grace’s Insect medium and Sf-900™ II SFM (1×) (Gibco, USA) supplemented with 100 μg/ml penicillin, 100 IU/ml streptomycin sulfate (Gibco, USA), and 5% FBS. Highly virulent CSFV reference SM, HCLV strain, and 106 CSFV field isolates (sub-genotypes 1.1, 2.1, 2.2, and 2.3) and bovine viral diarrheic virus 1 (BVDV1) strain 32 were stored in our laboratory. These field isolates were collected between 1990 and 2017 from 23 provinces of China (30 (link)–32 (link)) and adapted in PK-15 cells following isolation (31 (link), 33 (link)). The growth and replication of CSFV in PK-15 cells were confirmed by indirect fluorescent antibody assay (IFA) using CSFV-specific mAb WH303 (34 (link)) as the primary antibody and Alexa Fluor 488 donkey anti-mouse IgG (H+L) (Life Technologies, MA, USA) as the secondary antibody (33 (link)). Full E2 and E^rns genes of various CSFV strains were synthesized at Jilin Comate Biotech Company (Changchun, Jilin, China).

Head kidney-derived macrophages were obtained as described previously^{9 (link)}. In short, total head kidney leukocytes were cultured for 6 days at 27 °C, at a density of 17.5 × 10⁶ cells/75 cm² flask in complete NMGFL-15 medium (incomplete -NMGFL15 supplemented with 5% pooled carp serum (PCS) and 10% bovine calf serum (Invitrogen Life Technologies) with 100 U/ml of penicillin G, 100 µg/ml of streptomycin sulfate (Gibco) and 50 µg/ml Gentamycin (Sigma-Aldrich) to obtain macrophages.
To polarize, macrophages were harvested by gentle scraping after incubation on ice for 15 min. Cells were pelleted at 450×g for 10 min at 4ºC before resuspension in cRPMI + (RPMI 1640 culture medium with 25 mM HEPES and 2 mM L-glutamine, supplemented with L-glutamine (2 mM), penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml, Gibco) and heat-inactivated PCS (1.5% v/v)). Depending on the assay, macrophages were polarized for 6 h or 24 h with 30 µg/ml LPS (Escherichia coli, L2880, Sigma-Aldrich) with or without 100 ng/ml recombinant Ifn-γ for M1 macrophages, or with 0.5 mg/ml dibutyryl cAMP (N⁶,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate sodium D0627, Sigma-Aldrich, referred to as cAMP) or 100 ng/ml recombinant Il-4/13b1 for M2 macrophages, or with an equal volume of medium as unstimulated controls. Cells were cultured at 27˚C in the presence of 5% CO₂.