Sf9 cells

pFastBac Dual His6 MBP N10 TEV LIC cloning vector (5 C) was purchased from Addgene (Addgene plasmid no. 30123; https://www.addgene.org/30123). All restriction enzymes, the pFastBac1 vector, and DH10Bac cells were purchased from Thermo Fisher Scientific. Sf9 cells were purchased from ATCC. Q5^® High-Fidelity DNA Polymerase, the Monarch^® DNA Gel Extraction Kit, and amylose resin were purchased from NEB (https://international.neb.com/). The DNA purification kit was purchased from Promega. The anti-6xHis antibodies were purchased from Abcam. All other chemicals and materials were purchased from Bio-Rad Laboratories, GE Healthcare, Sigma-Aldrich, or Roche Diagnostics, unless otherwise indicated.

Porcine embryo kidney (PS) cells were grown in RPMI 1640 (Gibco) supplemented with 5% heat-inactivated fetal calf serum (FCS) at 37°C in a CO₂ (5%) incubator. A549 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated FBS (Gibco). Unless otherwise stated, replicon transfected cells were maintained in media supplemented with 2% FCS and 1% Penicillin-Streptomycin (Invitrogen). Sf9 cells (ATCC) were cultured in BioWhittaker Insect-Xpress supplemented with 2% FCS, 1% Penicillin-Streptomycin, and 2.5 μg/mL amphotericin B. Cells were grown at 28°C as monolayers or in suspension with agitation at 100 rpm. Transfection of TBEV replicons were performed in Sf9 monolayer cultures. IRE/CTVM19 was maintained in ambient air at 28°C in L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth (TPB), 20% FCS, 2 mM l-glutamine, and Penicillin-Streptomycin (55 (link)).

Sf9 cells (ATCC) were cultured in BioWhittaker Insect-Xpress supplemented with 2% FCS, 100units/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B. Cells were grown at 28°C as monolayers or in suspension with agitation at 100 rpm. Baculoviruses were generally amplified in monolayer cultures but large scale infections for capsid isolation were done in suspension. Virus stocks were titred using plaque assay on Sf9 monolayers.

mAb binding to recombinant DuCV capsid protein expressed in baculovirus was examined by the seeding of Sf9 cells (ATCC CRL-1711) in a 96-well culture plate in DMEM/F12 (Hyclone) containing 10% FBS and incubating the cells at 28°C. Cells were infected at 80% confluence with the recombinant baculovirus containing the DuCV cap-ΔNLS gene at a multiplicity of infection (MOI) of 0.1. After 72 h of incubation at 28°C, cells were fixed with pre-chilled acetone/methanol (1:1) for 20 min at room temperature, followed by saponin permeabilization for 10 min. The plate was washed three times with PBST and incubated with a blocking buffer at 37°C for 30 min. Cell wells were washed, and the diluted murine ascitic mAb was added. After incubation at 37°C for 1 h, cell wells were washed, and DyLight 488 AffiniPure Goat Anti-Mouse IgG (H+L; Earthox, CA, USA) was added at a dilution of 1:1000. After incubation at 37°C for 1 h, cells were washed three times and mounted with DAPI. Fluorescence was recorded under a fluorescence microscope (Olympus Corporation, Japan).

HEK 293T cells (human embryonic kidney cells, female) were maintained in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1x MEM non-essential amino acids (Thermo Fisher Scientific), and 100 U mL⁻¹ of Penicillin-Streptomycin (Thermo Fisher Scientific). MDCK-SIAT1 cells (Madin-Darby canine hidney cells with stable expression of human 2,6-sialtransferase, female, Sigma-Aldrich) were maintained in DMEM medium supplemented with 10% FBS, 1x MEM non-essential amino acids, and 100 U mL⁻¹ of Penicillin-Streptomycin. Sf9 cells (Spodoptera frugiperda ovarian cells, female, ATCC) were maintained in Sf-900 II SFM medium (Thermo Fisher Scientific).