Tryptose phosphate broth

The Madin Darby canine kidney (MDCK), human embryonic kidney (HEK) 293T, chicken DF-1 and adenocarcinomic human alveolar basal epithelial cells (A549) (obtained from the Central Services Unit at TPI) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma), supplemented with 10% fetal bovine serum (FBS) (Life Science Production) and 1× penicillin-streptomycin (Gibco). Primary chicken kidney (CK) cells were prepared as previously described (97 (link)) and were maintained in Eagle’s minimum essential medium (EMEM) (Sigma) containing 7% bovine serum albumin (BSA) (Sigma), 1× penicillin-streptomycin (Gibco), and tryptose phosphate broth (Sigma). All cell lines and primary cells were maintained at 37°C and 5% CO₂.

Ixodes ricinus-derived tick cell lines IRE11 [17 (link)], IRE/CTVM19 and IRE/CTVM20 [4 (link)], and I. scapularis-derived tick cell lines ISE6 [18 (link)] and ISE18 [19 (link)], were maintained in L15 medium supplemented with 20% bovine fetal serum, antibiotic/antimycotic mix (all Biosera, Nuaille, France) and 10% tryptose phosphate broth (Sigma-Aldrich) in 10 ml flat sided cell culture tubes (Nunc, Rochester, USA) in a cell incubator at 28 °C as described in [20 (link)]. Confluent tick cell cultures were harvested by pipetting into sterile 2 ml microtubes and spun at 600× g for 5 min at room temperature. The supernatant was removed and cells were resuspended in phosphate-buffered saline, pH 7.4 (PBS) and spun at 600× g for 5 min. The PBS washing step was repeated two more times. Upon supernatant removal, the cell pellets were stored at − 80 °C until further use.

Two Aedes spp. cell lines were used in this study, Aag2 and C6/36 cells. The Ae. aegypti cell line, Aag2 (ATCC, VA), was used for in vitro studies. Aag2 cells were grown at 28 °C with 5% CO2 in Schneider’s Drosophila Medium for transfection studies and DMEM high glucose media for the remaining studies. Both types of media were supplemented with 10% heat-inactivated fetal bovine serum (Gemini, CA), 1% penicillin-streptomycin, and 1% tryptose phosphate broth (Sigma, MO). Lipid-depleted serum was made by incubating serum with fumed silica overnight followed by removal of silica-lipid complexes by centrifugation^{26 (link)}. Lipid-depleted serum was added to cell culture components to make lipid-depleted media. In addition, the Ae. albopictus cell line, C6/36 (obtained from Erol Fikrig), was used to grow DENV stocks using the same media. The dengue strain used was DENV-2 New Guinea C. C6/36 cells were infected at an MOI of 1.0 virus stock was stored at −80 °C until use.

Cells of the C6/36 (ATCC CRL-1660) and U4.4 cell lines (A. albopictus) (26 (link)) and the Aag2 cell line (A. aegypti) (48 (link)) (kindly provided by G. P. Pijlman, Wageningen University, the Netherlands) were maintained at 28°C in L-15 Leibovitz medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% nonessential amino acids (Gibco), 2% tryptose phosphate broth (Sigma), and 1% penicillin-streptomycin (Gibco).

The wild-type PC22A strain of PEDV was used for gilt infection and piglet challenge at a dose of 1 × 10⁵ plaque forming units diluted in Minimal Essential Media [MEM (Life Technologies, Carlsbad, CA, USA)]. Briefly, PC22A was isolated and cultured in Vero cells as described previously [32 (link), 33 (link)]. Cells were grown in growth medium containing Dulbecco’s Modified Eagle’s Medium [DMEM (Life Technologies, Carlsbad, CA, USA)] supplemented with 5% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 1% antibiotic–antimycotic (Life Technologies, Carlsbad, CA, USA). Virus was grown in Vero cells in maintenance medium containing DMEM supplemented with 10 μg/mL trypsin (Life Technologies, Carlsbad, CA), 0.3% tryptose phosphate broth (Sigma Aldrich, St. Louis, MO, USA), and 1% antibiotic–antimycotic. Cells were kept in a humidified incubator at 37 °C and 5% CO₂. PC22A was passaged three times in Vero cells before passaging once for generation of inoculum in a gnotobiotic pig. The virulence of pig passaged PC22A was confirmed in adult and neonatal pigs as described previously [13 (link), 34 (link), 35 (link)]. Cell-culture adapted PC22A was used as a positive control in the virus neutralization (VN) Ab assay.