Ccl 149

The cell culture was performed as described previously^{48 (link)}. Rat lung epithelial derived L2 cells were purchased from ATCC (CCL-149). Cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 75 cm² flask, and under a 5% CO₂ and 95% air mixture at 37 ^oC in a humid chamber. When growth density was reached 80%, cells were washed twice with PBS, and detached with 0.25% (w/v) trypsin-0.53 mM EDTA solution for 5 to 15 min. The aliquot of cells was added to a new flask or wells for next experiments.

Murine Hepatitis Virus-1 was obtained from the American Type Culture Collection (ATCC VR-261) and expanded through passage in L2 cells (ATCC CCL-149) followed by lysis of cells by freeze thaw after 24 hours of infection. Mice were infected with either 2500 PFU or 5000 PFU of MHV-1 diluted in sterile phosphate-buffered saline (PBS) in 30µL volume via intratracheal (i.t.) instillation as previously described (19 (link)). Mice were monitored daily for weight and disease symptoms and were euthanized if weight loss exceeded 30% of day 2 post-infection (PI) weight or if a combination of weight loss and clinical symptoms met humane euthanasia criteria.