Mammarenaviral Infection Is Dependent on Directional Exposure to and Release from Polarized Intestinal Epithelia

Warner, Nikole L.; Jokinen, Jenny D.; Beier, Juliane I.; Sokoloski, Kevin J.; Lukashevich, Igor S.

VeroE6 (C1008) cells and Caco-2 (HTB-37) cells were purchased from American Type Culture Collection (ATCC) and grown in minimal essential media using Dulbecco's modified eagle medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Fisher Scientific, Hampton, NH, USA) and 1% antibiotic-antimycotic (Life Technologies, Carlsbad, CA, USA) in a humidified chamber at 37 • C under 5% CO 2 . Cells were infected with LCMV-Armstrong (strain 53b), LCMV-WE (strain 54), Mopeia virus (MOPV, strain AN20410), or the Mopeia/Lassa reassortant virus, clone ML-29 [33, 34] . All viral stocks were generated using low multiplicity of infection (MOI) and stocks with titers ranging from 1 × 10 7 PFU/mL to 1 × 10 8 PFU/mL were stored at −80 • C until needed [17] . Viral titers were determined using a standard plaque assay with minor modifications [35] . Briefly, VeroE6 cells were seeded in the wells of a 12-well cell culture plate, and incubated until 80-90% confluent. Virus samples were serially diluted, and used to infect the Vero cells. Infection was carried out for 1 h in 37 • C. After this period, the cells were washed with DMEM without phenol red, and a semi-solid overlay containing 1X MEM, 5% FBS, and 0.5% Avicel (FMC BioPolymer, Philadelphia, PA, USA) (LCMV and ML-29) or 0.5% Agarose (MOPV). Cells were then incubated in a 37 • C humidified chamber with 5% CO 2 for 5 days. The plaque assay cells infected with LCMV and ML-29 had the overlay media removed, were fixed with 4% paraformaldehyde solution for 15 min and cells were stained with 1% Crystal Violet solution to identify virus-infected cell foci. For titration of MOPV, virus-infected cells were covered with semisolid overlay of 0.5% agarose/5% FBS overlay. A 0.04% neutral red, 0.5% agarose, 5%FBS solution was added to wells after 4 days incubation. Both plaque assays have a limit of detection of approximately 80 PFU/mL.