2.1. Cells and Virus Stocks

The amplification of the virus stock, the production of infected cells, and the monitoring of infectivity of potentially inactivated samples was performed in VERO 76 cells (ATCC^® CRL-1587™, American Type Culture Collection, Manassas, USA). The cells were maintained in medium containing Dulbecco’s minimal essential medium (DMEM) supplemented with 3% fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM glutamine, 0.5 mM pyruvate, and 1× non-essential amino acids (all from Pan Biotech, Aidenbach, Germany) at 37 °C and 5% CO₂.

MORV strain 3017/2004 had been isolated and sequenced in our laboratory [14] and the used stock was passaged ≤3 times. For the amplification of the stock, cells were infected with a multiplicity of infection (MOI) of 0.01, the virus-containing supernatant was harvested three days post infection and filtered through a 0.1 µm sterile filter unit (Millipore, Burlington, MA, USA). It was further concentrated to 5 × 10⁶ foci forming units (FFU) per mL via ultrafiltration (see below). The viral titer was determined by immunofocus assay (IFA) as described elsewhere [15,16,17], and the stock was stored at −80 °C until use. This concentrated stock was employed for the validation of the efficacy of the different inactivation methods. For the validation of the efficacy of cellular inactivation methods, cells were infected with a MOI of 0.01 and harvested three days post infection. The supernatant was removed and the cells were trypsinized until detachment, which was monitored visually. The infection rate of the cells was determined by indirect immunofluorescence staining [18] and cells were only used if an infection rate above 50% was reached. In IFA as well as in indirect immunofluorescence staining, MORV was detected with the Old-World arenavirus NP-specific monoclonal antibody 2LD9 [19].