Rift Valley Fever Virus Propagation

RVFV strain MP12 used for C6/36 cells, insects, and mice (Richard Elliott and Benjamin Brennan, Institute of Infection, Immunity and Inflammation, Centre for Virus Research, University of Glasgow, Glasgow, UK) was propagated using Vero-E6-cells (Collection of Cell Lines in Veterinary Medicine (CCLV), #CCLV-RIE 929, Friedrich-Loeffler-Institute (FLI), Riems, Germany) on a 96-well plate in Dulbecco’s Modified Eagle’s Medium (DMEM, #DMEM-HXA, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) / 5% fetal bovine serum (FBS; #FBS-HI-12A FBS, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at a temperature of 33 °C in a humidified atmosphere and a CO₂-content of 5%. Supernatant of infected cells was harvested after 3 days with cells showing a cytopathic effect (CPE), characterized by cell lysis in 80% of cells. Additionally, mice and sheep were infected with the virulent RVFV 35/74 strain provided by the virus stock of the FLI, Riems, Germany. The strain was propagated in a cell culture flask of BHK21 cells using Minimum Essential Medium (MEM) supplemented with penicillin–streptomycin and 2% FBS (FLI Bio-Bank, FLI, Riems, Germany) at a temperature of 37 °C and 5% CO₂. Supernatant was harvested, when cells showed 80% CPE. A TCID₅₀ assay, calculated as described by Spearman and Kaerber^{81 (link)}, was used to determine the virus titer.