Rift Valley fever virus (RVFV) is an arthropod‐borne, zoonotic disease endemic to sub‐Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative‐sense, single‐stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non‐endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID50) assay Basic Protocol 4: Quantification of Rift Valley fever virus by focus‐forming assay Basic Protocol 5: Storage and disinfection Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC‐5 cells Alternate Protocol 2: Propagation of RVFV in mosquito‐derived cells Alternate Protocol 3: TCID50 detection using fluorescence visualization Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection Support Protocol 2: Calculation of the virus titer by plaque assay or focus‐forming assay Support Protocol 3: Calculation of the TCID50 titer by the method of Reed and Muench Support Protocol 4: Calculation of the antibody volume for the focus‐forming assay

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