Abstract
Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.