Virus stocks and shares of SBV isolate NL-F647 were obtained after infection of Vero E6 cells in a MOI of 0.01. Genome segment-specific quantitative RT-PCR Mammalian cells (Vero E6) or insect cells (C6/36 for RVFV and KC for SBV) were seeded in 6-very well cell culture plates at 2??105 cells/well or 6??105 cells/well, respectively, and permitted to attach for 2C4?h. to concurrently visualize bunyavirus progeny virions and their genomic articles at single-molecule quality in the framework of singly contaminated cells. Using Rift Valley fever pathogen and Schmallenberg pathogen 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. (family (family test with Welchs correction (not assuming equal variances). *values are shown for each genome segment. e Relationship between the intracellular content of vRNPs and the packaging efficiency of individual cells. Ruxolitinib Phosphate A generic system to score the intracellular balance and the packaging efficiency was created. A frequency of 0.33 for each genome segment was considered as theoretically balanced. The balance score was calculated as the summatory of the absolute deviations from the theoretical frequency, normalized from 0 to 1 1, assigning the least balanced composition of the data set a score of 0. The packaging efficiency score was calculated taking into account the frequency of empty, incomplete and complete virus particles, normalized from 0 to 1 1, assigning the most efficient packaging value of the data set a score of 1 1. Scores are color coded from light green (lowest) to dark green (highest). f Proposed model on the efficiency of genome packaging based on the intracellular vRNP content. A balanced vRNP content in the cytoplasm seems to be a pre-requisite for relatively efficient genome packaging. Intracellular vRNP content correlates with genome packaging efficiency Seeking for an explanation to the high variability in genome packaging efficiency within cell populations, we looked into the vRNP content in the cytoplasm of the individual cells. Quantification of RVFV vRNPs in infected mammalian cells not only exposed a highly heterogenous cell-to-cell composition, but also an overall imbalanced content leaning towards higher abundances of the S (42%) and L (34%) segments compared to the M segment (24%). Quantification of SBV Ruxolitinib Phosphate vRNPs in infected mammalian cells demonstrated that the overall vRNP content of the cytoplasm approached a theoretical balance, with abundances near the 33% for all three genome segments. Although the cytoplasm of some SBV-infected cells deviated from the Ruxolitinib Phosphate average composition, the cell-to-cell heterogeneity in this population was less pronounced (Fig.?4a, b). Next, we evaluated whether an imbalanced cytoplasmic content could be associated with a particular genome composition of the virions. The correlation analysis made evident that indeed, if a specific genome segment is more abundant intracellularly, it will be incorporated into a virus particle more often, and vice versa. A strong positive correlation (Pearsons coefficients of at least 0.5660 and medium supplemented with 10% FBS and 1% antibiotic/antimycotic at 28?C. Cell culture media and supplements were purchased from Gibco, unless specified otherwise. Viruses Virus stocks of RVFV strain Clone 1346 were obtained after infection of Vero E6 or C6/36 cells at a MOI of 0.005. Virus stocks of SBV isolate NL-F647 were obtained after infection of Vero E6 cells at a MOI of 0.01. Genome segment-specific quantitative RT-PCR Mammalian cells (Vero E6) or insect cells (C6/36 for RVFV and KC for SBV) were seeded in 6-well cell culture plates at 2??105 cells/well or 6??105 cells/well, respectively, and allowed to attach for 2C4?h. Cells were subsequently infected at a MOI of 0.01 and after incubation for 3.5?h, the inoculum was removed and substituted with fresh medium. At defined time points (varied per experiment), IGFBP1 samples from the culture supernatant and cells were collected. In addition to the in vitro experiments, plasma samples were obtained from another study (lambs #158, #160 and #162) in which lambs were experimentally infected via intravenous route with a 105 tissue culture infectious dose (TCID)50 dose of RVFV strain 35/7428. From 1C2?mL of cell lysate, 200?L of culture supernatant or 200?L of plasma, total nucleic acid extractions were performed with the NucliSENS easyMAG system (bioMrieux) according to the manufacturers instructions. Subsequently, viral cDNA was synthesized with the SuperScript IV First-Strand Synthesis System for RT-PCR (Invitrogen) using a combination of S, M and L segment-specific primers (Supplementary Table?1), according to the manufacturers instructions. After the reverse transcription reaction, quantitative PCR amplifications were performed with the Power SYBR Green PCR Master Mix using.