Epon resin

Microcarriers were placed in a mixture of (1∶1) propylene oxide/Epon resin (Sigma) and then left overnight in pure resin for impregnation of the samples. Microcarriers were then embedded in Epon resin (Sigma), which was allowed to polymerize for 48 hours at 60°C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Wetzlar, Germany), as previously described [21] (link). Sections were deposited on formvar/carbon-coated nickel grids and stained with 5% uranyl acetate, 5% lead citrate. Observations were made with a JEOL 1011 transmission electron microscope.

To visualize the cellular compartments following infection, as well as the presence of viral particles both inside the cells and in the extracellular space, cellular pellets were prepared as follows for EM analysis. Vero E6, HuH7 and MyglaSWRecB cells were seeded in culture flasks and infected 24 h later with PUUV, TULV or PHV at a MOI of 2. After 5 days, cells were washed in PBS before being recovered by trypsinization, centrifuged for 5 min at 1000 rpm, then pelleted cells were suspended in a fixation buffer (4% FA, 1% glutaraldehyde, pH 7.3). Pellets were kept at least 48 h at 4°C and then a few days at room temperature before being treated for EM.
Samples were washed in PBS and post-fixed by incubation for 1 h with 2% osmium tetroxide (Agar Scientific). Cells were then fully dehydrated in a graded series of ethanol solutions and propylene oxide. They were impregnated with a 1∶1 mixture of propylene oxide/Epon resin (Sigma) and left overnight in pure resin. Samples were then embedded in Epon resin (Sigma), which was allowed to polymerize for 48 hours at 60°C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Leica Microsystem). Sections were stained with 2% uranyl acetate (Agar Scientific), 5% lead citrate (Sigma), and observations were made with a transmission electron microscope (JEOL 1011).

Cells were fixed in 4% paraformaldehyde, 1% glutaraldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.2) for 24 h. Samples were then washed in PBS and post‐fixed by incubation with 2% osmium tetroxide (Agar Scientific, Stansted, UK) for 1 h. Afterwards, cells were fully dehydrated in a graded series of ethanol solutions and propylene oxide. Impregnation step was performed with a mixture of (1∶1) propylene oxide/Epon resin (Sigma) and then left overnight in pure resin. Samples were then embedded in Epon resin (Sigma) to polymerise for 48 h at 60°C. Ultra‐thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Sections were stained with 2% uranyl acetate (Agar Scientific), 5% lead citrate (Sigma) and observations were made with a JEOL 1400 plus transmission electron microscope (JEOL, Tokyo, Japan).