Differences in tropism for endothelial cells between hemorrhagic fever-causing arenaviruses. (A) Multiplex immunofluorescence and ISH staining of kidney sections from infected or mock-infected animals. Single channel images of DAPI, CD31 (vascular endothelial cells), fibrin, viral RNA, and TF are shown as well as their merge. Images are representative of observations made in 3 separate animals for each condition. Scale bar corresponds to 50 μm. (B) Whole-slide images obtained by confocal scanner. Three slices in the Z dimension were transformed by maximum intensity projection. Left image shows a whole lung from a LASV Josiah-infected animal with the 3 staining merged. The 3 images in the center correspond to the white inset and allow appreciation of the staining localization at a cellular level. It includes a medium caliber vessel surrounded by alveoli. Scale bars = 50 μm. The 2 images on the right are from QuPath software and illustrate the process of cell detection based on DAPI staining and cell classification based on CD31 and vRNA positivity. Color-coding: blue = unclassified; green = CD31⁺ vRNA^–; yellow = CD31⁺ vRNA⁺; red = CD31^– vRNA⁺. (C) Quantification of the classification obtained in the previous analysis. Three organs for each condition were quantified. (D) Infectious particles titers in the supernatant of HUVEC infected with 3 different hemorrhagic fever-causing arenaviruses, harvested at 5 different time points after infection. Error bars correspond to the standard error of the mean of 3 replicates, asterisks signal a statistically significant difference between titers at 4 DPI according to a two-tailed Mann-Whitney test (P < .05).