Fig 1.
Illustration of approaches for studying virus cell-to-cell transmission in vitro and in vivo.
The alphaviruses CHIKV and SINV are used as examples (panel E includes HIV). (A) Immunofluorescence microscopy illustrating ILEs. MEFs were infected with a CHIKV-GFP reporter virus for 11 h, fixed, permeabilized, and stained with antibodies to the viral envelope proteins and tubulin (micrographs are pseudo-colored and the GFP channel is not displayed). Scale bar, 20 μm. Arrowheads point out ILEs. (B) SEM illustrating ILEs. Vero cells were infected with SINV for 9 h, processed for SEM and imaged using a Zeiss Supra 40 field emission SEM. Scale bar, 10 μm. Adapted from [12]. (C) Neutralizing antibody-resistant cell-to-cell transmission. Producer MEFs were infected with CHIKV-GFP; target MEFs were pre-stained with Cell Tracker Orange CMRA dye. Producer and target cells were then co-cultured in the presence of neutralizing antibody against CHIKV for 12 h. The co-culture media were collected to titer free virus particles, and the cells were fixed and analyzed by flow cytometry. Adapted from [13]. (D) Live cell imaging showing cell-to-cell transmission in presence of neutralizing antibody. The co-culture system described in C was incubated with or without neutralizing antibody. Cell contact formation and infection of target cells were monitored by live cell imaging using a DeltaVision Core Microscope. Images show a time range from 8.25–10 h postinfection. Scale bar, 20 μm. Arrows point out ILEs. (E) Cell-to-cell transmission in vivo assayed by flow cytometry and/or intravital imaging. In the CHIKV example, mice were pre-treated with neutralizing antibody before being injected in the footpad with CHIKV-Venus-infected MEFs prestained with CMRA-dye (adoptive transfer). Spread of infection can be assessed either by analysis of ipsilateral ankle tissues by flow cytometry [13] as illustrated for CHIKV or by intravital microscopy to follow infected producer cells (as has been done for HIV-1 [28]). Diagram was created with BioRender.com. CHIKV, chikungunya virus; ILE, intercellular long extension; MEF, mouse embryonic fibroblast; SEM, scanning electron microscopy; SINV, Sindbis virus.