An In Vitro Assay to Study Arenavirus Attachment to the Host Cell

Begin with an adherent culture of mammalian kidney epithelial cells that express viral entry receptors on their surface.

Incubate the cells on ice to inhibit endocytosis and prevent the internalization of cell-surface receptors.

After incubation, remove the growth medium and wash the cells with an ice-cold buffer.

Add an ice-cold suspension of arenavirus particles to the cells and incubate on ice.

Viral surface glycoproteins specifically bind to their receptors on the host cell surface, initiating the first step of arenavirus entry.

At low temperature, only surface attachment occurs while the virus internalization remains blocked.

After incubation, remove the unbound virus particles and wash with an ice-cold buffer to retain only surface-attached viral particles.

The cells are now ready for quantifying surface-bound viral particles.

To make complete DMEM, to a 500 milliliter bottle of DMEM, add 50 milliliters of heat inactivated fetal bovine serum, and 5 milliliters each of 100X penicillin-streptomycin, and 100X HEPES buffer. At the end of the day, seed two point five times ten to the fourth Vero E6 cells per well, in triplicate for each virus, in a 48-well tissue culture plate in a final