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 volume of 500 microliters of complete DMEM. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5% carbon dioxide for 10 to 18 hours.

Under a Class 2 biosafety cabinet, using ice-cold complete DMEM, dilute the JUNV Candid 1 virus samples to be tested to 200 to 2,000 focus forming units or plaque forming units. Plaque forming units will be referred to as PFUs in the remainder of the protocol. For example, to inoculate with 50 microliters of 2,000 PFUs in duplicate, add 4,800 PFUs into a total volume of 120 microliters of medium.

Store on ice until ready to add to the Vero cultures. Remove the plates from the 37 degree Celsius incubator and place on ice or at 4 degrees Celsius for 15 minutes to inhibit the ability of the plated cells to carry out endocytosis. Next, aspirate the complete DMEM from each well, and use 100 microliters of ice-cold PBS to rapidly wash each well twice.

Then add 50 microliters of the pre-diluted virus samples to the appropriate wells. Wrap the plate in plastic and immediately place it back on ice or at 4 degrees Celsius for 60 to 90 minutes to allow virus attachment to occur. Keep the wash buffer and virus samples at 4 degrees Celsius throughout this step.

After the incubation, aspirate the virus inoculum and use 200 microliters of ice-cold PBS to wash each well three times.