Quantifying Kaposi’s Sarcoma-Associated Herpesvirus Infectivity Using an Infectious Units Assay

Abstract

Source: Zhu, Q. et al. Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposi's Sarcoma-Associated Herpesvirus. J. Vis. Exp. (2025)

This video demonstrates a green fluorescent protein (GFP) based infectious units assay to quantify the infectivity of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) in mammalian cancer cells.

Protocol

1. Infectious units assay (IU assay)

   NOTE: SLK cells are permissive to Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) entry and support early lytic gene expression. This assay quantifies infectious units based on green fluorescent protein (GFP) expression in infected SLK cells following exposure to serially diluted virus-containing supernatants.

   1. Seed 1 x 10⁴ SLK cells per well in a 96-well plate 24 h before infection. Ensure cells are evenly distributed and reach ~60%-70% confluence on the day of infection.
   2. Label nine 1.5 mL microcentrifuge tubes for serial two-fold dilutions: Tube 1 (undiluted, 1:1), Tube 2 (1:2), Tube 3 (1:4), Tube 4 (1:8), Tube 5 (1:16), Tube 6 (1:32), Tube 7 (1:64), Tube 8 (1:128), and Tube 9 (1:256).
   3. Add 200 µL of virus supernatant to Tube 1. Add 100 µL of pre-warmed, serum-free Dulbecco's modified Eagle medium (DMEM) to each of Tubes 2 through Tube 9. To begin the dilution series, transfer 100 µL from Tube 1 to Tube 2 and mix thoroughly by pipetting up and down. Then transfer 100 µL from Tube 2 to Tube 3 and repeat this process sequentially through Tube 9, mixing thoroughly at each step.NOTE: Each tube should contain a final volume of 100 µL. Perform all dilutions in triplicate.
   4. Aspirate the culture medium from SLK cells. Add 100 µL of each virus dilution to the designated wells. Use 27 wells in total (3 wells per dilution from 1:1 to 1:256).
   5. Centrifuge the 96-well plate at 1,500 x g for 1 h at 30 °C to promote viral adsorption. Transfer the plate to a 37 °C CO₂ incubator for an additional 1 h.
   6. Remove the inoculum and gently add 100 µL of complete DMEM to each well. Incubate the plate for 24 h at 37 °C.
   7. Harvest the cells and analyze GFP expression by flow cytometry. Record the number of GFP-positive cells per well.
   8. Calculate infectious units per mL (Infectious units (IU)/mL) using the following formula:IU/mL = [(number of GFP-positive cells per well)/ inoculum volume (mL)] x dilution factor. Determine the final virus titer using the last dilution with detectable GFP-positive cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete Dulbecco's modified Eagle's medium (DMEM)			Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10%(v/v) fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), and 100U/ml penicillin-streptomycin (Pen-Strep; GenClone).
LSR II 18 flow cytometer	BD Bioscience	N/A