KSHV Viral Titer Quantification Protocol

rKSHV.219 was multiplied by the induction of iSLK.219 cells with doxycycline as previously described (Myoung and Ganem, 2011 (link)). Briefly, iSLK.219 cells were induced in both the presence of doxycycline (Sigma, United States) and the absence of hygromycin, puromycin, and G418 (Sigma, United States) at approximately 90% confluency. Four days post-induction, cell supernatants were collected, cleared twice by centrifugation (2,500 × g, 30 min at 4°C), virus was passed through 0.45 μm pore size membrane filters, and then pelleted by ultracentrifugation (25,000 × g, 3 h at 4°C) using a CP-80WX (Hitachi, Japan). Virus pellets were then resuspended overnight in serum-free DMEM collection media and stored at −80°C. KSHV titer (GFU/mL) was determined in HEK293T cells by counting GFP-positive cells as described previously (Zhao et al., 2015 (link)).
Titer (GFU/mL) = [(green fluorescent cells/field)] × [(fields/well)/volume virus (mL) × (dilution factor)].