Viral replication in PBMCs and MDMs

Virus replication of the CL757 was evaluated in rhesus macaque PBMCs and MDMs. Rhesus macaque MDMs were purified from freshly isolated PBMCs by incubation with anti-nonhuman primate CD14 magnetic beads (Miltenyi Biotec, Auburn, CA) followed by positive selection with magnetically activated cell sorting (MACS) separation columns (Miltenyi Biotec). A total of 3 x 10⁵ cells per well of CD14⁺ cells were cultured in a 48-well plate for 4 days in RPMI 1640 medium containing 10% FCS, 10% human serum type AB (Sigma, St. Louis, MO), and 20 ng/ml of macrophage colony-stimulating factor (R&D systems, Minneapolis, MN). Wells were washed two times with Hanks Balanced salt solution (HBSS) and cultured in fresh medium for three additional days. PBMCs (5 x 10⁵ cells/ well) were dispensed into 48-well plate and then inoculated with each virus at MOI of 0.01. MDMs were incubated with virus at an MOI of 0.01 for 1 hour and then washed twice with HBSS and cultured in fresh medium. Virion-associated RT activity of the culture supernatant was monitored periodically [46].