Finally, we compared five assays to determine co-receptor usage of these primary isolates: (1) the relative infection by primary C19 and C27 HIV-1 isolate on U87.CD4.CCR5 cells and U87.CD4.CXCR4 cells (Fig. 1c, d), (2) the TCID50 values derived on CCR5+ and CXCR4+ cells, (3) the relative inhibition by AMD3100 and MVC on PBMCs (Fig. 4b, d), (4) predicted co-receptor usage from the clones (Additional file 1: Fig. S1), and (5) the actual co-receptor usage of Env clones in the quasispecies (Fig. 5c, d). This evidence concerns the gene CXCR4 and infection.