The methodologies used in this study are listed in Table 1 and differences between assay protocols in supplementary Table S1 and supplementary Figure S1. Detailed protocols are available at the EUROPRISE website www.europrise.org. The conventional PBMC based assay [24] (link), [25] (link), [26] (link), [27] (link) with readout based on p24 antigen production involves multiple rounds of virus replication, has a moderate reproducibility and sensitivity, is time-consuming and cumbersome to perform but involves the most physiological target cell. An alternative readout can be the measurement of viral RNA, which shortens the time by several days [28] (link), [29] (link). Intracellular (IC) p24 antigen determination in infected PBMC cultures may be run as a single round assay with increased sensitivity, reproducibility and speed but it is not easy to perform [30] (link). The method of measuring ICp24 was also applied to other target cells, like macrophages [31] (link). Plaque reduction assays use either U87.CD4 or GHOST(3) cells engineered to express coreceptors for HIV [32] (link), [33] (link). In U87.CD4 cells the syncytium-inducing capacity of HIV is exploited, while infected GHOST(3) cells turn green due to the activation of the GFP gene linked to the HIV-2 LTR. These assays are single round, highly reproducible, easy to perform, with sensitivity comparable to the PBMC assay, but require a shorter time. The fusion assay is based on fusion of effector cells expressing the native HIV-1 envelope on their surface (PM1 persistently infected with HIV-1) with target cells expressing the appropriate receptors (initially NIH-3T3 mouse fibroblasts or HeLa human epithelial cells stably expressing human CD4, CCR5 and/or CXCR4). The readout is measurement of ß-galactosidase activity [34] (link). Pseudovirus (PSV)-based assays exist in a number of variant assay formats using different target cells [35] , [36] (link), [37] (link), [38] (link). A selected molecular clone is tested in a single round assay with luciferase readout that results in short-term assays with high reproducibility and sensitivity. Plasmid production and producer cell line culture history are crucial criteria and influences the results. Due to this a fairly large inter-laboratory variation has been documented [23] . Finally, assays using recombinant viruses have also been included [39] (link), [40] (link), [41] (link). This assay type was run with two different starting materials, env sequences were amplified either from culture supernatants or from cloned plasmid.
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