Buffy coats from healthy donors from the Red Cross Blood Transfusion Center at the University Hospital of Antwerp were used for isolation of PBMC by LymfoPrep (Axis-Shield, Oslo, Norway) centrifugation and adjusted to 1610 6 /ml in culture medium, consisting of RPMI 1640, 15% fetal calf serum (FCS), 0.03% L-glutamine and 50 mg/ml gentamycin (Lonza, Verviers, Belgium), 2 mg/ml polybrene (Sigma-Aldrich, Bornem, Belgium). Cells were stimulated with 0.5 mg/ml phytohemagglutinin (PHA, Oxoid, Hampshire, UK) for 2 days and 1 day with 200 U/ml interleukin-2 (IL-2; Gentaur, Brussels, Belgium) in a 7% CO 2 incubator at 37uC and then used for neutralization assays. The following cell lines were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZMbl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. and Ghost (3) . All isolates were classified by phylogenetic analysis of their envelope genes. All virus stocks were prepared and titrated on PHA/IL-2 stimulated PBMC. These strains have been extensively used for at least 10 years at ITM and are considered equivalent to neutralization resistant tier 2 viruses [8, 9] . Corresponding envelope PV constructs were obtained by DNA amplification of the complete env starting from PBMC co-cultures or by RT-PCR using plasma and subsequent cloning into an expression vector (pSV7d or pcDNA4/TO) [10] . These included the ITM strains VI 191 (A), VI 829 (C), VI 882 (C), VI 1358 (C), VI 824 (D), VI 1888 (CRF01), VI 1090 (CRF02), CI 20 (CRF02) and CA 18 (CRF02). The env expressing plasmids 92RW009 (A), SF162 (B) and 92BR025 (C) were provided by the EU Programme EVA Centre for AIDS Reagents, NIBSC, UK (AVIP Contract Number LSHP-CT-2004-503487). Sequencing of the PV constructs and phylogenetic analyses of the complete gp160 confirmed the identity of the PV and its corresponding virus. 