Tropism Testing Who needs a Phenotype Arevir Meeting May 2011 Swiss - PowerPoint PPT Presentation

Tropism Testing – Who needs a Phenotype… Arevir Meeting May 2011

Swiss 3 ‐ step System Element 1 (TrackC): (ca. 85% of analyses) Element 2 (Geno2Pheno): -RNA-DNA conversion (cDNA); (ca. 25% of analyses) -PCR-amplification of V3 region by PCR; -RNA-DNA conversion; -Hybridization to known DNA probes; -DNA-amplification of V3 region by PCR; -Capillary-based nucleic acid duplex tracking -Sequencing of the env V3-region of the assay “TrackC”: viral genome; - Sequence based interpretation of V3 Amino Acid information using Geno2Pheno

Element 3 (PhenX-R): (ca. 10% of analyses) -RNA-DNA conversion; amplification of env; -Cloning into replicative HIV cassette (plasmid lacking env); -Transfection into permissive human cell line; -4-days virus replication in presence of drug; -Read-out: HIV tropism; virus fitness; dualtrop/mixes

Amplification Env Env Env Viral RNA from Patient Env “ PhenX-R ” RT Env Env Env Provirus-Cassette for the Env Gene RT (=> complete Virus!) Treatment in vitro w/ Corec.-Antagonists

Replicative phenotypic Tropism test • Why? – To verify ambiguous Geno ‐ results – To assess minority populations – To discriminate dualtropic and mixed viruses – To determine viral fitness

rPhenotypic Tropism Testing • When? – When FPR does not allow conclusive decision – Disagreement between TrackC/G2P – For unusual V3 lengths