Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing

Abstract

Objectives: R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. Methods:We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population.We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate=5.75) to identify the HIV-1 quasispecies. Results: CXCR4-using viruseswere detected in all but1of the11patients byUDS,andaccountedfor0.2%-100%of the virus populations. The frequency of CXCR4-using viruses was<20% in six subjects and 100% in four subjects. Virus populations containing100%CXCR4-using variants during PHI persisted forat least1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. Conclusions: UDS and the phenotypewere concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.