Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection.

Objectives: HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment.
Methods: Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis.
Results: Variants predicted to use CXCR4 were detected (frequency ≥0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3-56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants.
Conclusion: Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.