Restriction_of_sequence_diversity_in_the_V3_region_of_the_HIV_1_envelope_gene_during_antiretroviral_treatment_in_a_cohort_of_recent_seroconverters

Thirty study subjects diagnosed with primary HIV-1 infection (PHI) were selected from the Short Pulse Antiretroviral Therapy at Seroconversion (SPARTAC) study (http://www1.imperial.ac.uk/departmentofmedicine/divisions/infectiousdiseases/infectious_diseases/hiv_trials/hiv_treatment/spartac/). Viral RNA was extracted from EDTA plasma collected at enrolment and after ~ 60 weeks from all subjects, and longitudinally over ~ 60 weeks from three subjects. In vitro transcribed RNA from two cloned and sequenced env genes (E6 and H8) as well as a mixture of both (E6:H8) with a molar ratio of 9:1 served as control samples for total processing error rates. Viral RNA and in vitro transcribed RNA were reverse transcribed and the env V3 region was amplified by nested RT-PCR. 500ng of amplified DNAs were sequenced without further purification using the Genome Sequencer FLX Instrument and GS FLX Titanium series reagents (Roche/454 Life Sciences) according to the manufacturer’s instructions.