Background:  To what extent viral heterogeneity can be transmitted during primary HIV infection is a matter of debate. HIV-1 heterogeneity upon transmission may be due to co-infection, early superinfection, or within-host evolution. Here we aimed at analyzing HIV env diversity in a large number of primary HIV infection patients. Moreover, we identified transmission clusters by anonymously linking HIV pol sequences from primary HIV infection patients with participants of the Swiss HIV Cohort Study (SHCS).
Methods:  Duplicate real-time polymerase chain reaction (RT-PCR) from plasma extracts, cloning (16 clones/sample), and sequencing of the env C2V3C3 domain was performed (total 1248 sequences). All primary HIV infection patients obtained baseline genotypic resistance testing (RT/protease). From 100 patients, pol sequences were anonymously compared with those from the SHCS genotypic drug-resistance database (4276 patients, 6500 sequences). Possible transmitters were identified by sequence clustering with bootstrap values >98% and genetic distances <1.5% and confirmed by clonal analysis of the env region. Phylogenetics were done using PHYLIP and Mega 4 (maximum likelihood/neighbor joining).
Results: From 150 patients consecutively enrolled in the ZPHI-study (clinicaltrials.gov) env diversity of 78 patients was determined so far (median of estimated time after infection: 6 weeks (range 2 to 24). Of the total, 68 patients (87%; 53 patients with negative/indeterminate Western blot) had documented acute (<12 weeks) and 10 patients had recent infection (12 to 24 weeks). Patients were primarily male (86%) infected with HIV-1 subtype B (80%). Baseline env nucleotide sequence diversity ranged from 0.11% to 2.74% (median 0.48%) and neither correlated with viral load (median 2.6x10⁵ copies/mL; range 2.1x10² to 4.0x10⁷) nor with estimated time of infection. Diversity of 10 patients (13%) was >1% despite short time after infection (6 weeks, range 4 to 13; 9 of 10 acute; 7 of 10 negative Western blot; 9 of 10 male). Overall, pol sequences segregated into 28 distinct clusters which subsequently were confirmed by env sequences derived from plasma samples from the SHCS repository. The identified transmission clusters ranged in size from 2 to 10 patients and comprised a total of 82 patients (42 primary HIV infection, 40 SHCS).
Conclusions:  In the ZPHI study, we found 13% of patients with relatively high viral diversity shortly after HIV transmission. This suggests that transmission of multiple viral variants may occur more often than previously thought. A very high number of our primary HIV patients (42%) could be linked to possible transmitters. To identify such clusters is key to study viral determinants associated with HIV-1 transmission.