Hsieh AYY
Hassan AS
Nazziwa J
Lindquist L
Karlson S
Hare J
Kamali A
Karita E
Kilembe W
Price MA
Bj├Ârkman P
Kaleebu P
Allen S
Hunter E
Gilmour J
Rowland-Jones SL
Sanders EJ
Esbj├Ârnsson J
Virus Evol. 2026;12veaf099
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is one of the fastest-evolving human pathogens. Understanding HIV-1 transmission, within-host adaptation, and evolutionary dynamics is pivotal for development of interventions and vaccines. HIV-1 infection is generally caused by a single transmitted founder virus (TFV), and TFV sequences are typically obtained using single genome amplification (SGA). However, suboptimal sample quality can cause sequencing failures, representing considerable losses considering the scarcity of acute HIV-1 infection (AHI) samples. Sequencing failures may be mitigated by molecular cloning (MC), which can be less vulnerable to sample quality but more susceptible to polymerase chain reaction (PCR) errors. Here, we explore the feasibility of supplementing SGA with MC data using samples from clinical and research cohorts to determine whether sequence diversity and evolutionary rate estimates are comparable between the techniques. METHODS: Plasma samples were selected from participants with documented AHI from an East African research cohort (the International AIDS Vaccine Initiative, 2006-2011) and a clinical cohort from Sweden (1983-2011). SGA and MC sequencing were done on the HIV-1 env V1-V3 region (~940 base pairs). Within-host sequence diversity was determined from maximum likelihood phylogenetic trees, and evolutionary rate by Bayesian phylogenetic analysis. Highlighter plots, Hamming distances, and assessment of star phylogenies were used to quantify TFVs. RESULTS: One hundred participants (median age 30.3 years, 15% female), contributing 350 samples from four longitudinal time points, 10-540 days post-infection, met the inclusion criteria. SGA succeeded on 90% of research cohort and 48% of clinical cohort samples. Comparative analysis of linked SGA and MC data from 10 samples indicated that approximately eight sequences were necessary for diversity estimates. Consistently higher sequence diversity was observed among MC relative to SGA sequences (median [IQR]: 0.009 [0.003, 0.015] and 0.004 [0.001, 0.012] substitutions/site, P = .002), whereas evolutionary rates were comparable between the two methods (0.016 [0.012, 0.019] and 0.011 [0.008, 0.020] substitutions/site/year, P = .232). Five participants with samples obtained within 45 days post-infection were eligible for TFV quantification, and all found to have one TFV using both techniques. CONCLUSION: MC data is a suitable supplement for SGA-based HIV-1 studies to preserve the value of precious samples for analysis of evolutionary rate, but not for sequence diversity.
