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Single genome amplification and molecular cloning of HIV-1 populations in acute HIV-1 infection : implications for studies on HIV-1 diversity and evolutionary rate

Hsieh, Anthony Y.Y. ; Hassan, Amin S. LU ; Nazziwa, Jamirah LU orcid ; Lindquist, Lovisa LU ; Karlson, Sara LU orcid ; Hare, Jonathan ; Kamali, Anatoli ; Karita, Etienne ; Kilembe, William and Price, Matt A. , et al. (2026) In Virus Evolution 12(1).
Abstract

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)... (More)

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.

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Contribution to journal
publication status
published
subject
keywords
acute HIV-1 infection, HIV-1, HIV-1 diversity, HIV-1 evolution, molecular cloning, phylogenetics, single genome amplification, transmitted founder virus
in
Virus Evolution
volume
12
issue
1
article number
veaf099
publisher
Oxford University Press
external identifiers
  • scopus:105028030665
ISSN
2057-1577
DOI
10.1093/ve/veaf099
language
English
LU publication?
yes
id
1e79b36b-68bd-4efa-b2b0-5324cb659dff
date added to LUP
2026-02-25 14:59:08
date last changed
2026-02-26 10:18:49
@article{1e79b36b-68bd-4efa-b2b0-5324cb659dff,
  abstract     = {{<p>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.</p>}},
  author       = {{Hsieh, Anthony Y.Y. and Hassan, Amin S. and Nazziwa, Jamirah and Lindquist, Lovisa and Karlson, Sara and Hare, Jonathan and Kamali, Anatoli and Karita, Etienne and Kilembe, William and Price, Matt A. and Björkman, Per and Kaleebu, Pontiano and Allen, Susan and Hunter, Eric and Gilmour, Jill and Rowland-Jones, Sarah L. and Sanders, Eduard J. and Esbjörnsson, Joakim}},
  issn         = {{2057-1577}},
  keywords     = {{acute HIV-1 infection; HIV-1; HIV-1 diversity; HIV-1 evolution; molecular cloning; phylogenetics; single genome amplification; transmitted founder virus}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Oxford University Press}},
  series       = {{Virus Evolution}},
  title        = {{Single genome amplification and molecular cloning of HIV-1 populations in acute HIV-1 infection : implications for studies on HIV-1 diversity and evolutionary rate}},
  url          = {{http://dx.doi.org/10.1093/ve/veaf099}},
  doi          = {{10.1093/ve/veaf099}},
  volume       = {{12}},
  year         = {{2026}},
}