Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single Genome Amplification and Sequencing

Departments of Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA 35294; Institute of Genetics, University of Nottingham, Nottingham NG7 2UH, UK; Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA 30329; Zambia Emory HIV Research Project, Lusaka, Zambia, Department of Internal Medicine and UNC Center for AIDS Research, University of North Carolina, Chapel Hill, NC USA 27599; Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, Los Alamos National Laboratory, Los Alamos, NM, USA 87545; and Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 3JT, UK

^* To whom correspondence should be addressed. Email: bhahn{at}uab.edu.

	Abstract

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating human immunodeficiency virus type 1 (HIV-1) transmission and in developing vaccines, drugs or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units from plasma virion RNA by single genome amplification (SGA) followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation and insertion-deletion) of less than 8 x 10^-5. Applying this method to the analysis of plasma virus from 12 Zambian subjects who were sampled within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80 day period spanning seroconversion was 1.7 x 10^-5 substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the SGA approach with more conventional bulk PCR amplification methods on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical of large scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.