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Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing

American Society for Microbiology
Journal of Virology
Authors:
Benjamin N Bimber at Oregon Health Science University
Benjamin N Bimber
  • Oregon Health Science University
Dawn M Dudley at University of Wisconsin–Madison
Dawn M Dudley
Michael Lauck
Michael Lauck
Michael Lauck
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Ericka A Becker at Johns Hopkins Medicine
Ericka A Becker
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Abstract

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
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JOURNAL OF VIROLOGY, Nov. 2010, p. 12087–12092 Vol. 84, No. 22
0022-538X/10/$12.00 doi:10.1128/JVI.01378-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Whole-Genome Characterization of Human and Simian Immunodeficiency
Virus Intrahost Diversity by Ultradeep Pyrosequencing
䌤
Benjamin N. Bimber,
1
‡ Dawn M. Dudley,
2
‡ Michael Lauck,
2
Ericka A. Becker,
1
Emily N. Chin,
2
Simon M. Lank,
1
Haiying L. Grunenwald,
5
Nicholas C. Caruccio,
5
Mark Maffitt,
5
Nancy A. Wilson,
1
Jason S. Reed,
1
James M. Sosman,
6
Leandro F. Tarosso,
4
Sabri Sanabani,
4
Esper G. Kallas,
4
Austin L. Hughes,
3
and David H. O’Connor
1,2
*
Wisconsin National Primate Research Center, University of Wisconsin—Madison, Madison, Wisconsin 53706
1
; Department of
Pathology and Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin 53706
2
; Department of
Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
3
; Division of Clinical Immunology and
Allergy, University of Sao Paulo, Sao Paulo, Brazil
4
; Epicentre Biotechnologies, 726 Post Road, Madison,
Wisconsin 53713
5
; and University of Wisconsin Carbone Cancer Center, Madison, Wisconsin 53705
6
Received 1 July 2010/Accepted 30 August 2010
Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodefi-
ciency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance,
receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV
genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete
characterization of the viral population than is possible with conventional methods, revealing low-level drug
resistance and detecting previously hidden changes in the viral population. While this work applies pyrose-
quencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
The viral population within each human immunodeficiency
virus (HIV)-infected individual is highly diverse and constantly
evolving (2, 3). However, our understanding of the viral pop-
ulation is based largely on the consensus sequence of the dom-
inant circulating virus because the full diversity of the viral
population is extremely difficult to characterize. One recent
study showed that despite viral fitness recovery in vitro, recov-
ery was not correlated with changes observed in the consensus
sequence of HIV. Instead, increased fitness correlated with
general viral heterogeneity (5). This finding suggests that by
limiting our studies to consensus sequences, we are missing
many aspects of viral evolution that influence fitness, drug
resistance, and immune evasion, among other characteristics.
Studies that examined minor viral variants have provided new
insights into HIV transmission and pathogenesis, with direct
implications for HIV treatment (7, 13). Unfortunately, tradi-
tional techniques to identify rare variants, such as molecular
cloning, single-genome amplification, or quantitative real-time
(qRT)-PCR, are either labor intensive or restricted to the
detection of single variants, limiting their widespread use
(8, 11, 12, 14).
New second-generation technologies have radically altered
DNA sequencing. Recent work by our group and others has
employed pyrosequencing for targeted ultradeep sequencing
of short regions of the viral genome, including CD8
⫹
T-lym-
phocyte epitopes and regions of known drug resistance muta-
tions, demonstrating a practical method to identify extremely
low-frequency viral variants (4, 15). While sequencing short
regions is appropriate in certain circumstances, the region of
interest must be identified in advance, and the effect of
mutations in that region on the remaining genome is ig-
nored. Studying the heterogeneity of HIV across the entire
genome may provide insights into interactions between mi-
nor variants, improve our understanding of HIV evolution,
and ultimately provide insights into disease pathogenesis.
In this study, we combined pyrosequencing with a transpo-
son-based fragmentation method to allow powerful ultradeep
sequencing of the full-length HIV and simian immunodefi-
ciency virus (SIV) genomes, demonstrating a new and highly
practical approach to study the complexity of the viral popu-
lation within a host and identify minor variants on a genome-
wide scale. While this study applied pyrosequencing to immu-
nodeficiency viruses, this approach could be applied to any
viral pathogen.
Genome-wide pyrosequencing of SIV. We first applied this
approach to sequence virus from an Indian rhesus macaque
experimentally infected with SIVmac239. We designed four
overlapping reverse transcription PCR amplicons of approxi-
mately 2.5-kb each to span nucleotides 1269 to 10235 of the
SIVmac239 genome, which includes all coding regions (Fig.
1A and B). We then performed reverse transcription PCR
on plasma viral RNA isolated at two time points in chronic-
stage infection, weeks 54 and 82. The reverse transcription
PCR products were randomly fragmented using modified
transposons (Nextera; Epicenter Biotechnology) and were
then pyrosequenced. As a control, we sequenced an SIVmac239
viral stock. Sample preparation and pyrosequencing are de-
scribed in greater detail at the following URL: https://xnight
.primate.wisc.edu:8443/labkey/files/WNPRC/WNPRC_Laboratories
/oconnor/public/publications/%40files/2010%20JV%20Bimber
-Dudley%20et%20al%20Supplemental%20Material.pdf?renderAs
⫽DEFAULT.
* Corresponding author. Mailing address: University of Wisconsin—
Madison, 555 Science Drive, Madison, Wisconsin 53711. Phone: (608)
890-0845. Fax: (608) 265-8084. E-mail: doconnor@primate.wisc.edu.
‡ These authors contributed equally to the manuscript.
䌤
Published ahead of print on 15 September 2010.
12087
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Article
  • Nov 1978
Formulae are developed for the distribution of allele frequencies (the frequency spectrum), the mean number of alleles in a sample, and the mean and variance of heterozygosity under mutation pressure and under either genic or recessive selection. Numerical computations are carried out by using these formulae and Watterson's (1977) formula for the distribution of allele frequencies under overdominant selection. The following properties are observed: (1) The effect of selection on the distribution of allele frequencies is slight when 4Ns </= 4, but becomes strong when 4Ns becomes larger than 10, where N denotes the effective size and s the selective difference between alleles. Genic selection and recessive selection tend to force the distribution to be U-shaped, whereas overdominant selection has the opposite tendency. (2) The mean total number of alleles in a sample is much more strongly affected by selection than the mean number of rare alleles in a sample. (3) Even slight heterozygote advantage, as small as 10(-5), increases considerably the mean heterozygosity of a population, as compared to the case of neutral mutations. On the other hand, even slight genic or recessive selection causes a great reduction in heterozygosity when population size is large. (4) As a test statistic, the variance of heterozygosity can be used to detect the presence of selection, though it is not efficient when the selection intensity is very weak, say when 4Ns is around 4 or less. A model, which is somewhat similar to Ohta's (1976) model of slightly deleterious mutations, has been proposed to explain the following general patterns of genic variation: (i) There seems to be an upper limit for the observed average heterozygosities. (ii) The distribution of allele frequencies is U-shaped for every species surveyed. (iii) Most of the species surveyed tend to have an excess of rare alleles as compared with that expected under the neutral mutation hypothesis.
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