Analysis of nevirapine resistance mutations in cloned hiv type 1 variants from hiv-infected ugandan infants using a single-step amplification-sequencing method (ampliseq)
AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 24, Number 9, 2008 Mary Ann Liebert, Inc.
Analysis of Nevirapine Resistance Mutations
in Cloned HIV Type 1 Variants from HIV-Infected
Amplification-Sequencing Method (AmpliSeq)
William Ian Towler, Jessica D. Church, James R. Eshleman, Mary Glenn Fowler,
Laura A. Guay, J. Brooks Jackson, and Susan H. Eshleman
We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma sampleswere obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by pop-ulation sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplifi-cation-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 vari-ants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealednumerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistancemutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
INGLE DOSE (SD) NEVIRAPINE (NVP) and other NVP-con- variants. This information can be obtained by sequencing vi-
taining regimens are used in resource-limited settings for
ral genomes after cloning6 by single genome sequencing
prevention of HIV-1 mother-to-child transmission. These
(SGS),7 by ultradeep pyrosequencing,8 or by parallel allele-
regimens are effective, but are associated with the emergence
specific sequencing (PASS).9 Of these methods, the first three
and persistence of NVP-resistant HIV-1 variants in some
(traditional cloning/sequencing, SGS, and ultradeep py-
women and in some infants who are HIV infected despite
rosequencing) provide sequence information. In contrast,
prophylaxis.1 Low-level NVP-resistant HIV-1 variants can
PASS provides information for individual codons (point mu-
persist 2 and may affect treatment outcome.3
tations). For each method, the sensitivity of mutation detec-
Several methods can be used to analyze NVP resistance
tion depends on the number of HIV-1 templates analyzed.
mutations. However, each method has some limitations.
Pyrosequencing and PASS require specialized equipment,
HIV-1 genotyping methods based on population (bulk) se-
but can be used to screen large numbers of templates, en-
quencing are relatively insensitive for the detection of low-
hancing the sensitivity of mutation detection.
level HIV-1 variants with resistance mutations. Point muta-
In this report, we used a single-step amplification-se-
tion assays, such as allele-specific polymerase chain reaction
quencing reaction (AmpliSeq)10 to analyze the genetic link-
(PCR), the LigAmp, and the oligonucleotide ligation (OLA),
age of NVP resistance mutations and genetic complexity at
are more sensitive than routine genotyping assays, but may
HIV-1 reverse transcriptase codons associated with NVP re-
be labor intensive if multiple mutations are analyzed.4,5 Vari-
sistance in six 6-week-old Ugandan infants who were HIV-
ations in the HIV-1 nucleotide sequence at oligonucleotide
1 infected despite SD NVP prophylaxis and who had two or
or primer binding sites can interfere with hybridization, lig-
more NVP resistance mutations detected by population se-
ation, and/or priming in these assays.
quencing (Table 1). The samples were collected in three clin-
In addition to the limitations mentioned, the assays de-
ical trials conducted in Kampala, Uganda: (1) the HIVNET
scribed above do not provide information about the genetic
012 trial,11,12 (2) the Repeat Pregnancy Study,13 and (3) the
linkage of NVP resistance mutations in individual HIV-1
Breast Feeding Study (“Pathobiology of Breast Milk among
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
TOWLER ET AL.
aThe table shows the source of samples analyzed (Clinical study), the mutations detected by
the ViroSeq system, and the HIV-1 subtype (pol
bNote that mutations K103R and V179D are associated with NVP resistance only when they
HIV-1 Infected Ugandan Women Receiving Intrapartum
TABLE 2. NVP RESISTANCE MUTATIONS DETECTED
Nevirapine”). HIV-1 genotyping was performed using the
ViroSeq HIV Genotyping System (ViroSeq, Celera Diagnos-
tics, Alameda, CA) using 0.1 ml of infant plasma. The HIV-
1 subtype of each sample was determined by phylogeneticanalysis of pol
DNA cloning was accomplished as follows. Using the
ViroSeq system, RNA was extracted from plasma, and the
HIV-1 RNA was reverse transcribed to generate comple-
mentary DNA. Pol
region DNA (encoding the 3Ј portion of
, HIV-1 protease, and the 5Ј portion of HIV-1 re-
verse transcriptase) was amplified using Research Use Only
PCR mix provided by Celera Diagnosics, which was identi-
cal to the PCR mix included in the ViroSeq system, except
that dTTP was substituted for dUTP. Amplification was per-
V106A ϩ Y188C
formed without dUTP, because dUTP-containing PCR prod-
ucts cannot be cloned using commercially available compe-
tent cells. The PCR products were purified using spin
columns, and were incubated with Taq polymerase and
dNTPs for the addition of terminal adenosine-phosphate
groups, and transformed into TOP 10 electrocompetent Esch-
cells (Invitrogen Corp., Carlsbad, CA). Colonies
K103N ϩ Y181C
were isolated on plates containing ampicillin. S-Gal (Sigma
Corp., St. Louis, MO) was used to indicate the presence of a
DNA insert in the plasmid vector. White colonies were se-
K101E ϩ Y181C
AmpliSeq uses commercially available DNA sequencing
reagents supplemented with additional dNTPs for the com-
bined amplification/sequencing reaction. Two oligonu-
cleotides are added to each reaction, with one in molar ex-
cess. During the initial reaction cycles, the low-concentration
oligonucleotide and the dNTPs are incorporated into ampli-
fication products. In later reaction cycles, DNA sequencing
termination products (generated by priming from the high-
concentration oligonucleotide and incorporating ddNTPs)
predominate. To perform the analysis, a small inoculum
(barely visible on a pipette tip) of each bacterial colony wasadded directly to a 20
l reaction containing 4
aFifty HIV-1 clones were analyzed by AmpliSeq from each infant
Ready Mix (Applied Biosystems, Foster City, CA), 1ϫ Se-
sample. The number of clones with 0, 1, or 2 mutations is shown.
quencing Buffer (Applied Biosystems), 0.15 mM dNTPs, 0.05
Mutations shown in bold were detected using the ViroSeq HIV-1
Genotyping System (population sequencing). The following amino
M forward primer (5Ј-AGATTTCAGGGAACTCAATAA-
acid polymorphisms (not shown in this table) were also detected in the
AAGAACTCA-3Ј), and 0.25
M reverse primer (5Ј-GGTTC-
samples at codons in HIV-1 reverse transcriptase that are the sites of
TTTCTGATGCTTTTTGTCTGGTGT-3Ј). These primers were
NVP resistance mutations: 147770: V179A (1 clone), V106S ϩ V108S (1
designed to bind to regions of HIV-1 that are conserved in
clone). 147858: V181H (1 clone). 274581: L100V ϩ A98C (1 clone).
288407: K103R (50 clones), V179E (26 clones). 289005: V179I (49 clones),
HIV-1 subtypes A, C, and D. The AmpliSeq reaction was per-
V179M (1 clone), K103E ϩ A98S (2 clones). 324698: K101R (1 clone).
formed using an Applied Biosystems 9700 thermal cycler as
bNote that the mutations K103R and V179D are associated with
follows: 95°C for 5 min, followed by 25 three-step cycles of
NVP resistance only when they occur together.
ANALYSIS OF NVP RESISTANCE IN HIV-1 CLONES
94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by
mutations (Table 2). For each infant, NVP resistance mu-
40 three-step cycles of 95°C for 15 s, 50°C for 15 s, and 60°C
tations that were identified in the population sequence
4 min. The reactions were ethanol precipitated, resuspended
were also identified in one or more of the 50 clonal se-
l of HiDi formamide (Applied Biosystems), and ana-
quences. Clones from five of the six infants also had NVP
lyzed on an ABI PRISM 3100 Genetic Analyzer (Applied
resistance mutations that were not identified by population
Biosystems). Fifty sequences were analyzed for each infant
sequencing. Clones with two genetically linked NVP resis-
tance mutations were identified in five of the six infants
HIV-1 sequence data generated using the AmpliSeq
method were analyzed using BioEdit Sequence Alignment
We next analyzed the nucleotide sequences of the 50
Editor.14 Sequences included nucleotides encoding HIV-1 re-
clones from each infant to determine which codons were
verse transcriptase amino acids 95–195. Sequences were ex-
present at positions of NVP resistance mutations (Table 3).
amined for the following mutations: A98G
, L100I, K101E/P,
Up to six different amino acids were detected at a single po-
, V108I, E138K,
sition (e.g., at codon 179). Furthermore, at 5 of the 10 posi-
V179D/E/F, Y181C/I/V, Y188C/H/L,
tions analyzed, we detected two different codons encoding
tations shown in bold are associated with reduced NVP sus-
the same amino acid (e.g., GCA and GCG for Ala at codon
ceptibility; mutations in bold and italic are associated with
98). In one infant, all 50 clones had K103R, and 24 of those
high-level phenotypic NVP resistance and/or reduced viro-
clones had V179D; these two mutations are associated with
logic response to a clinical regimen.15 DNAStar MegAlign
NVP resistance only when they occur together.15,16 Exclud-
(DNAStar, Inc., Madison, WI) was used to generate sequence
ing K103R and V179D in that infant, the portion of clones
alignments. Phylogenetic trees based on 500 bootstraps were
that had NVP resistance mutations varied from one posi-
generated using DNAStar MegAlign (DNAStar, Inc., Madi-
tion to another (from 0 to 140 clones out of 300 clones ex-
son, WI). The 50 clonal HIV-1 sequences from each infant
grouped together with the corresponding maternal se-
The methods described in this report can be performed us-
ing DNA remaining from HIV-1 genotyping, which is ideal
We first analyzed the amino acid sequences from the 50
for analysis of samples that are limited in volume, such as
clones isolated from each infant sample for NVP resistance
those from pediatric studies. We were able to obtain read
TABLE 3. CODONS DETECTED IN CLONES AT POSITIONS IN HIV-1 REVERSE TRANSCRIPTASE ASSOCIATED WITH NVP RESISTANCEa
Arg Glu Glu
Lys Asn Asn Arg
b Glu Val
TGC AGC CTA TTA
AGA GAA GAG
AAG AAA AAC AAT AAG
GAA GTA GCA
GTA GTG ATA
ATT GAA ATG TAT TGT
CAT TAT TGT GGA GCA
aFifty HIV-1 clones were analyzed from each of the six infant samples (300 clones total). Sample numbers are indicated on the left. NVP re-
sistance mutations occur at codons 98, 100, 101, 103, 106, 108, 179, 181, 188, and 190 in HIV-1 reverse transcriptase. The table shows the nucle-otide sequences and corresponding amino acids detected in clones from each infant sample at each position. The number of clones with eachnucleotide sequence is indicated. Reference sequences/amino acids (those found in reference strain HXB2) are in italics. Sequences/muta-tions associated with NVP resistance are shown in bold.
bNote that mutations K103R and V179D are associated with NVP resistance only when they occur together.
TOWLER ET AL.
lengths of approximately 300 bases with a single AmpliSeq
(DHHS). NIH, DHHS (U01-AI-068613), (2) the International
reaction, enabling us to analyze the two major regions where
Maternal Pediatric Adolescent AIDS Clinical Trials Network
NVP resistance mutations occur (i.e., at codons 98–108 and
of the NIAID (U01-AI-068632), (3) the HIV Network for Pre-
codons 179–190). AmpliSeq has been optimized for read
vention Trials (HIVNET) and sponsored by NIAID, NIH,
lengths of approximately 500 bases using purified human
DHHS, through contract U01-AI-046749 with Family Health
and bacterial genomic templates.10 The methods described
International, contract U01-AI-046745 with Johns Hopkins
in this report also produce cloned plasmids, which can be
University, and (4) the Centers for Disease Control and Pre-
used for further characterization of specific HIV-1 variants.
vention, Atlanta, GA, including contract number 200-2004-
The samples used in this study were selected from six in-
M-09279 to Johns Hopkins University. None of the authors
fants who had two or more NVP resistance mutations de-
has a commercial or other association that might pose a con-
tected by population sequencing. Clonal analysis revealed
flict of interest with the following exception: Dr. James Esh-
that five of the six infants had some HIV-1 variants with two
leman (spouse of Susan H. Eshleman) is a co-inventor of the
or more genetically linked mutations. HIV-1 variants with
AmpliSeq assay and Johns Hopkins University has filed a
multiple NVP resistance mutations may have altered fitness,
patent application with the US-Patent and Trademark Office.
different patterns of cross-resistance to other nonnucleoside
The inventors may receive royalty payments if the applica-
reverse transcriptase inhibitors compared to HIV-1 variants
with single NVP resistance mutations.15 With the exceptionof one infant who had the K103R polymorphism in all clones
and V179D in a high portion of clones, HIV-1 variants withtwo genetically linked mutations represented a small por-
1. McConnell MS, Stringer JS, Kourtis AP, Weidle PJ, and Esh-
tion of the viral population (one to four clones out of 50 an-
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of mother-to-child transmission of HIV-1: Does develop-
genetically linked mutations, each of the individual muta-
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tions was also detected separately in some clones. Because
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2. Flys TS, Donnell D, Mwatha A, et al.
: Persistence of K103N-
there is some possibility that recombination occurred during
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3. Johnson J, Li JF, Wei X, et al.
: Low-frequency mutations sub-
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Analysis of HIV-1 clones from these infants also revealed
tance and greatly strengthen the relationship between resis-
a high degree of genetic complexity at codons that influence
tance mutations and virologic failure. 14th Conference on
NVP susceptibility. Among the clones from six infants, we
Retroviruses and Opportunistic Infections, Los Angeles, CA,
detected at least four different codons at 5 of the 10 positions
February 25–28, 2007, Abstract #639.
analyzed, and 5 of the 10 positions analyzed had two dif-
4. Ellis GM, Mahalanabis M, Beck IA, et al.
: Comparison of oli-
ferent codons that encoded the same amino acid. This high
gonucleotide ligation assay and consensus sequencing for
degree of diversity, which was also seen at codons in HIV-
detection of drug-resistant mutants of human immunodefi-
1 reverse transcriptase that are not associated with NVP re-
ciency virus type 1 in peripheral blood mononuclear cells
sistance, underscores the challenges faced in designing oli-
and plasma. J Clin Microbiol 2004;42:3670–3674.
gonucleotide reagents for point mutation assays and other
5. Palmer S, Boltz V, Martinson N, et al.
: Persistence of nevi-
assays that rely on hybridization of olignonucleotides to
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ine therapy for prevention of maternal-to-fetal HIV-1 trans-mission. Proc Natl Acad Sci USA 2006;103:7094–7099.
6. Eshleman SH, Jones D, Flys T, Petrauskene O, and Jackson
JB: Analysis of HIV-1 variants by cloning DNA generated
The GenBank accession numbers for the population se-
with the ViroSeq HIV-1 Genotyping System. Biotechniques
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: Multiple, linked
human immunodeficiency virus type 1 drug resistancemutations in treatment-experienced patients are missed by
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The authors thank the mothers and infants who partici-
8. Hoffmann C, Minkah N, Leipzig J, et al.
: DNA bar coding
pated in the HIVNET 012 trial, the RP study, and the Breast
and pyrosequencing to identify rare HIV drug resistance
Feeding study. The authors also thank the study teams for
mutations. Nucleic Acids Res 2007;35:e91.
providing the samples used in this study, and thank the lab-
9. Cai F, Chen H, Hicks CB, Bartlett JA, Zhu J, and Gao F: De-
oratory staff at Makerere University and Johns Hopkins Uni-
tection of minor drug-resistant populations by parallel al-
versity for assistance with sample shipment and processing.
lele-specific sequencing. Nat Methods 2007;4:123–125.
This study was supported by (1) the HIV Prevention Trials
10. Murphy KM, Berg KD, and Eshleman JR: Sequencing of ge-
Network (HPTN) sponsored by the National Institute of Al-
nomic DNA by combined amplification and cycle sequenc-
lergy and Infectious Diseases (NIAID), National Institute on
ing reaction. Clin Chem 2005;51:35–39.
Drug Abuse (NIDA), National Institute of Mental Health
11. Guay LA, Musoke P, Fleming T, et al.
: Intrapartum and
(NIMH), and Office of AIDS Research, of the National Insti-
neonatal single-dose nevirapine compared with zidovudine
tutes of Health (NIH), Dept. of Health and Human Services
for prevention of mother-to-child transmission of HIV-1 in
ANALYSIS OF NVP RESISTANCE IN HIV-1 CLONES
Kampala, Uganda: HIVNET 012 randomised trial. Lancet
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K101P and K103R/V179D mutations in human immunode-
12. Jackson JB, Musoke P, Fleming T, et al.
: Intrapartum and
ficiency virus type 1 reverse transcriptase confer resistance
neonatal single-dose nevirapine compared with zidovudine
to nonnucleoside reverse transcriptase inhibitors. Antimi-
for prevention of mother-to-child transmission of HIV-1 in
crob Agents Chemother 2006;50:351–354.
Kampala, Uganda: 18 months follow-up of the HIVNET 012randomised trial. Lancet 2003;362:859–868.
13. McConnell MS, Bakaki P, Eure C, et al.
: Effectiveness of re-
peat single-dose nevirapine for prevention of mother-to-
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14. Hall TA. BioEdit: A user-friendly biological sequence align-
The Johns Hopkins Medical Institutions
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Nucleic Acids Symp Ser 1999;41:95–98.
15. Stanford University: Stanford University HIV Drug Resis-
tance Database. http://hivdb.stanford.edu/cgi-bin/NNR-TIResiNote.cgi: Accessed 04/08.
Erzengel Ambriel B110 Helles Rosenpink/Tiefmagenta Geboren: 31. Mai 2012, 12 Uhr mittags, brit. Sommerzeit Der Erzengel des Tierkreiszeichens Zwillinge Die Zwillinge und der kosmische Christus Die Verbindung mit der schützenden Energie von Liebe und Freundlichkeit Verknüpft mit B67 Mebahel, Tagesengel des 31. Mai Ambriel ist bekannt als der Prinz des Ordens der Throne, der Oberste der
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