JOURNAL OF VIROLOGY, June 1995, p. 3778–3788
Copyright ? 1995, American Society for Microbiology
Vol. 69, No. 6
Analysis of Envelope Sequence Variants Suggests Multiple
Mechanisms of Mother-to-Child Transmission of
Human Immunodeficiency Virus Type 1
LAURENCE BRIANT,1† CHRISTOPHER M. WADE,2JACQUELINE PUEL,1
ANDREW J. LEIGH BROWN,2AND MIREILLE GUYADER1*
Laboratoire de Virologie, Centre Hospitalo-Universitaire Purpan, 31059 Toulouse, France,1
and Centre for HIV Research, Institute of Cell, Animal and Population Biology, Division of
Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JN, United Kingdom2
Received 5 July 1994/Accepted 6 March 1995
In order to elucidate the molecular mechanisms involved in human immunodeficiency virus type 1 (HIV-1)
mother-to-child transmission, we have analyzed the genetic variation within the V3 hypervariable domain and
flanking regions of the HIV-1 envelope gene in four mother-child transmission pairs. Phylogenetic analysis and
amino acid sequence comparison were performed on cell-associated viral sequences derived from maternal
samples collected at different time points during pregnancy, after delivery, and from child samples collected
from the time of birth until the child was approximately 1 year of age. Heterogeneous sequence populations
were observed to be present in all maternal samples collected during pregnancy and postdelivery. In three
newborns, viral sequence populations obtained within 2 weeks after birth revealed a high level of V3 sequence
variability. In contrast, V3 sequences obtained from the fourth child (diagnosed at the age of 1 month)
displayed a more restricted heterogeneity. The phylogenetic analysis performed for each mother-child sequence
set suggested that several mechanisms may potentially be involved in HIV-1 vertical transmission. For one
pair, child sequences were homogeneous and clustered in a single branch within the phylogenetic tree,
consistent with selective transmission of a single maternal variant. For the other three pairs, the child
sequences were more heterogeneous and clustered in several separate branches within the tree. In these cases,
it appeared likely that more than one maternal variant was responsible for infection of the child. In conclusion,
no single mechanism can account for mother-to-child HIV-1 transmission; both the selective transmission of
a single maternal variant and multiple transmission events may occur.
Extensive genetic diversity is a characteristic feature of hu-
man immunodeficiency virus type 1 (HIV-1) (2, 19, 33, 39).
Considerable genomic diversity has been observed among in-
dependent isolates from epidemiologically unlinked infections
and has also been reported to occur to a lesser degree in vivo,
among viral species from a single patient (4, 5, 22, 29, 44, 45).
Studies of sequence variation within the V3 hypervariable re-
gion of the envelope gene have described the heterogeneous
viral population in long-term-infected individuals and have
shown that sequences collected from an individual shortly after
infection appear to be less variable than those found in the
donor at the time of transmission (45, 51). This observation
supports the hypothesis that sexual or parenteral HIV-1 infec-
tion may be initiated by a limited number of molecular vari-
ants. Recent reports based on V3 sequence analyses of moth-
er-child transmission pairs have also suggested that a very
limited number of variants or even one particular variant could
initiate infection within the child (31, 42, 50). This genotype
may represent a minor maternal form, perhaps escaping a
critical immune surveillance mechanism or having particular
phenotypic properties. However, the molecular and biological
properties of viruses transmitted perinatally to children have
not yet been determined; whether cell-free or cell-associated
virus is transmitted and at what time transmission occurs re-
main unclear. The detection of HIV nucleic acids in fetal
tissues (8, 46) has indicated that transmission to the child may
occur at an early stage of gestation. On the other hand, clinical
studies have demonstrated that a high proportion of perina-
tally infected children show no sign of infection at delivery (12,
28). This would suggest transmission to the child either at a
late stage of pregnancy or at delivery. In contrast, the early
diagnosis of HIV infection in newborns (within the first few
days following birth) has been interpreted to reflect infection
early in pregnancy. Striking differences have also been re-
ported regarding the evolution of disease in young perinatally
infected children. AIDS has been shown to develop more rap-
idly in 20% of perinatally infected children than in adults (3,
38). Such rapid progression may be associated with infection of
the child at an early stage of pregnancy.
In order to elucidate the molecular characteristics of HIV-1
variants involved in mother-to-child transmission, we have as-
sessed the genetic diversity of proviral DNA sequences span-
ning the V3 loop and flanking regions (313 bp) in four peri-
natally infected children and their respective mothers. Since
transmission may have occurred at any time during pregnancy
or at delivery, we have analyzed maternal variants detected at
different time points throughout pregnancy and following de-
livery and compared these with variants detected in the child
over a period of 16 months. The analysis of longitudinal sam-
ples collected from mothers during pregnancy is essential to
determine whether positive selection of maternal variants oc-
curs in HIV-1 vertical transmission. In one mother-child pair
we have observed transmission of a single maternal variant,
which may indicate a selective process. However, we also re-
* Corresponding author. Present address: Centre d’Immunologie,
Parc Scientifique et Technologique de Luminy, Case 906, 13288 Mar-
seille cedex 09, France. Phone: (33) 91 26 94 94. Fax: (33) 91 26 94 30.
† Present address: Centre de Tri des Mole ´cules anti-HIV, CRBM-
CNRS, 34060 Montpellier, France.
port the apparent infection of children by multiple maternal
subtypes, with evidence provided for both early and late trans-
mission events. Vertical transmission of HIV-1 is clearly com-
plex, and further characterization of the transmitted viral spe-
cies may be required to provide further insight and a better
understanding of this transmission route.
MATERIALS AND METHODS
Patients. Sequence variation within the V3 domain and flanking regions in
four HIV-1-infected mother-newborn pairs was assessed. All four mothers gave
birth at La Grave Hospital in Toulouse, France, and were monitored over a
1-year period from the beginning of pregnancy. The four mother-child pairs were
selected because they provided the best series of samples from the mother and
child. The clinical status of the patients studied, the time of sampling, levels of
cell and plasma viremia, and immunological data are presented in Table 1.
Evidence of infection in newborns was provided by coculture and/or PCR using
specific primers for the gag and pol genes (18).
Cell and plasma viremia. Maternal cell viremia and plasma viremia were
assessed according to the standard methods of the French Agence Nationale de
Recherches sur le SIDA collaborative group. Briefly, fresh peripheral blood
mononuclear cells (PBMCs) and plasma were collected from blood samples after
separation on Ficoll-Hypaque gradient (Pharmacia). Cells were washed twice in
RPMI-1640 (Whittaker). Cells and plasma were then diluted separately from 50
to 5 ? 106and cocultivated with 2 ? 106fresh phytohemagglutinin-stimulated
PBMCs from an HIV-negative donor in RPMI-1640 medium containing 15%
fetal calf serum, 0.3 mg of glutamine per ml, 20 IU of human interleukin 2 per
ml, and antibiotics. Four replicates of each sample were analyzed. Twice a week,
the supernatant was collected and reverse transcriptase activity was monitored.
Cell and plasma viremia titers were calculated according to the Karber method
and expressed as 50% tissue culture infective doses per 106cells (21).
Nucleic acid extraction. Total cellular DNA was obtained from patients’ un-
cultured PBMCs. To avoid any risk of contamination, nucleic acid extraction was
carried out in laboratories free of PCR products, cultured HIV isolates, or
TABLE 1. Times of sampling and clinical data from mother-child pairs studied
2 mo p-del
3 mo p-del
4.5 mo p-del
aTime points are expressed as months of pregnancy and age for mothers and children, respectively, unless indicated otherwise. p-del, postdelivery.
bThe average normal CD4?cell count for an adult is 1,000 cells per mm3. For children under the age of 11 months, the normal CD4?cell count is between 1,700
and 2,880 cells per mm3(mean, 2,200) (49). For children between 1 and 6 years of age, the average CD4?cell count is between 1,000 and 1,800 cells per mm3.
cTCID50, 50% tissue culture infective dose.
dND, not determined.
VOL. 69, 1995 SEQUENCE VARIATION IN HIV MOTHER-TO-CHILD TRANSMISSION3779
cloned HIV sequences. DNA purification was performed separately for each of
the pairs, and as a control, DNA was also extracted from uninfected PBMCs at
the same time. After separation on Ficoll-Hypaque (Pharmacia), cells were
washed twice in RPMI-1640 (Whittaker) and lysed for 2 h at 56?C in 10 mM Tris
(pH 8.3)–50 mM KCl–2.5 mM MgCl2–0.45% Nonidet P-40–0.45% Tween 20–80
mg of proteinase K per ml. After lysis, proteinase K was inactivated by heating
the mixture for 10 min at 95?C.
PCR amplification, cloning, and sequencing of the V3 region. The region
encoding the V3 loop and flanking sequences (positions 6615 to 6928 in the
HIV-LAI genome ) was amplified in a nested PCR (32). One microgram of
total cellular DNA was amplified in a 100-?l reaction mixture containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM each deoxynucleoside
triphosphate, 0.4 ?g of each primer, and 1.5 U of Taq DNA polymerase (Perkin-
Elmer Cetus) and overlaid with 25 ?l of mineral oil. The first amplification step
was performed for 25 cycles with the outer primers E1, 5?-TACAATGTACA
CATGGAATT-3?, and E2, 5?-TTACAGTAGAAAAATTCCCC-3? (positions
6551 to 6570 and 6955 to 6974, respectively, in the HIV-LAI genome ). Ten
microliters of the first amplification product was then used as the template in a
second amplification step performed for 30 cycles with primers E3, 5?-GTATCG
GAATTCCTGCTGTTGAATGGC-3? (positions 6592 to 6618), and E4, 5?-TT
AGCAAGCTTCTGGGTCCCCTCCGAGGA-3? (positions 6907 to 6935), con-
taining EcoRI and HindIII restriction sites, respectively (underlined nucleotides).
Primers E3 and E4 have been previously described (35), and all four primers
show a high degree of identity with published reference sequences. Thermal
cycling was performed by using a Perkin-Elmer Cetus 9600 thermal cycler with
denaturing at 94?C for 1 min, annealing at 55?C for 1 min, and extension at 72?C
for 1 min. This was followed by a final extension step for 2 min at 72?C. Ten
percent of the secondary PCR product (313 bp) was analyzed by electrophoresis
on a 2% agarose gel, with the expected band being visualized following ethidium
bromide staining. Amplified products were digested with EcoRI and HindIII
restriction enzymes, purified by using the Glassmax purification system (Life
Technologies, Bethesda Research Laboratories), and cloned in EcoRI-HindIII-
digested M13 vector. The ligated vector was used to transform DH5?F? compe-
tent cells. One to 17 individual positive M13 clones were sequenced for each
sample by the dideoxy chain termination method (41) using [?-35S]dATP (T7
sequencing kit; Pharmacia). PCR amplification, cloning, and sequencing were
performed separately for each pair in the following order: pair A, pair B, pair D,
and pair C. Appropriate negative controls and DNA from uninfected cells were
included in each reaction mixture. In addition, all negative controls from the first
round of amplification were included in the second amplification step.
Sequence analysis. The nucleotide sequences from each mother-child pair
were aligned by using the CLUSTAL V algorithm (20) as implemented in version
2.2 of the Genetic Data Environment package (kindly provided by the Harvard
Genome Laboratory). The final alignment was improved manually by preferring
gaps to transition differences and transition differences to transversion differ-
ences and by the insertion of gaps to maintain the reading frame. Translation of
nucleotide sequences to amino acids was also undertaken with this package.
Distance-based phylogenetic analyses were performed with programs taken from
version 3.52c of the Phylogeny Inference Package (PHYLIP ). Nucleotide
sequence distances for all pairwise sequence comparisons were estimated by
using the generalized two-parameter (maximum-likelihood) model which uses
the transition probability formulas of Kishino and Hasegawa (25), incorporating
unequal rates of transition and transversion and allowing for different frequen-
cies of the four nucleotides (program DNADIST). Phylogenies were recon-
structed by both the neighbor-joining method (40) (program NEIGHBOR) and
the Fitch-Margoliash (16) (program FITCH) distance method. Phylogenies were
also reconstructed by the maximum likelihood method (13) using the modified
PHYLIP program FASTDNAML (kindly provided by Gary Olsen of the Uni-
versity of Illinois at Urbana-Champain and the Ribosomal Database Project)
(data not shown). Settings for the transition/transversion ratio were estimated
from each data set. Bootstrap resampling (14) was employed on the neighbor-
joining trees (programs SEQBOOT and CONSENSE) to assign approximate
confidence limits to the branches. Two thousand bootstrap replications were
performed. Alternative phylogenetic hypotheses were evaluated statistically by
the Kishino-Hasegawa-Templeton likelihood ratio test (25), following the assign-
ment of log likelihoods (program DNAML) to artificially generated topologies
(program RETREE) (unpublished data).
Nucleotide sequence accession numbers. Nucleotide sequences reported in
this study have been assigned the GenBank accession numbers U24717 to
U24999 and U25001 to U25025.
Clinical statuses of patients. (i) Mothers. Mothers A, B, and
D were of European origin and were formerly intravenous
drug users. Mother C was of African origin (Angolan), had
lived in Zaire, and is most likely to have been infected hetero-
sexually. Nevertheless, since the HIV status of her partner was
unknown, a risk factor could not reliably be identified for this
woman. The clinical status of the mothers and children, the
times of sampling, levels of cell and plasma viremia, and im-
munological data are presented in Table 1. At the commence-
ment of pregnancy, mothers A, B, and D had been infected for
at least 25 months, 5 years, and 7 months, respectively. Diag-
nosis of infection was performed at 5 months of pregnancy for
mother C. All mothers were asymptomatic at the beginning of
pregnancy (Centers for Disease Control and Prevention
[CDC] stages II and III) (6), although mothers B and D be-
came symptomatic (oral hairy leukoplakia and multidermal
herpes zoster, respectively) during late pregnancy, at which
point they were reclassified as CDC stage IVc2. The CD4?cell
counts among the mothers varied between 276/mm3and 858/
mm3with no significant variation in the mothers during preg-
nancy (Table 1). However, a significant decrease in the CD4?
cell count was observed to occur in mother D 1 year after
delivery (32 CD4?cells per mm3), although this was not ac-
companied by further modification in clinical status (data not
shown). Mothers A, B, and D were negative for plasma p24
antigen throughout pregnancy, but mother C was positive at all
time points (128, 8, and 6 pg/ml for time points MC1 (time
point 1 for mother C), MC2, and MC3, respectively). Cell
viremia was observed throughout pregnancy in all four moth-
ers studied (Table 1). In contrast, plasma viremia was not
present in mothers B and D and was only transiently observed
at low levels in mothers A and C (Table 1). In all cases,
pregnancies were free of complications and all children were
delivered vaginally. The mothers did not receive any antiret-
roviral therapy before or during pregnancy.
(ii) Newborns. Children A, B, and C were diagnosed as HIV
positive (PCR and coculture positive) during the first 2 weeks
of life (at 6, 5, and 12 days of age, respectively). Child D was
negative by PCR and coculture at birth but became HIV pos-
itive at 1 month of age. All children were originally classified as
CDC stage P2A (7). Children B, C, and D remained asymp-
tomatic for the duration of the study (24 months) (Table 1). In
contrast, child A displayed neurological signs at 1 month of age
and was reclassified as CDC stage P2B at 3 months (Table 1).
This child developed AIDS within the first year of life, with
evidence of cryptosporidiosis and multidermal herpes zoster
and was reclassified as CDC stage P2D at 16 months of age. All
children received zidovudine treatment (50 to 70 mg three
times a day). Children A, B, and C were treated with zidovu-
dine from the age of 7 weeks, whereas child D received anti-
retroviral therapy from the age of 14 weeks.
Sequence diversity. We have analyzed genetic variation in
226 complete sequences spanning the V3 region and flanked 5?
and 3? by 102 and 114 bp, respectively. Four incomplete se-
quences lacking only a few bases at the 5? and/or 3? end which
did not lack any variable region were also included.
The within-sample genetic diversity and between-sample ge-
netic distance were calculated for each pairwise comparison
between sequences from the four mother-child pairs (Fig. 1
and 2). To compare the diversity between the mothers’ samples
and those of the infants, we have plotted each value as a
histogram (Fig. 1). The overall means for all sequences ob-
tained from each patient (Fig. 1e) lay between 3.5 and 5.2% for
the mothers’ samples, with the between-mother distances (5.3
to 8.2%; mean, 7.2%) (Fig. 2e) being greater, as expected. The
within-child diversity had a greater range (0.74 to 8.8%) (Fig.
1e), and the between-child distances were mostly greater (7.6
to 10.8%; mean, 8.8%) than for the mothers (Fig. 2f). Com-
paring the overall within-patient diversity for the mothers with
that of their infants (Fig. 1) revealed two pairs (pairs B and D)
where the infants’ samples were substantially less diverse than
their mothers’ (Fig. 1b and d), one where they were similar
(Fig. 1a, pair A) and one (Fig. 1c, pair C) where substantially
greater diversity was found in the child. The within-sample
3780BRIANT ET AL.J. VIROL.
genetic distances showed considerable variation within each
patient. For three of the four pairs, the first child sample
showed less diversity than the mother’s sample closest to de-
livery (Fig. 1, pairs A, B, and D). However, exactly the opposite
was true for pair C (Fig. 1c). In the mothers’ samples, a pro-
gressive increase of genetic distance with time was found to
occur in pair A (Fig. 2a; entries off-diagonal), with the distance
from sample 1 to sample 6 (time interval, 7.5 months) almost
twice that from sample 1 to sample 2 (interval, 1 month).
Again, no such trend was observed with the other samples (Fig.
2b through d). Examination of individual values showed that
divergence between samples from the same patient can reach
levels similar to those found when comparisons between dif-
ferent patients are made. Clearly, a more detailed form of
analysis is required to identify any evolutionary pattern that
may exist in these data.
Phylogenetic analyses. The sequences of the four mother-
child pairs were classified according to HIV-1 global subtype
classification by phylogenetic comparison with reference se-
quences from the five designated env subtypes (33). The se-
quences of all four mother-child pairs (including sequences
from mother C, who originated from Africa) were of subtype B
(European or North American) (data not shown). The rarity
of a B clade virus in the African-derived isolates makes it more
likely that the infection of mother C was acquired in Europe.
(i) Mother-child pair A. Analysis of V3 sequences from the
first transmission pair (pair A) led to the generation of a ‘‘star’’
phylogeny in which the different lineages radiated from a single
point. The neighbor-joining tree reconstructed from the data
set is presented in Fig. 3 (pair A). Similar topologies were
obtained by each of the three methods of tree construction
employed, as well as with the tree reconstructed on the basis of
the amino acid sequence data (data not shown). Internal
branches within the phylogeny were short, and bootstrap val-
ues across the tree were very low. As a consequence, there was
little statistical support for any specific grouping within the
tree, although three groups of child sequences were apparent.
The first group (Fig. 3, pair A, child group 1) appeared to
consist predominantly of early-time-point sequences (1 and 2.5
months of age), although sequences from the child’s 16-month
sample were also associated with the group. There appeared to
be some association of these sequences with a number of
second- and third-trimester sequences from the mother (4.5, 6,
and 7 months). The second child group (Fig. 3, pair A, child
group 2) consisted of three 2.5-month sequences which clus-
tered within the main group of maternal sequences detected
during pregnancy (3.5 and 4.5 months) but appeared to be
most closely related to a small number of maternal sequences
from the third trimester. The third group (Fig. 3, pair A, child
group 3) consisted predominantly of late-time-point sequences
(16 months) which appeared to be associated with sequences
from the 2-month-postdelivery sample of the mother.
(ii) Mother-child pair B. For the second transmission pair
(pair B), a clear division of all sequences into two distinct
groups was apparent from the neighbor-joining tree (Fig. 3,
pair B). All three tree-building methods gave similar trees
from the nucleotide sequences, and the division into two dis-
tinct groups was also clearly apparent from the amino acid
neighbor-joining tree (data not shown). The branch separating
the two groups was resolved in 100% of bootstrap replicates,
and child sequences were associated with both groups. The first
sequence group (Fig. 3, pair B, group 1) formed the predom-
inant lineage present within the maternal population through-
out pregnancy and included sequences from all time points
from 3.5 months into pregnancy until delivery. Only three child
sequences, isolated from the child at the age of 5 weeks, were
associated with this group. The majority of the child sequences
were associated with the second group (Fig. 3, pair B, group 2),
with sequence isolates from all child time points (5 days, 5
weeks, and 3.5 months). The maternal sequences associated
with this group were mainly from the 3-month-postdelivery
sample, whose sequences formed a tight cluster, although the
variant was present as a minor form throughout pregnancy. It
was interesting that the group 1 maternal variant which was
predominant at delivery had been replaced with the group 2
sequence type by 3 months postdelivery.
(iii) Mother-child pair C. Two child groups were also clearly
apparent in the neighbor-joining tree for the third transmission
pair (Fig. 3, pair C). The main child group (Fig. 3, pair C, child
group 1), consisting solely of sequences found at 1.5 and 2.5
months after birth, appeared to be descended from a lineage
present within the mother during the second trimester (5- and
6.5-month maternal samples) (mother group 1). The lineage
was represented by 4 of 7 sequences from the 5-month mater-
nal sample and in only 1 of 11 sequences from the 6.5-month
maternal sample. It was not represented in the 7.5-month
sample. A single child sequence from the 13-month sample was
also weakly clustered with this group. The level of bootstrap
support for the cluster incorporating the main group of child
and ancestral mother sequences was reasonably high, with the
cluster supported in 87.4% of replicates. Except for one se-
quence (designated by an arrow in Fig. 3, pair C), the cluster
was found in 96.1% of bootstrap replicates. The second minor
child group (Fig. 3, pair C, child group 2) consisted of four (of
FIG. 1. Within-sample and between-sample nucleotide sequence diversity. (a
to d) Within-sample diversity in mothers (dark grey columns) and children (light
grey columns). (e) Mean within-patient diversity. The number of sequences for
each time point is indicated at the top of each column.
VOL. 69, 1995 SEQUENCE VARIATION IN HIV MOTHER-TO-CHILD TRANSMISSION3781
five) late-time-point sequences (13 months of age). The group
was located within the main cluster of the maternal sequences
(Fig. 3, pair C, mother group 2), closest to those found late in
pregnancy (7.5 months), although only a low level of bootstrap
support for this was indicated. The overall level of bootstrap
support for the main mother group itself was high (96.1%,
excluding intermediate sequences). The phylogenies recon-
structed by alternative methods for mother-child pair C were
again consistent (data not shown).
(iv) Mother-child pair D. For pair D, the three methods of
phylogeny reconstruction employed all identified a single child
group (Fig. 3, pair D). The sequences from both the 1.5- and
11.5-month infant samples were clustered fairly tightly within
the phylogeny and showed an association with a number of
maternal sequences from the 8.5- and 4.5-month-postdelivery
samples. These sequences represented a minor form in both
maternal samples (2 of 9 sequences from the 8.5-month sample
and 2 of 10 sequences from the 4.5-month-postdelivery sam-
ple). Four maternal sequences, three from the 4.5-month-post-
delivery sample and one from the 2-month sample, were inter-
mediate between the mother-child group and the main
maternal cluster. The presence of these intermediate se-
quences reduced the bootstrap support, but if they were ex-
cluded the branch separating the main maternal group and the
mother-child group was found in 99.2% of bootstrap repli-
cates. Once again the phylogenies inferred by the three meth-
ods of phylogeny reconstruction employed were highly congru-
ent, and again the amino acid neighbor-joining tree was very
similar to that reconstructed on the basis of nucleotide se-
quence data (data not shown). Testing the relative likelihoods
of these phylogenetic hypotheses (23) confirmed the results of
the bootstrap analyses.
Amino acid sequence heterogeneity between mothers and
children. The amino acid sequence alignments for the four
mother-child pairs are presented in Fig. 4. For each pair, a
consensus sequence was constructed for each time point by
assigning the amino acid most frequently observed in the
clones to each position. When the phylogenetic analysis re-
vealed more than one group within a time point, sequences
from that time point were divided into clusters of related
FIG. 2. Nucleotide distances in the V3 loop and flanking regions of HIV-1 among mother-child transmission pairs. Nucleotide distances were estimated for each
pairwise sequence comparison by using the generalized two-parameter model (15). (a to d) Nucleotide distances between mother and child time point sequences. (e)
Mean nucleotide distances between all maternal sequences. (f) Mean nucleotide distances between all child sequences.
3782 BRIANT ET AL.J. VIROL.
clones. All consensus sequences were aligned in relation to the
first time point consensus of the mother. The phylogenetic
group to which each consensus sequence corresponds is re-
ported at the right edge of Fig. 4.
For pair A, the three groups of child sequences found in the
phylogenetic tree (Fig. 3, pair A) were associated with amino
acid substitutions within both V3 and the flanking regions
(positions 308, 320, 335, 339, 343, 346, and 360). Comparison
of amino acid sequences of the mother with those of the child
revealed a greater similarity between child group 1 sequences
and maternal sequences detected late in pregnancy (Fig. 4a,
MA4.C). Child group 2 sequences (Fig. 4a, CA2.C2) shared
amino acid variants with maternal sequences from various time
points (positions 320, 339, 346, and 360); however, no common
amino acid sequence pattern was apparent between child
group 3 sequences and the maternal sequence set.
For pair B, two distinct groups of sequences which corre-
spond to the phylogenetic groupings observed in the neighbor-
joining tree (Fig. 3, pair B) were apparent, on the basis of their
amino acid sequences (Fig. 4b). The two groups were charac-
terized by the following amino acid sequence patterns: group 1,
T-283, S-291, S-300, R-305, S-306, T-308, T-317, K-342, V-345,
and T-360; group 2, S-283, T-291, N-300, K-305, G-306, H-308,
A-317, R-342, A-345, and N-360.
For pair C, the two subgroups of child sequences (Fig. 3, pair
C) were characterized by distinct amino acid sequence patterns
(Fig. 4c). The major amino acid differences between the two
subgroups occurred at positions 289 (N or K) and 308 (D or S).
Child group 2 sequences were mostly characterized by the
presence of a basic lysine residue (K) at position 313. This
modified the GPGR motif at the crown of the V3 loop to
GPGK. A high degree of similarity was observed between
mother group 2 and child group 2 sequences.
For pair D, the child sequences, which clustered in a single
group within the phylogenetic tree (Fig. 3, pair D), were char-
acterized by the following amino acid sequence pattern: K-293,
S-306, K-313, A-317, D-320, D-324, K-341, N-346, G-349,
K-360 (Fig. 4d). A small number of maternal sequences
present as a minor form in the late-pregnancy–postdelivery
maternal samples shared this amino acid sequence pattern
(Fig. 4d, MD3.C2 and MD4.C2). The lysine residue observed
at position 313, which gave rise to the GPGK motif at the
crown of the V3 loop, was particularly characteristic of the
group. This was in contrast to the GPGR motif observed in the
majority of maternal sequences, which were also more heter-
ogeneous than those of the child. The amino acid sequences of
the main maternal group (Fig. 4d, group 2) diverged from
those of the child by many amino acid substitutions (positions
306, 313, 317, 320, 349, and 360).
(i) Pattern of potential N-linked glycosylation between
FIG. 3. Unrooted neighbor-joining trees for the four mother-child transmission pairs. Symbols at the tip of each branch denote the time point to which the sequence
belongs. Open symbols represent one individual child sequence, and shaded symbols represent one individual maternal sequence. All branch lengths are drawn to scale.
Bootstrap values are expressed as percentages for each branch and represent the percent occurrence of that branch per 2,000 bootstrap replicates. Symbols for pair
A: I, MA1; F, MA2; ?, MA3; å, MA4; O, MA5; !, MA6; ", CA1; H, CA2; J, CA3. Symbols for pair B: I, MB1; F, MB2; ?, MB3; å, MB4; !, MB5; ", CB1; H,
CB2; J, CB3. Symbols for pair C: I, MC1; F, MC2; ?, MC3; ", CC1; H, CC2; J, CC3. Symbols for pair D: I, MD1; F, MD2; å, MD3; !, MD4; ", CD1; H, CD2.
VOL. 69, 1995 SEQUENCE VARIATION IN HIV MOTHER-TO-CHILD TRANSMISSION3783
3784 BRIANT ET AL.J. VIROL.
mothers and children. Comparison of mother and child amino
acid sequences revealed common patterns of potential
N-linked glycosylation in the four mother-child pairs (Fig. 4).
Six potential glycosylation sites, located at positions 276, 295,
301, 331, 338, and 354, were perfectly conserved in all groups
of sequences from pairs A, B, and D. However, in pair C, the
potential glycosylation site at position 354 was absent in a small
number of child sequences (Fig. 4c, CC1.C). Two additional
sites at positions 289 and 360 differed between the mother and
child. The sites appeared only in child late-time-point se-
quences in pair A (Fig. 4a, CA3.C). For pair B, the sites were
present in the majority of mother and child amino acid se-
quences associated with phylogenetic group 2 (Fig. 4b), the
main group present within the child. By contrast, the sites were
absent in mother and child sequences associated with group 1
(Fig. 4b). For pair C, the potential N-linked glycosylation site
at position 289, present in the main maternal group, child
group 2, and child subgroup 1a sequences, was absent in se-
quences of maternal group 1 and child subgroup 1b. The site at
position 360 was detected in the majority of mother and child
sequences, with the exception of child subgroup 1a sequences
(Fig. 4c). For pair D, the potential N-linked glycosylation site
at position 360, which was absent in the child, was present in all
maternal sequences with the exception of sequences MD3.C2
and MD4.C2 (Fig. 4d). These sequences were most closely
associated with those of the child. The site at position 289 was
absent in both the mother and child.
(ii) Potential phenotype of transmitted viral species. The
potential phenotype of the amino acid sequence variants was
predicted on the basis of the global net charge of the V3 loop
and the degree of sequence divergence from the La Rosa
subtype B consensus (11, 30). The majority of sequences from
the mother-child pairs were predicted to be of the macro-
phage-tropic, non-syncytium-inducing (NSI) phenotype. How-
ever, a small number of sequences predicted to be of the
T-cell-tropic, syncytium-inducing (SI) phenotype were ob-
served to be present in pairs A and B. Five potential SI variants
were observed in pair A, and these consisted of a single early-
pregnancy maternal sequence (time point MA1) and four child
sequences (time points CA1, CA2, and CA3). For pair B, four
potential SI variants were observed to occur in the mother
(time points MB1, MB2 and MB4) and a single predicted SI
variant was observed at the first time point in the child (time
In order to examine the molecular mechanisms involved in
HIV-1 mother-to-child transmission, we have analyzed the ge-
netic relationships among cell-associated viral populations de-
tected during pregnancy in four HIV-infected mothers and in
their respective children. This analysis was performed with a
313-bp fragment containing the V3 region and the highly in-
formative flanking sequences. We have compared proviral
DNA sequences obtained from the mothers at different time
points during pregnancy and postdelivery with child sequences
obtained from birth.
Genetic diversity and possible multiple transmission. Se-
quence data obtained from the V3 region for two of the four
children sampled in this study (pairs A and C) revealed sub-
stantial levels of genetic heterogeneity in their cell-associated
viral populations. Such heterogeneity has not been a promi-
nent feature of earlier studies of mother-child transmission
(31, 42, 50). Child B also showed significant viral heterogeneity
when all samples from the child were included. Only child D
showed substantially less diversity than found in its mother.
The occurrence of such heterogeneity in the newly infected
infants is quite unlike the situation in newly infected adults,
several studies having shown very restricted levels of variation
in the env gene in the peripheral blood of hemophiliacs and
patients infected by sexual contact (4, 22, 45, 51). This has been
interpreted as evidence for selection for particular viral vari-
ants from the heterogeneous pool present in most individuals
later in infection. The striking difference between the situation
described for these children and that observed for adults sug-
gests that mother-child transmission may be a more complex
Phylogenetic analysis of the V3 sequence data has shed
more light on the circumstances of transmission for each of the
four mother-child pairs. For pair A, three groups of viral se-
quences within the child were identified, but as they were only
weakly defined it was not possible to draw any specific conclu-
sions regarding transmission. Nevertheless, the heterogeneity
of the viral populations of both the mother and the child does
not support the view that the infection of the child involved a
single viral variant. It is interesting that the mother showed a
high level of cell viremia throughout pregnancy (Table 1),
which could have facilitated the transmission of multiple vari-
ants to the child.
For pair B, both the statistical analysis of the reconstructed
phylogeny and the amino acid sequence alignments clearly
indicated the occurrence of two very distinct populations
within both the mother and the child. The simultaneous pres-
ence of both populations within the 1.5-month sample of the
child, which was responsible for the particularly high within-
sample diversity for this time point, clearly indicated that the
infection within the child was the result of the transmission of
two distinct maternal variants. We cannot infer from the data
whether the two variants were transmitted simultaneously or at
different times, as both maternal sequences with which the two
child groups were associated were present throughout preg-
The analysis of pair C also revealed an unusually high level
of genetic diversity within the child sequences obtained within
the first year of life (8 to 10%). Two statistically significant
groups of child sequences were apparent from the phylogenetic
tree, and specific maternal sequences were clearly associated
with each group. The major child lineage was associated with
maternal sequences from 5 and 6.5 months. Other child se-
quences were more closely related to maternal sequences ob-
tained later. PCR and culture diagnosis for child C, performed
at 12 days postdelivery, provided further evidence for early
The results of the phylogenetic analysis are compatible ei-
ther with early infection with multiple variants or with trans-
mission of virus on more than one occasion, possibly during
FIG. 4. Amino acid sequence alignments. Consensus sequences were deduced for each phylogenetic group present within a time point and aligned in relation to
the first time point consensus of the mother. The number of individual sequences within a consensus and the phylogenetic group to which each sequence corresponds
are shown at the right. Consensus sequences are presented in uppercase, whereas individual clone sequences are given in lowercase. Amino acids are numbered
according to their position in the HIV-LAI genome (33). Potential N-linked glycosylation sites conserved between mother and child sequences are indicated by shaded
boxes, and variable N-linked glycosylation sites are underlined. A ‘‘•’’ indicates the deletion of a codon, ‘‘n’’ indicates the number of clones represented by each
consensus, and ‘‘?’’ indicates that no consensus amino acid residue could be defined at a given position.
VOL. 69, 1995 SEQUENCE VARIATION IN HIV MOTHER-TO-CHILD TRANSMISSION3785
pregnancy and at delivery. Although transmission of more than
one variant between adults appears to be rare, we note that
there is evidence that it occurred for one patient of a Florida
dentist (27, 36) and also for a victim of rape (1).
Evidence for selective mother-to-child transmission. In con-
trast to the situation for the other three cases we have studied,
the cell-associated viral sequences detected in child D were
highly homogeneous at all time points. These sequences clus-
tered within a single group in the phylogenetic tree and were
closely related to a small number of maternal sequences de-
tected during late pregnancy and following delivery. This sug-
gests that the infection within this child was the result of the
transmission of a single maternal variant. The fact that these
maternal genotypes were found only in late pregnancy and
after delivery is consistent with a later infection, possibly at
delivery. This hypothesis is supported by the fact that HIV
infection within child D could not be detected at birth but was
first diagnosed at 1 month of age (Table 1). It is interesting that
the sequences observed to be present in this child were highly
homogeneous and remained closely associated with sequences
detected at the time of diagnosis for at least a year after birth,
whereas samples obtained from infected children in the other
transmission pairs at approximately 1 year of age were more
heterogeneous. These results therefore support the hypothesis
of selective transmission of a minor maternal variant in pair D,
as indicated in other mother-child transmission studies (31, 42,
Implications of V3 amino acid sequence variation in trans-
mission. For the three mother-child pairs in which multiple
variants appeared to be transmitted to the child (pairs A, B,
and C), comparison of amino acid sequences from the mothers
with sequences from their respective children did not identify
any overall pattern distinguishing between transmitted and
nontransmitted viral species. We did not observe the selective
loss of any glycosylation site within the V3 region in variants
transmitted to the children. In particular, the potential N-
linked glycosylation site proximal to the first cysteine of the V3
loop (position 295), absent in the infant sequence sets de-
scribed by Wolinsky et al. (50), usually remained conserved
between mother and child sequences in our study. This obser-
vation has also been reported by other investigators (31, 42).
Glycosylation site differences between mother and child were,
however, observed to occur in the V3 flanking regions at po-
sitions 289 and 360. The GPGR motif at the crown of the V3
loop showed a high degree of conservation within the maternal
and child sequences of pairs A, B, and C, probably because of
the functional importance of the region. However, a conver-
sion to GPGK was observed to occur in child C at 1 year of age.
In contrast, in pair D, where selective transmission of a
minor maternal variant was observed, specific amino acid se-
quence variations were identified between the mother and
child sequences. The N-linked glycosylation site at position
360, present in the majority of maternal sequences, was absent
from the sequences of the child. Moreover, the GPGR motif
which was present in most of the mother’s sequences was
converted to GPGK in all child sequences. These amino acid
sequence variants were also observed to be present in the
minor subset of maternal sequences highly related to the trans-
mitted subtype. Genetic variations within V3 have been found
to influence host immune responses to antibody (34, 52) and
antibody titers, as well as affinities for epitopes within V3.
These factors have been implicated as important in HIV-1
perinatal infection (10, 37). The mutations observed to occur
between the mother and child of pair D may suggest that the
transmitted variant had been subjected to immune selection
within the mother, perhaps conferring a selective advantage for
transmission. The observed sequence variations in pair D do
not themselves, however, localize the determinants accounting
for the immune escape of the virus. Other biological tests,
particularly assays based on analysis of maternal neutralizing
antibodies and maternal cellular immunity, would be required
to confirm the immune escape hypothesis.
The V3 region has also been shown to influence the ability
of the virus to replicate in macrophages and to grow in trans-
formed T-cell lines (7, 17, 24, 43). Several articles have re-
ported that the emergence of basic amino acids at specific
positions within V3 is associated with the phenotypic shift of
the virus from the NSI, macrophage-tropic form to the SI,
T-cell-line-tropic form (7, 9, 11, 30). Amino acid sequences
characteristic of the macrophage-tropic NSI phenotype pre-
dominated in the mother-child sequence sets. A few individual
maternal and child sequences in pairs A and B exhibited basic
amino acids at critical positions for tropism, potentially con-
ferring a T-cell-tropic, SI phenotype. Macrophage-tropic vi-
ruses have been shown to be transmitted sexually and to be
responsible for establishing chronic infection (51).
It is interesting that child A rapidly showed neurological
symptoms and developed AIDS within 24 months. A small
number of sequences with a potential SI phenotype were de-
tected in this child at 1 month of age and remained when the
child was 16 months old. Whether these variants were trans-
mitted from the mother or whether they emerged from se-
quence variations of transmitted viral species within the child
could not be determined. Overall, no association between HIV
sequence diversity and disease evolution has been established.
However, the rapid progression to AIDS in child A might be
associated with the presence of T-cell-tropic, SI variants soon
after birth, as has been reported for adult infections (26, 47,
48). This observation is in good agreement with the rapid
decline in CD4?T-cell count and progression to AIDS re-
ported previously to occur in patients showing a phenotypic
shift of the virus from the NSI phenotype to SI.
In conclusion, our analysis has shown that the genetical
processes involved in vertical HIV-1 transmission may be more
complex than those found to occur in transmission between
adults. In one case from our study, the selective transmission of
a single maternal variant appeared to have occurred. However,
in two other pairs, infection of the child by at least two mater-
nal variants was demonstrated. These multiple transmissions
may have occurred at different times during pregnancy. Anal-
ysis of mother-to-child transmission of HIV infection in rela-
tion to maternal HIV antibodies as well as comparison of
biological properties of variants transmitted and not transmit-
ted to the child could lead to a better understanding of viral
determinants involved in HIV-1 vertical transmission.
Special thanks are given to J. Tricoire from the division of Pediatrics
at CHU Purpan and A. Berrebi from the division of Gynecology-
Obstetrics at La Grave Hospital, who provided maternal and child
blood samples. We also acknowledge the help provided by Denis
Lobidel at the Centre for HIV Research, Edinburgh, United Kingdom.
We also thank J. F. Magnaval at CHU Purpan for helpful assistance.
L.B. is a fellow of the Agence Nationale de Recherches sur le SIDA.
This work was supported by the Agence Nationale de Recherches sur
le SIDA, the Medical Research Council AIDS Directed Programme,
and the Conseil Re ´gional de Midi-Pyre ´ne ´es.
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