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J Virol, July 1998, p. 5831-5839, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetically Related Human Immunodeficiency Virus
Type 1 in Three Adults of a Family with No Identified Risk Factor
for Intrafamilial Transmission
Laurent
Bélec,1
Ali Si
Mohamed,1
Michaela C.
Müller-Trutwin,2
Jacques
Gilquin,3
Laurent
Gutmann,1
Michel
Safar,4
Françoise
Barré-Sinoussi,2 and
Michel D.
Kazatchkine3,*
Laboratoire de
Virologie,1
INSERM U430 and Service
d'Immunologie,3 and
Service de
Médecine Interne,4 Hôpital
Broussais, and
Laboratoire de Biologie des Rétrovirus,
Institut Pasteur,2 Paris, France
Received 23 June 1997/Accepted 24 March 1998
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ABSTRACT |
A small number of cases of human immunodeficiency virus (HIV)
infection have been reported in individuals with no identified risk
factors for transmission. We report on the seroconversion of the
61-year-old mother and the subsequent finding of HIV seropositivity in
the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino
acid signature patterns of the V3, V4, and V5 hypervariable domains and
flanking regions in the HIV-1 gp120 env gene of 26 clones
derived from proviral DNA in peripheral blood mononuclear cells of the
members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the
patients were genetically related and formed an intrafamilial cluster
of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between
1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of
viral clones from the three patients were closely related. In the C2-V3
region, two minor clones derived from the son's virus showed less
nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived
from the viruses of both parents or the seven other predominant clones
derived from the virus from the son (mean, 5.4%). The top of the V3
loop of the last two clones and of all viral clones from the parents
exhibited an unusual GPGG sequence. This is the first report of
genotypic relatedness of HIV-1 in three adults of the same family in
the absence of identified risk factor for transmission between the
members of the family. Our findings suggest that atypical transmission
of HIV may occur.
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INTRODUCTION |
A small number of cases of human
immunodeficiency virus (HIV) infection have been reported in adults and
children with no identified risk factors for transmission or an
unproven mode of transmission despite thorough investigation (5,
6, 14, 34, 45). Two reports of such cases have included detailed genetic analyses of variable env sequences of the viral
envelope in five patients of a dentist infected with HIV-1
(34) and in a child possibly contaminated through
unrecognized exposure to blood by an HIV-1-infected child living
in the same home (14). Although epidemiologic evidence
argues against casual transmission of HIV (4, 12, 17, 19,
42), these few cases raise the possibility that atypical
transmission by means other than those usually evidenced does occur.
We report on a cluster of HIV-1 infection in three adults of the same
family whom we found to be infected with genetically related viruses
and in whom no risk factor for intrafamilial viral transmission was
identified.
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CASE REPORTS |
Patient 1 was a 31-year-old man identified to be seropositive for
HIV-1 in December 1994. The patient had used intravenous drugs between
1984 and 1989 and claimed to have been free of drugs since then. The
patient lived in his parents' home. He was admitted to our hospital on
4 December 1994 because of high fever, productive cough, hemoptysis,
and respiratory distress. The chest X ray and thoracic computed
tomogram showed extensive bilateral bullous lesions and a voluminous
cavity in the apex of the right lung. Pneumocystis carinii
pneumonia was diagnosed upon analysis of the bronchoalveolar lavage
fluid. A diagnosis of tuberculosis was also suspected, although
Ziehl's staining of bronchoalveolar lavage fluid gave negative
results. The patient was treated simultaneously with high-dose
trimethoprim-sulfamethoxazole and a quadruple combination of
antituberculous drugs. Cultures and PCR for Mycobacterium
tuberculosis remained negative. At the time of admission, the CD4
T-cell count was 106/liter and the HIV RNA level in plasma
was 209,700 equivalent (Eq) copies/ml as assessed retrospectively
(Chiron, Emeryville, Calif.). Analysis of the pol 215 codon of proviral
DNA from the patient's peripheral blood mononuclear cells (PBMC) by
selective PCR (26) revealed a mixed sensitive and resistant
genotype to zidovudine with a predominance of sensitive variants. In
the next 16 months, the patient developed a disseminated form of
Kaposi's sarcoma (KS). He died in April 1996.
Patient 2, the mother of patient 1, is a 61-year-old woman who had had
no medical history until December 1994, except for mild hypertension
and hypothyroidism. She was admitted to our hospital on 10 December
1994 with acute febrile meningitis. Cerebrospinal fluid analysis showed
30 lymphocytes/ mm3 and hyperalbuminorachia (1.34 g/liter). No bacteria, including M. tuberculosis and
Treponema pallidum, and no viruses (herpes simplex
virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus,
human herpesvirus 6) were found by PCR or isolated upon culture of
cerebrospinal fluid and blood samples from this patient. However,
because of her son's recent history, the patient was given
antituberculous drugs. Antimycobacterial therapy was discontinued after
18 weeks because PCR for M. tuberculosis and cultures for mycobacteria remained negative. A first HIV screening serologic test
performed on 12 December was negative. However, p24 antigenemia was
found to be positive (64 pg/ml). On 19 December, Western blot analysis
revealed an incomplete HIV-1 pattern, with the presence of bands at 24, 41, and 55 kDa. One week later, the Western blot showed a pattern
typical of HIV-1 infection. Therefore, we interpreted the
patient's meningitis as being associated with acute primary HIV
infection. Analysis of the pol 215 codon in the patient's PBMC
revealed a similar genotype for zidovudine resistance to that observed
in her son. Patient 2 recovered from her initial symptoms within 2 weeks. She received a combination of zidovudine and zalcitabine for 11 months; the therapy was then switched to a combination of zidovudine
and didanosine. In April 1995, the HIV RNA level in plasma was below
the threshold of detection (<10,000 Eq copies/ml) and the CD4 T-cell
count was 600 × 106/liter.
Patient 3, the father of patient 1 and the husband of patient 2, is a
66-year-old baker with a medical history of hypertension for the last
20 years. In April 1995, patient 3 volunteered for an HIV test, which
was found to be positive for HIV-1. The CD4 T-cell count was 220 × 106/liter. The patient had no history suggestive of
an acute retroviral syndrome. His plasma viral load at the time of
diagnosis of the HIV infection was 683,700 Eq copies/ml. Analysis of
the pol 215 codon revealed a similar genotype for zidovudine resistance
to that observed in patients 1 and 2. Patient 3 received a combination of zidovudine and zalcitabine for 13 months. He remains clinically asymptomatic with a stable CD4 T-cell count of 300 × 106/liter.
Epidemiological investigation.
Extensive discussions conducted
by three independent physicians, not involved in the patients' care,
with both parents of patient 1 did not reveal any risk factor for HIV
in patients 2 and 3, including sexual transmission, blood transfusion,
usage of intravenous drugs, and identified contact with the blood of patient 1. Patient 1 lived at home, except for the periods when the
disease required him to stay in hospital. While in hospital in the
terminal phase of his illness, patient 1 received frequent visits from
his parents. The patient died at hospital. The parents and their son
had separate rooms and did not sleep together. They did not
share toothbrushes or razors, but they did share eating utensils. The parents have not been involved at any time in the nursing
care of their son. They claimed not to have been in contact with blood
or other body fluids of their son, including stools, urine, vomitus,
saliva, and nasal secretions. They did not use gloves at home. Patient
1 had no other skin lesions besides KS lesions. There was no bleeding
associated with the KS lesions. There were no skin lesions on the hands
of patients 2 and 3. Patient 1 had a poor dental condition and
presented with oral lesions of KS. He did not present with nasal
bleeding. He had had a severe cough and hemoptysis, while at home,
prior to his first referral to hospital. Epidemiological investigation
also did not evidence how transmission had occurred between the
parents. Patient 2 works in her husband's bakery, which is located on
the first floor of their lodging. Patients 2 and 3 claimed not to have
had sexual intercourse for more than 5 years. Patient 3 had consulted
one of us in previous years for sexual dysfunction associated with 
-methyldopa therapy.
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MATERIALS AND METHODS |
Blood samples were obtained on 2 January 1995 from patient 2, on
29 March 1995 from patient 1, and on 1 June 1995 from patient 3. Plasma
and PBMC were frozen until use. We also analyzed PBMC obtained from all
three patients 1 to 3 months after the initial sampling and plasma from
two unrelated HIV-seropositive patients from the same ward, obtained at
the same time as the 2 January 1995 sample from patient 2.
PBMC (2 × 106) were incubated overnight in lysis
buffer containing proteinase K. DNA was extracted by the
phenol-chloroform method. Amplification of proviral
env DNA fragments in the hypervariable V3, V4, and V5 loops
and their flanking interspersed constant (C) regions was performed by
nested PCR with oligonucleotide primers derived from HIV-1 consensus
env sequences, as described previously (10). The
final amplified product of 667 bp was allowed to migrate on a
low-melting-point agarose gel at 6.0%. Relevant bands were eluted, and
DNA was extracted by the phenol-chloroform method. The amplified
products were inserted into the pMOS blue T-vector plasmid (Amersham
Life Science, Little Chalfont, United Kingdom). Clones carrying HIV-1
env sequences were identified by blue-white screening of
recombinants and confirmed by direct-colony env PCR.
The nucleotide sequences were determined with the T7 sequencing kit
from Amersham, using the dideoxynucleotide chain termination method.
DNA sequences were aligned with CLUSTAL software (22) and
corrected manually. Twenty-five env sequences of European isolates and 28 env sequences of local field isolates from
Paris (8) were used as nucleotide and amino acid
reference sequences for the HIV-1 subtype B C2-V3 region. The "local
control" sequences that were used originated from patients in
three hospitals in different areas of Paris from that where the family
lives. The distance between those hospitals and the area where the
family lives is less than 5 miles. We also used C2-V3 sequences
from the previous studies of atypical HIV transmission reported by Ou
et al. (GenBank accession numbers are as follows: M90847 for
the dentist [US1.Dent], M90854 for patient A [US1.PtA], and
M90964 [US1.LC9] and M90939 [US1.LC35] for two local controls) (34), and by Fitzgibbon et al. (GenBank
accession numbers are as follows: L12752 for child 1 [US12.CHA],
L19695 for child 2 [US12.CHB], and L12753 [US12.LC1] and
L12754 [US12.LC2] for two local controls) (14).
C2-V3 consensus sequences were generated from control European and
Parisian HIV-1 sequences. Nine env sequences from Europe
isolates were used as nucleotide and amino acid reference
sequences for the V4-C4-V5 region. We also used V4-C4-V5 sequences
from those previously published by Ou et al. (GenBank accession
numbers are as follows: M91084 for the dentist [US1.Dent], M91090 for
patient A [US1.PtA], and M91132 [US1.LC1] and M91133
[US1.LC2] for two local controls) (34). Sibling
sequences (sets of viral sequences from the same patients) were
not included in the reference sets. A V4-C4-V5 consensus sequence
was generated from the control European HIV-1 sequences.
The genetic distances between the HIV-1 env sequences from
the same patient and from those of one patient to those of another patient or set of reference sequences were defined as the average percentage of sequence divergence of all available pairs of C2-V3 and
V4-C4-V5 nucleotide sequences. Only single-nucleotide differences were
scored, and positions with gaps were excluded. The nonparametric U test
of Mann-Whitney was used to evaluate the significance of the
differences in the genetic variability between env
sequences. The patients' sequences were also compared with all
sequences accessible in the GenEMBL database by using the FASTA search
tool (35).
Phylogeny construction and evaluation were performed with the Phylip
software package (11), the neighbor-joining algorithm (36), the matrix distance Fitch and Margoliash method
(13), and the Fitch and Wagner maximum parsimony method.
The hypervariable env amino acid sequences deduced from the
patients' viruses were subjected to signature pattern analysis (14, 24, 34) with VESPA software (24). This
method permits us to define particular sites in sequences at which
residues are shared among certain groups of virus (24, 39).
The amino acid C2-V3 or V4-C4-V5 sequences of patients' viruses were
then scanned for amino acids that occurred at homologous positions in
less than 50% of the reference set, defined by all the previously
selected C2-V3 (n = 48) and V4-C4-V5 (n = 9) sequences of HIV-1 subtype B isolates from Europe and from Paris.
Nucleotide sequence accession numbers.
The nucleotide
sequences presented in this report have been deposited in the GenBank
database under accession no. U87171 to U87221.
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RESULTS |
Nucleotide env sequences were obtained from 26 clones
of PCR products amplified from the PBMC of the patients. After
truncating and gap-stripping, the sequences spanned 243 bp in the C2-V3
region, comprising the V3 loop flanked at the 5' and 3' ends by 87 and 51 bp, respectively; the sequences spanned 265 bp in the V4-C4-V5 region. All sequences belonged to the European/North American group M,
subtype B.
Mean intrapatient diversity ranged between 1.1 and 2.6% in the C2-V3
region and between 0.7 and 3.5% in the V4-C4-V5 region (Table
1). In the C2-V3 region, intrapatient
diversity was higher in patient 1 than in patients 2 and 3 (P < 0.0003). Genetic diversity in the V4-C4-V5 region
was higher in patient 1 than in patients 2 and 3 (P < 0.0004) and higher in patient 3 than in patient 2 (P < 0.0001). The mean nucleotide distances in C2-V3 between clones PA7 and
PA12, and all clones from viruses of patients 2 and 3 were 3.9 and
3.5%, respectively; the distances were significantly smaller than the
mean divergence calculated between clones PA1, PA4, PA11, PA14, PA15,
PA12, and all clones derived from viruses of patients 2 and 3 (5.4%;
P < 0.0001). Means of inter-patient diversity ranged
between 1.2% and 5.0% in the C2-V3 region, and between 2.2% and
7.5% in the V4-C4-V5 region (Table 1). In contrast, means of genetic
diversity between the patients' cloned sequences and control
sequences ranged between 16.8 and 17.2% for the C2-V3 regions
and between 13.8 and 14.7% for the V4-C4-V5 regions. Means of genetic
diversity in the C2-V3 region between patients' clones and local field
isolates ranged between 15.1 and 15.7%. The results suggest that the
viruses from patients 1 through 3 are genetically related. Moreover,
the genetic diversity in C2-V3 was higher between patients'
env sequences and European control sequences (mean, 17.0%; range, 10.0 to 29.3%) than between European
sequences (mean, 13.2%; range, 3.2 to 30.1%)
(P < 0.0001); similarly, genetic diversity in C2-V3
was higher between patients' env sequences and sequences from Parisian viruses than between the Parisian env
sequences themselves (P < 0.0001). Similar results
were obtained upon analysis of the V4-C4-V5 region (data not shown).
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TABLE 1.
Nucleotide diversity in the C2-V3 and V4-C4-V5 regions of
the gp120 env gene of clones derived from
patients' proviruses
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Upon comparison of the patients' data with the GenEMBL database by
using the FASTA search tool, the sequences of the patients' viruses
did not appear related to previously published HIV-1 sequences; the
less divergent sequences in C2-V3 (HIV-1 clone LC02; 10.6% divergence) as well as in V4-C4-V5 (HIV-1 clone RT1.21; 13.7% divergence) originated from North America.
The genetic relatedness of the patients' viruses was then confirmed by
the analysis of phylogenetic trees. The sequences in C2-V3 (Fig.
1) and V4-C4-V5 (Fig.
2) of patients' viruses clustered with
each other and diverged from control sequences of strains from Europe
and Paris, as well as from published sequences of viruses from patients
with atypical HIV transmission. The branch dividing the familial
cluster from the other groups of sequences was resolved in 100% of
bootstrap replicates in the C2-V3 domain and in 96% in the V4-C4-V5
domain, supporting the monophyletic grouping of the sequences of
viruses from patients 1 through 3.
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FIG. 1.
Neighbor-joining phylogenetic tree of the coding
sequences for the HIV-1 env C2-V3 region of 26 clones
derived from proviruses of patients 1, 2, and 3 (isolates PA, SI, and
RO, respectively), sequences of field isolates from Paris (PAR),
published sequences from patients with atypical HIV transmission and
from their local controls (US) (14, 34), and consensus
sequences obtained from 25 unrelated controls from Europe (Europe CONS)
(FR.LAI, IT.Sala1, IT.115, IT.193, UK.CPHL1, UK.CPHL2, UK.CPHL6,
UK.CPHL7, UK.V77, UK.V87, CH.K11, CH.K16, DE.HAN, DE.31, BE.SIMI84,
NL.H466, NL.B130, NL.127M, NL.114M, and NL.H36 [32],
including 5 sequences from Paris [PAR.FC10, PAR.FL2, PAR.FL6, PAR.FR9,
and PAR.FR29]), from 28 controls from Paris (Paris CONS), and the
consensus sequence of HIV-1 subtype B (Subtype B CONS) (32).
An HIV-1 subtype A consensus sequence (A.SF1703) was used as an
outgroup. Vertical branches are for clarity only; the lengths of the
horizontal branches are proportional to the single-base changes.
Numbers at nodes represent the percentage of bootstrap samples for 100 replications, for which the corresponding cluster is depicted to the
right. Only bootstrap values above 70% are indicated. Phylogenetic
trees constructed by using the Fitch and Margoliash algorithm and by
using the maximum-parsimony method resulted in similar branching
patterns.
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FIG. 2.
Phylogenetic tree analysis (obtained by using the Fitch
and Margoliash algorithm) comparing the coding sequences for the HIV-1
env V4-C4-V5 region of the 26 clones derived from proviruses
of patients 1, 2, and 3 (isolates PA, SI, and RO, respectively),
published sequences of viruses of patients with atypical HIV
transmission and their local controls (US) (34), the
consensus sequence obtained from nine unrelated controls from Europe
(Europe CONS) (FR.LAI, IT.Sala1, UK.CPHL1, UK.CPHL2, UK.CPHL6,
UK.CPHL7, DE.HAN, DE.31, and BE.SIMI84), and the consensus sequence of
HIV-1 subtype B (Subtype B CONS). Phylogenetic trees constructed by
using the neighbor-joining method and by using the maximum-parsimony
method resulted in similar branching patterns.
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Phylogenetic analysis of the C2-V3 sequences showed two separate
lineages (Fig. 1), one comprising all sequences of viruses from patient
1, including two predominant variants, among which sequences of clones
PA7 and PA12 were minor variants, and the other comprising all
sequences of viruses from patients 2 and 3. Although there was a
distinctive branch favoring a separate lineage for the C2-V3 sequences
of the viruses from patient 1 within the family cluster, it was not
strongly supported by the bootstrap value (which was only 56% in the
neighbor-joining tree). Within the subcluster of variants of viruses
from patients 2 and 3, the C2-V3 sequences did not discriminate between
the patients.
Phylogenetic analysis of the V4-C4-V5 sequences clearly differentiated
two lineages: the first subcluster comprised all sequences of viruses
from patient 1, with 100% of bootstrap replicates; the second
subcluster comprised all sequences of viruses from patients 2 and 3, with 92% of bootstrap replicates (Fig. 2). Within the subcluster of
viruses from patient 1, there was a strong grouping of sequences of
clones PA7 and PA12, with 100% of bootstrap replicates. In addition,
all sequences of viruses from patient 2 formed a distinct subgroup in
the subcluster of sequences from patients 2 and 3.
The phylogenies inferred by using the neighbor-joining, the Fitch and
Margoliash, and the maximum parsimony methods were highly congruent.
When hypervariable sites in the V4-C4-V5 domain were removed, the
branching patterns were reproduced (data not shown). Taken together,
the data demonstrate a strong genetic linkage among env
sequences of viruses from patients 1 through 3 and an additional
subgrouping of env sequences for viruses from patients 2 and
3.
Cross-contamination between patients' samples upon amplification was
ruled out by the following lines of evidence. First, samples from
patients 1, 2, and 3 were not amplified, cloned, and sequenced in the
same week. Second, we have used a second blood sample obtained from
each of the patients at 1 to 3 months after the initial sample, cloned
the PCR product from the env gene, and sequenced three
clones from the samples from each patient in a different laboratory
from that in which the original sequencing work was performed. Minor
changes from the initial sequences were observed (data not shown). The
newly obtained sequences in C2-V3 and V4-C4-V5 fitted within the
phylogenic subgroup corresponding to the family cluster (Fig.
3). The only difference in C2-V3 observed with the second samples from the patients, compared with the initial samples (Fig. 1), was that one clone from patient 2 belonged to the PA
subcluster (clones obtained from patient 1) and vice versa (Fig. 3A).
This difference, however, was not significant. Thus, the clones from
the second samples behaved similarly to the clones from the initial
samples upon phylogenetic analysis. The results of the phylogenetic
analysis of the second samples in V4-C4-V5 (Fig. 3B) were similar to
those of the analysis of the initial samples (Fig. 2), with distinct
subgrouping according to the patients. The fact that similar features
have been found in the initial samples and the second samples, which
have been independently analyzed, rules out the possibility that our
results correspond to cross-contamination between samples. Third, we
further sequenced the amplification product of viruses from PBMC of two
unrelated HIV-1-seropositive patients (patients 272 and 374) obtained
at the same time as the first sample from patient 2. Unexpectedly, HIV-1 from patient 374 was found to be of subtype A. C2-V3 as well as
V4-C4-V5 sequences from the viruses derived from patients 272 and 374 were clearly distinct from the family cluster (Fig. 3).
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FIG. 3.
Phylogenetic tree analyses comparing the coding
sequences for the HIV-1 env C2-V3 region as obtained by the
neighbor-joining method (A) and the env V4-C4-V5 region as
obtained by the Fitch-Margolias method (B) for the 9 clones derived
from proviruses of patients 1 (clones PAB1, PAB2, and PAB3), 3 (clones
ROB2, ROB3, and ROB4), and 2 (clones SIB4, SIB5, and SIB6) obtained at
a second blood sampling; published sequences of viruses from patients
with atypical HIV transmission and their local controls (US) (14,
29); the consensus C2-V3 sequence from 28 controls from Paris
(Paris CONS); the consensus C2-V3 and V4-C4-V5 sequences obtained from
unrelated controls from Europe (Europe CONS); and the consensus C2-V3
and V4-C4-V5 sequences of HIV-1 subtype B (Subtype B CONS)
(32), as previously defined in the legends to Fig. 1 and 2,
respectively. An HIV-1 subtype A consensus sequence (A.SF1703), and the
V4-C4-V5 sequence of HIV-1 of patient 374 were used as outgroups for
the phylogenetic analysis in C2-V3 and V4-C4-V5, respectively.
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The relatedness of patients' viruses was further assessed by analyzing
the deduced amino acid sequences of the V3 and V4 hypervariable regions
of gp120. The deduced amino acid sequences obtained from patients 1 through 3 were aligned (Fig. 4 and
5). The top of the V3 loop harbored the
GPGR motif in five of nine clones derived from patient 1. GPGR is the
most common motif in HIV-1 subtype B sequences (32). The
other clones harbored the GPGG and GPDR motifs. Both clones with the
GPGG motif belonged to the phylogenetic subcluster of clones PA7 and
PA12 (Fig. 1). The GPGG motif also represented the amino acid sequence
of the top of the V3 loop in most clones derived from the viruses from
patients 2 and 3. As previously reported for the V4 region of HIV-1
(32, 40), multiple insertions of 3 nucleotides were observed
in the V4 loop, thus maintaining the reading frame downstream. Two V4
motifs, YSNGTW and KGSNYTGVND, that are not present in HIV-1 subtype B, were frequently observed, indicating a high degree of relatedness among
the V4 loop sequences of the viruses from these three patients. Of 13 residues that constituted the signature pattern of patient 1, 11 were
found with high frequencies (0.96 to 1.0) in clones derived from
patients 2 and 3 (Table 2). Only one
amino acid residue in clones from patients 2 and 3 was present with a
100% frequency in the 13 amino acids of the reference set of unrelated isolates of HIV-1 subtype B at the homologous positions of the signature pattern of patient 1, supporting the conclusion that the
viruses of patients 1 through 3 were placed together within the
cluster. Only 3 of 10 positions of the common signature pattern of the
three patients corresponded to residues in the same position in the
signature pattern reported by Ou et al. (34), and only 2 positions corresponded to residues in the signature pattern reported by
Fitzgibbon et al. (14). The amino acid patterns in the
V4-C4-V5 region were similar for the three patients (Table 2).
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FIG. 4.
Multiple alignments of the inferred amino acid sequence
of the HIV-1 env C2-V3 region coding sequences of clones
from patients 1, 2, and 3 (isolates PA, SI, and RO, respectively). The
consensus C2-V3 amino acid sequence of European and North American
HIV-1 subtype B (Subtype B CONS), that from 25 unrelated controls from
Europe (Europe CONS), and that from 28 local controls from Paris (Paris
CONS) are shown at the top. The macrophage (Mac CONS) and T-cell
(T-cell CONS) consensus sequences of the V3 loop defined by Chesebro et
al. (9) are shown at the bottom. Positions 11, 24, 25, and
32, in the consensus T-cell CONS defined as amino acid positions that
may encode basic residues in T-cell-tropic isolates, are indicated in
bold and underlined (15, 31). Positions 21 and 25, which has
been identified as important in macrophage tropism by Westervelt et al.
(44), are indicated by the symbol £. The V3 loop is boxed;
its top is in bold. The V3 loop charge of each sequence is indicated on
the right. Numbers within the box refer to amino acid position in the
consensus sequence of the V3 loop of HIV-1 subtype B env
gp120 (32), counting from the cysteine at the amino
terminus. Amino acids are numbered according to their position in the
HIV-1 LAI sequence (32). Dots indicate sequence homology,
and dashes indicate gaps introduced for optimal alignment. Stop codons
in the sequence are indicated by Z. Asterisks designate the
noncontiguous amino acids of the signature pattern in the C2-V3 region
for patient 1 (Table 2). Single-letter abbreviations for the amino acid
residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
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FIG. 5.
Multiple alignments of the inferred amino acid sequence
of the HIV-1 env V4 loop coding sequences and its flanking
regions of clones from patients 1, 2, and 3 (isolates PA, SI, and RO,
respectively). The consensus V4-C3 amino acid sequence of European and
North American HIV-1 subtype B (Subtype B CONS) is shown at the top.
The V4 loop is boxed. Amino acids are numbered according to their
position in the HIV-1 LAI sequence (32). The question marks
indicate hypervariable sites at indicated positions. Asterisks
designate the noncontiguous amino acids of the signature pattern in the
V4-C4 region for patient 1 (Table 2). The amino acid region located
after the frame shift in clones RO5, RO8, and SI14 was kept blank.
Other symbols are defined in the legend to Fig. 4.
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TABLE 2.
Amino acid signature patterns of viruses of patients 1 through 3 with reference to unrelated HIV-1 subtype B local controls
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DISCUSSION |
We report on a family in which the parents of an HIV-positive
adult man became HIV positive with a virus that was phylogenetically linked to their son's virus. We identified no risk factor for intrafamilial viral transmission and no unproven mode of transmission despite conducting a thorough investigation. Our attention was initially drawn by the unexpected seroconversion of the mother of a
31-year-old HIV-infected drug addict and the subsequent finding of HIV
seropositivity in his father. We initiated a molecular analysis of
cell-associated viruses of the three family members. A similar
genotypic pattern of the pol 215 codon was observed, suggesting some
similarity between the viral populations of the three patients. We then
documented the genotypic relatedness between the viruses of the
patients by three separate approaches, including assessment of genetic
distance, phylogenetic analysis, and analysis of amino acid signature
patterns within the hypervariable regions of the gp120 env
gene. Analysis of the nucleotide sequences in the hypervariable
env regions of viruses of the three family members showed an
interpatient diversity of 1.2 to 5.0% and 2.2 to 7.5% in the C2-V3
and V4-C4-V5 regions, respectively. Interpatient diversities below
5.0% in C2-V3 region (3, 7, 14, 29, 30, 34, 46) and below
8.5% in the V4-C4-V5 region (3, 34) have been reported
previously in the setting of epidemiologically linked sexual and
parenteral HIV transmission. Interpatient diversity in the V4-C4-V5
domain was in a similar range to that estimated in the analysis of an
epidemiologically linked cluster of infection reported by Ou et al.
(34). In contrast, nucleotide sequences yielded more than
13.5% divergence between the C2-V3 and V4-C4-V5 sequences studied and
sequences of unrelated local field isolates. Similar degrees of genetic
divergence have been reported in nonepidemiologically linked HIV
infections (2, 3, 14, 18, 34, 46, 47). These findings
strongly suggested that the viruses from the three members of the
family originated from a common source of infection.
The genetic relatedness of the viruses was further confirmed by the
finding that all sequences clustered tightly within the same
monophyletic group; the sequences diverged significantly from selected
reference sequences, as well as from sequences from patients with
atypical HIV transmission (14, 34). In addition, phylogenetic analysis demonstrated that within the familial HIV cluster, variants of the virus from patient 1 belong to a subcluster that is phylogenetically distinct from a subcluster comprising the
variants of viruses from patients 2 and 3. In the subcluster of
variants from patient 1, the C2-V3 and V4-C4-V5 sequences of two minor
clones, PA7 and PA12, were strongly subgrouped. Obviously, one cannot
totally exclude that the fact that PA7 and PA12 appear as minor strains
is not due to a sampling bias. In the C2-V3 region, these clones showed
less nucleotide divergence than did the clones derived from the viruses
from both parents and the other predominant clones of the virus from
the son. Analysis of the amino acid sequences of the latter two clones
further revealed the presence of an unusual GPGG motif at the top of
the V3 loop, reported to occur with a frequency of only 2.1% among the
published HIV-1 sequences of North American and European isolates
(32), that was shared with viruses from patients 2 and 3. Furthermore, the majority of the viruses from patients 1 to 3 showed
several similar amino acid changes in the C2-V3 region compared with
the HIV-1 B consensus sequence. Signature patterns were almost
identical for the clones from the three patients but distinct from
reference sequences, supporting the notion that the viruses from these
patients harbored very similar quasispecies (20, 32), with
marked similarities in highly functionally relevant domains of the
env gp120 gene including the V3 loop (15, 27,
41). Indeed, the viruses from all three patients had the same
predicted macrophage-tropic, non-syncytium-inducing (NSI) phenotype;
i.e., all studied V3 sequences showed a serine at position 11 and an
alanine at position 25, resulting in global electrostatic neutrality at
these positions, compatible with an NSI phenotype (15) and
highly characteristic of macrophage-tropic viruses (9, 44).
Each of the patients' variants exhibited more homology to the
consensus V3 sequence of macrophage-tropic variants than to the
consensus V3 sequence of T-cell-line-tropic variants (9)
(Fig. 4). Only one of the variants encoded a basic amino acid at one of
the four positions in the V3 loop that are associated with T-cell
tropism (31).
The mode of cross-infection of HIV among members of the family remains
unknown. The clinical features of HIV disease and the genetic analysis
of viruses suggest a chronology of infection where patient 1 was
infected before patient 3, who, in turn, was infected before patient 2. Sequences obtained from patient 2 demonstrated a limited nucleotide and
amino acid genetic diversity, as previously documented in primary
infections (1, 25, 32, 48, 49). The relatively high
homogeneity of the viral populations in patient 3 is compatible with a
recent infection (46). In contrast, the C2-V3 and C4-V4-V5
domain nucleotide sequences of the variants from patient 1, who had
most probably been infected while using intravenous drugs, i.e., at
least 5 years before sampling, were significantly more heterogeneous
than those derived from patients 2 and 3. Analysis of the
intraindividual variations in the V4-C4-V5 domain confirmed the higher
divergence of viral strains from patient 1 than of those from patients
2 and 3 and further revealed that strains from patient 3 were
significantly more divergent than those from patient 2. The results are
consistent with patient 1 having been infected for a longer time than
patients 2 and 3 and with patient 3 having been infected before patient
2. In addition, patient 3 already presented with a high HIV load in
plasma and low CD4 cell counts 4 months after patient 2 had
seroconverted. Since the majority of the variants from patients 2 and 3 were genetically related among themselves and were related to minor variants from patient 1, one may speculate that patient 1 first contaminated patient 3 and later contaminated patient 2; in the latter
case, the same minor variants from patient 1 would have represented the
common source of infection of patients 2 and 3, and therefore should
have been selected twice during intrafamilial transmission. There is
evidence that minor strains are commonly transmitted from HIV-positive
donors to the recipients (49, 50). Alternatively, patient 1 may have infected patient 3, who, in turn, contaminated patient 2; in
this case, minor variants of the virus from patient 1 should have been
initially selected during transmission to patient 3; major variants of
the virus from patient 3 would then have been the source of infection
in patient 2.
Despite extensive epidemiological investigation, no risk factor for
cross-infection was identified among family members. The risk of
transmission of HIV in the household in the absence of sexual or
percutaneous exposure has been extensively investigated (4, 12,
17, 19, 28, 33, 37, 38, 42, 43). It is known that viral
transmission has not occurred after tens of thousands of days of
sharing eating utensils, towels, combs, toilets, bathtubs, and beds and
of hugging and kissing between family members (16). A
meta-analysis of several studies in the United States and Europe has
revealed no case of infection in the follow-up of more than 1,700 person-years after household contact with HIV-infected people
(42). However, a small number of cases of infection has been
reported in which no risk factor could be identified despite extensive
investigation (5, 6, 14, 34, 45). The contact of cutaneous
or mucous membranes with infected blood or body fluids has been
considered to be the explanation for most such cases of HIV
transmission (38, 43). The estimated risk of transmission
after mucous and cutaneous exposure to HIV-infected blood in
prospective studies of health care workers is below 0.1% (21,
23). Transmission of the virus from the son to one of his parents
through casual contact as the parents were caring for their son is
highly unlikely to have occurred on two independent occasions. These
findings raise the issue of whether the virus exhibited distinct
phenotypic properties or the recipients exhibited specific genetic
susceptibility to infection. Taken together, our observations suggest
that atypical transmission of HIV may occur.
| |
ACKNOWLEDGMENTS |
We are indebted to Xavier Jeunemaître, Françoise
Ferchal, Catherine Letondal, Elisabeth Menu, Sylvie Corbet, and
Marie-Charlotte Hallouin for assistance with nucleotide sequencing or
analyses; Sentob Saragosti and Marie-Laure Chaix for providing
sequences of field isolates in Paris; and Denis Beaumont and Eric
Gressier (Laboratoire Cedric, CNAM, Paris, France) for the opportunity to use the "veryfastDNAml" software.
M.C.M.-T. is a recipient of grants from Agence Nationale de Recherches
sur le SIDA (ANRS) and from Fondation pour la Recherche Médicale (SIDACTION), France. This work was supported by
Institut National de la Santé et de la Recherche
Médicale, ANES, and SIDACTION, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology and INSERM U430, Hôpital Broussais, 96, rue Didot,
75674 Paris Cedex 14, France. Phone: (33) 1 43 95 95 83. Fax: (33) 1 45 45 90 59. E-mail:
michel.kazatchkine{at}brs.ap-hop-paris.fr.
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Abstract
Introduction
