CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,
1071-412X/01/$04.00?0 DOI: 10.1128/CDLI.8.5.884–890.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Sept. 2001, p. 884–890Vol. 8, No. 5
Molecular Investigation of Transmission of Human
Immunodeficiency Virus Type 1 in a Criminal Case
ROBERTO MACHUCA,1* LOUISE B. JØRGENSEN,1PETER THEILADE,2AND CLAUS NIELSEN1
Retrovirus Laboratory, Department of Virology, Statens Serum Institut,1and Institute of Forensic Medicine,
Department of Forensic Pathology, University of Copenhagen,2Copenhagen, Denmark.
Received 4 December 2000/Returned for modification 15 March 2001/Accepted 23 May 2001
Very few criminal cases involving human immunodeficiency virus type 1 (HIV-1) transmission have been
described. We report on an HIV-1 transmission case with a child being infected by an HIV-1-positive man. The
objective was to determine through molecular epidemiology and phylogenetic analyses whether HIV-1 from the
HIV-1-positive man could be the source of infection in the HIV-1-positive child, as claimed by the authorities.
We conducted genetic analysis of three different parts of the HIV-1 genome (gag, pol, and env) by PCR,
direct-sequencing, and phylogenetic analyses. We used maximum likelihood, maximum parsimony, and neigh-
bor-joining methods for the phylogenetic analyses to investigate whether the sequences from the man and the
child were related. We found that the viral sequences from the man and the child formed separate clusters in
all of the phylogenetic analyses compared to the local controls. A unique amino acid deletion was identified in
the C2-V3-C3 region of the env gene in the virus from the man and the child. These results were used in the
criminal court to elucidate whether the virus from the man was related to the virus from the child. In summary,
the results from the phylogenetic analyses, the sequence distances between the virus from the man and the
virus from the child, and the identification of the unique molecular fingerprint in the env gene together
indicated that the virus from the man and the virus from the child were epidemiologically linked.
Only a small number of criminal cases involving transmission
of human immunodeficiency virus (HIV) have been reported
in which the use of molecular epidemiology was used to inves-
tigate the possible source of infection. Three previous cases
have been described. In the “Florida dentist case,” sequence
analysis of the V3-loop of the external envelope glycoprotein
gp120 was used in the investigation. However, this case never
made it to trial, since the case was settled out of court, but from
analysis of the V3-loop, Ou et al. concluded that HIV type 1
(HIV-1) from the dentist was the source of infection in the
In The Netherlands, a man was convicted of infecting his
ex-girlfriend by injecting blood from an HIV-infected drug
user (23). That study investigated the V3-loop of gp120 by
nested PCR and showed that the index case and the victim
clustered tightly in the phylogenetic analysis. In addition, a
mutation was found in codon 215 of the RT gene associated
with zidovudine resistance in HIV from the victim, reflecting
that the index subject had been in zidovudine therapy and had
developed resistance against zidovudine. These sequences,
phylogenetic analysis, and the molecular fingerprint strongly
supported the allegations concerning the HIV-1 transmission
from the drug abuser’s blood to the victim.
The third transmission case used molecular epidemiology to
elucidate a rape case with HIV-1 transmission to the victim (1).
In this Swedish rape case, different regions of the HIV-1 ge-
nome were investigated. Analyses of sequences from the pol
and gag genes demonstrated that the HIV-1 strains carried by
the male and the female were genetically very closely related.
Analysis of the gag region showed that the two individuals
shared an unusual out-of-frame deletion of three nucleotides.
From these results, the authors of that study concluded that it
was highly likely that the HIV-1 strains from the two individ-
uals were epidemiologically closely linked.
Here we report a possible Danish transmission case, where
a sexually abused 12-year-old child contracted infection with
HIV-1. The case became known after 10 boys revealed that
their recreation center coach (“the man”), who trained them in
wrestling, had abused them sexually. The authorities were in-
formed, and the situation was reported to the police. It was
later reported that the abuse had been going on since 1994.
All boys were examined at the Institute of Forensic Medi-
cine in Copenhagen in December 1998 and in the beginning of
1999, and one of them tested as HIV positive. It turned out
that the man had had sexual intercourse with the boy in ques-
tion and, moreover, had been sexually abusing him during the
period from January 1995 through the summer of 1996, i.e.,
when the boy was only 12 to 13 years old. The other boys were
all about the same age when the victimization took place;
however, none of them tested positive for HIV.
The man was therefore charged with violation of the Penal
Code, §222, for having performed intercourse with a child
under the age of 15 and, since it was assumed that the man was
aware of his own HIV-positive status, was also charged with
violation of §252 of the Penal Code, for recklessly having
caused danger to another person’s life or health.
The man was charged with 22 cases according to the Penal
Code, §225 and §252. The man was sentenced to 6 years of
imprisonment in March 2000 at the Copenhagen High Court.
Genetic analysis of the HIV-1 strains carried by the man and
the child was performed to determine whether viral sequences
from the child were significantly more closely related to se-
* Corresponding author. Mailing address: Department of Virology,
Retrovirus Laboratory, Statens Serum Institut, Artillerivej 5, DK-2300
Copenhagen S, Denmark. Phone: 45-32683115. Fax: 45-32683082. E-
quences from the man than to sequences from relevant con-
We describe here the molecular epidemiological investiga-
tion, the genotypic results, and the phylogenetic analyses re-
lated to this case.
MATERIALS AND METHODS
Patient samples. A sample of whole blood was obtained from both the child
(pt843) and the man (pt844). Each sample was prepared separately, and periph-
eral blood mononuclear cells (PBMC) and plasma were stored. Furthermore, we
had access to frozen PBMC from the man obtained 21 months earlier (prot165).
All sample preparations from each individual patient were done separately for
DNA extractions (Roche DNA isolation kit for blood) and RNA extractions
(Qiagen QIAamp Viral RNA kit). PBMC samples from 14 unrelated HIV-1-
infected individuals from the same geographic area as the child and the man
(Copenhagen, Denmark) were used as local controls. Because it was possible
that the man had been infected during the early 1990s in Thailand with subtype
B, we included two Danish samples from individuals infected with subtype B in
Thailand. The man had not received antiretroviral treatment prior to the esti-
mated time of sexual abuse leading to the HIV-1 transmission to the child 3 years
earlier. Because of low viral load in the sample from the man (pt844), we were
not able to perform PCR analysis on the HIV RNA from this sample. Thus, the
early sample, prot165, from the man was only analyzed for proviral DNA from
the PBMC. Sample pt843 from the child was analyzed for both proviral DNA and
The local control sequences, which we selected, were from different patient
groups with respect to transmission mode. Nine sequence samples were from
patients with homosexual transmission, one sample was from an intravenous-
drug user, five samples were from patients with heterosexual transmission, and
one sample was from a patient with hemophiliac transmission.
PCR and direct DNA sequencing. We performed PCR and subsequent se-
quencing of three regions in the HIV-1 genome: the C2-V3-C3 region of the env
region, the p17gagregion of the gag gene, and the reverse transcriptase (RT)
region of the pol gene.
For amplification of the C2-V3-C3 region of the env gene, we used the primers
JA167 [5?-TAT C(C/T)T TTG AGC CAA TTC C(C/T)A TAC A-3?] and JA170
[5?-GTG AGT TAT T(A/G)C A(A/G)T AGA AAA ATT C-3?] for the first PCR
and primers JA168 [5?-ACA ATG (C/T)AC ACA TGG AAT TA(A/G) GCC
A-3?] and JA169 [5?-AGA AAA ATT C(C/T)C CTC (C/T)AC AAT TAA A-3?]
for the nested PCR, yielding a 410-bp amplification fragment (17).
For the amplification of the p17gagregion, we used primers JA152 (5?-ATC
TCT AGC AGT GGC GGC CGA ACA G-3?) and JA155 (5?-CTG ATA ATG
CTG AAA ACA TGG GTA T-3?) for the first PCR and primers JA153 (5?-CTC
TCG ACG CAG GAC TCG GCT TGC T-3?) and JA154 (5?-CCC ATG CAT
TCA AAG TTC TAG GTG A-3?) for the nested PCR. A 558-bp fragment was
We amplified the RT region of the pol gene using a nested PCR with primers
L3 (5?-GAC CAG AGC CAA CAG C-3?) and L4 (5?-ATC ACT AGC CAT
TGC TCT CCA-3?) for the first PCR and primers A (5?-TTC CCA TTA GTC
CTA TT-3?) and L31 (5?-CCA GCT GTC TTT TTC TGGCAG CAC TAT-3?)
for the nested PCR. A 768-bp fragment was amplified (5).
All PCR products were analyzed on a 1.5% agarose gel and visualized by
ethidium bromide staining. The PCR products were purified, prior to sequenc-
ing, using the Qiaquick PCR purification kit (Qiagen GmbH) according to the
manufacturer’s instructions. Direct sequencing on the nested PCR products was
performed on both DNA strands of all PCR fragments using the Big Dye
Terminator Cycle Sequencing Kit (Perkin-Elmer) and analyzed on the ABI
PRISM 377 sequencer.
All nucleotide sequences obtained were translated and aligned using the soft-
ware DNASTAR (Lasergene, Madison, Wis.).
Reference sequences of all the known subtypes were obtained from the Los
Alamos database. We included 101 complete envelope genes of subtype B ob-
tained from the Los Alamos database used for the phylogenetic analysis shown
in Fig. 2.
Phylogenetic analyses. Phylogenetic-tree analyses of nucleotide sequences
were conducted with three different programs from the PHYLIP package, ver-
sion 3.57c. provided by J. Felsenstein (6): DNAML (maximum likelihood [ML]),
DNAPARS (maximum parsimony [MP]), and NEIGHBOR (neighbor joining
[NJ]). The DNADIST program provided the distance matrix needed for the NJ
method. For DNADIST and DNAML, the value for the transition/transversion
ratio was set at 1.5 as described by Holmes et al. (13). The trees were constructed
using Treeview from the PHYLIP package. Bootstrap analyses were performed
on ML and NJ trees with the DNABOOT program of PHYLIP; in each case
1,000 resamplings were performed.
Analyses of the sequences from the man and the child. The
direct DNA sequencing procedure was performed on both
DNA strands to verify all nucleotide positions. Sequences were
obtained from both DNA samples (prot165 and pt844) from
the man and from HIV RNA and proviral DNA from the child
(pt843r and pt843).
The alignment of the sequences from the p17gaggene
showed that the sequences from the man’s first sample
(prot165) diverged by 0.58% from the later sample of proviral
DNA (pt844), while the sequences obtained from viral RNA
and proviral DNA from the child (pt843) diverged by 0.29%.
The sequence distance between the samples from the man and
the child diverged by 2.66 to 2.95%. Sequences obtained from
the child (pt843) diverged by 3.87 to 13.00% and sequences
from the first sample from the man (prot165) diverged by 3.89
to 12.90% from the local control sequences, respectively. Di-
vergence among the controls was 4.48 to 13.06%.
Results from the nucleotide alignment of the C2-V3-C3
region of the env gene showed that the sequences from HIV
RNA and proviral DNA from the pt843 sample from the child
diverged by 0.34%, while the sequences from the two samples
obtained within a 21-month interval from the man (prot165
and pt844) diverged by 0.69%. Upon comparing the sequences
obtained from plasma viral RNA and the proviral DNA in the
samples from the child (pt843) and the sample from the man
closest to the estimated time of the sexual abuse (prot165), we
found that they diverged by 7.18 to 8.33%. A comparison of
these sequences with sequences from local controls showed a
divergence between the child’s sequences and the controls of
8.71 to 16.63%. The man’s sequences diverged by 8.76 to
15.11% compared to the controls. The divergence between the
controls was 7.11 to 19.58%.
When an alignment of 169 sequences, including 101 se-
quenced envelope genes of subtype B from the Los Alamos
database and 68 from our own database, was performed, a
unique amino acid deletion was observed in codon 269 (strain
HIV-IIIB) of the env gene (20). This molecular fingerprint was
only found in HIV from the man and the child (alignment not
shown). An alignment of the C2-V3-C3 region of the local
controls and of the virus of the man and the child was per-
formed and confirmed the previous result concerning the
unique deletion in codon 269 (Fig. 1).
Analyses of the amino acid alignment from the pol gene
(data not shown) showed that no mutations associated with
antiretroviral resistance were detected in either the early
(prot165) or the late (pt844) sample from the man or the child
(pt843). Sequence analysis of the proviral DNA from the sam-
ples prot165 and pt843 showed that sequences of the man and
the child diverged by 2.72%. In addition, the child’s sequences
(pt843) diverged by 3.56 to 5.53% and the man’s sequences
(prot165) diverged by 2.85 to 3.80% compared to the local
controls. The divergence between the local control sequences
was 3.18 to 4.58%. Furthermore, we did not find any significant
amino acid pattern between the two patient samples (pt844-
prot165 and pt843) in the pol gene.
VOL. 8, 2001MOLECULAR INVESTIGATION OF HIV-1 TRANSMISSION 885
Phylogenetic analyses. The genotypic relatedness between
HIV-1 from the man and the child was analyzed using different
phylogenetic methods, i.e., NJ, ML, and MP. Phylogenetic
analyses performed with reference strains of all the known
subtypes showed that the viruses from the three samples
(pt843, pt844, and prot165) were of subtype B in the env, gag,
and pol genes.
We tested the relationship of the local controls by perform-
ing phylogenetic analysis (NJ) on the C2-V3-C3 region of these
samples, together with 101 subtype B sequences obtained from
the Los Alamos database. These analyses demonstrated that
the local controls were distributed throughout the phylogenetic
tree (Fig. 2).
We used different parts of the HIV-1 genome: the p17gag,
env, and pol genes for the analyses of the relationship between
HIV-1 from the man and that from the child. The phylogenetic
analysis by NJ of the p17gaggene (Fig. 3A) showed that se-
quences obtained from RNA and DNA from the child’s sample
pt843 clustered with a bootstrap value of 100%, and sequences
from samples prot165 and pt844 from the man clustered to-
gether with a bootstrap value of 93%. These four sequences
formed a separate cluster with a bootstrap value of 87%. Boot-
strap values of ?70% have been shown to correspond to a
probability of ?95% that the corresponding clade is true (10).
Because other research groups have reported that the C2-
V3-C3 region of the env gene is suitable for analysis of trans-
mission cases (1, 14, 18, 21, 22), we selected this region for
investigation of the env gene. The phylogenetic analysis of the
C2-V3-C3 region (Fig. 3B) performed by NJ analysis showed
that the four sequences from the man and the child clustered
together. This is in accordance with the results obtained from
the p17gaggene. The bootstrap values for the two individuals’
separate sequences were 100%, while the bootstrap value be-
tween the sequences from the two individuals was only 48%.
For maximum resolution in the phylogenetic analysis, it has
been suggested that the sequence data should be combined in
this case, the p17gagand C2-V3-C3 regions, before performing
the phylogenetic analysis (1). In performing these analyses
(Fig. 3C), we found that the four sequences from the two
individuals clustered together with 100% bootstrap values for
their individual sequences and with 79% between their se-
Finally, we investigated the pol gene and performed phylo-
genetic analysis on the RT region (Fig. 3D). We selected the
prot165 sample from the man and the pt843 sample from the
child and analyzed these two sequences, together with eight
other control sequences. These results confirmed our previous
findings, and the two sequences clustered with a bootstrap
value of 83%.
Results from the analyses performed by ML and MP for the
three genes are not shown, but these analyses showed that the
virus amplified by PCR from samples pt843 (RNA and DNA)
clustered together with the samples prot165 and pt844, sup-
porting the phylogenetic data obtained by the NJ method.
Use of molecular epidemiology for investigation of trans-
mission cases is often complicated by the fact that the deter-
mination always is very difficult, since the molecular analysis is
performed on the HIV-1 virus in samples obtained with dif-
ferent time spans from the estimated time of transmission that
vary from case to case. The HIV-1 variation also evolves indi-
vidually depending on the individual patient and the course of
The fact that the estimated time of transmission of HIV-1 in
this study was more than 4 years prior to the discovery of the
HIV-1 infection of the child makes the investigation of the
molecular epidemiology more difficult and the interpretation
of the phylogenetic analyses harder. Because of these difficul-
ties, we selected three different genes for our analysis (gag, pol,
and env). By doing so we tried to bypass any randomly phylo-
genetic relationship in a certain gene that might have disturbed
the investigation. In choosing this approach, establishing a
genetic relation was further challenged and demanded a true
genetic relationship between the man’s virus and the child’s
FIG. 1. Alignment of the amino acid sequences derived from the C2-V3-C3 region. The child’s sequences correspond to the pt843t and pt843rt
samples, and the sequences from the man correspond to the pt844t and v3prot165 samples. An asterisk indicates the described deletion between
the child and the man. A minus sign indicates an amino acid that corresponds to the consensus sequence. Deletions in the amino acid alignment
are indicated by a period. “X” corresponds to an amino acid mixture.
886 MACHUCA ET AL.CLIN. DIAGN. LAB. IMMUNOL.
FIG. 2. Phylogenetic tree derived from sequences from the C2-V3-C3 region. A total of 101 subtype B C2-V3-C3 sequences obtained from the
Los Alamos database were included in the analyses, together with the 16 local control sequences (in boldface) and the four sequences from the
man and the child (in boldface and underlined). The NJ method was used in constructing the tree. An HIV-1 subtype A sequence, U455, was used
as an outgroup. The two lines (?) on the outgroup indicate that the branch has been shortened.
VOL. 8, 2001MOLECULAR INVESTIGATION OF HIV-1 TRANSMISSION 887
virus, which would be elucidated by the phylogenetic analyses.
In addition, three different phylogenetic tools were used to
analyze the genetic relatedness of HIV-1 from the two sub-
jects: NJ, ML, and MP. However, it is important to note that
determination of the transmission direction between the child
and the man could not be resolved by these molecular inves-
tigations due to the fact that either of these individuals could
have been infected by a third party. This molecular investiga-
tion should only be used in conjunction with additional evi-
The data on sequence divergence from the described genes
(gag, pol, and env) showed that the divergence differed depend-
ing on the gene in question. Other investigations on related
transmission cases have shown that the divergence differs from
case to case (4, 13, 18, 21). We have previously shown that the
DNA sequence of the V3-loop was conserved during the first
FIG. 3. (A) Phylogenetic tree derived from sequences from the p17gagregion. (B) Phylogenetic tree derived from sequences from the C2-V3-C3
region. (C) Phylogenetic tree derived from sequences from the p17gagregion and the C2-V3-C3 region combined. (D) Phylogenetic tree derived
from sequences from the RT region of the pol gene. The NJ method was used in constructing all four trees. An HIV-1 subtype A sequence, U455,
was used as an outgroup. The two lines (?) on the outgroup indicate that the branch has been shortened. Only relevant bootstrap values have been
included in the tree. Local controls are in boldface. The four sequences from the man and the child are in boldface and underlined.
888 MACHUCA ET AL.CLIN. DIAGN. LAB. IMMUNOL.
24 weeks of infection in a donor-recipient pair (18). In a study
by Bobkov et al. it was found that the interpatient variation
ranged from 5.9 to 6.6% (4). Belec et al. showed in an inves-
tigation of intrafamilial transmission that the divergence in the
C2-V3 region was between 1.2 and 5.0% (2).
Goujon et al. investigated a transmission case analyzing the
pol gene (9). They found a very high degree of divergence
depending on the sequences in question. They further showed
that the divergence ranged from 11.33% within group M to
26.37% when group O was included, which shows that refer-
ence sequences and control sequences should be selected care-
fully. Our results for the pol gene show that related sequences
can differ substantially.
A previous investigation (14) concerning a nosocomial HIV
transmission shows, in comparison with the present study, that
the p17gaggenes can differ substantially and still be phyloge-
netic related. Katzenstein et al. found that the p17gaggenes of
two individuals differed by 0.9% (14), while the man and the
child differed by 2.66 to 2.95%. Of note is that the two trans-
mission cases have different time spans from infection to dis-
covery (i.e., the point at which patient samples were obtained).
This can explain the difference in divergence between the
transmission cases investigated, together with the fact that
virus infection evolves in a fashion specific to each infected
person, depending on the immunological status of that person
and the nature of the virus.
The results from the alignment of the C2-V3-C3 region
showed that the divergence was higher in this gene compared
VOL. 8, 2001MOLECULAR INVESTIGATION OF HIV-1 TRANSMISSION889
to p17gagand pol. Blanchard et al. found in their investigation
of a nosocomial transmission from a surgeon to a patient that
the gag sequence divergence was higher compared to the env
sequences (3). This was, however, caused by the fact that the
individuals were infected by a recombinant HIV-1 A/F sub-
Because the V3 region has been described as the principal
neutralizing epitope, many studies have investigated the vari-
ation within the envelope C2-V3-C3 region (7, 8, 19). This has
led to speculation on how suitable this region is in resolving
transmission cases (11, 12). Leitner et al. investigated which
regions were most accurate in the reconstruction of a true
transmission case by using phylogenetic methods on sequences
derived from the p17gagregion or the C2-V3-C3 region and
sequences from the two regions in combination (15). These
authors found that the most accurate phylogenetic tree com-
pared to the true tree was constructed by using sequences from
p17gagand C2-V3-C3 in combination, and they also found that
analysis of the C2-V3-C3 region was more accurate than anal-
ysis of the p17gagregion.
When the time spans from the estimated time of infection to
the collection of blood samples in this study (21 months and 3
years respectively) were taken into account, the phylogenetic
analysis using either p17gagor p17gagplus C2-V3-C3 showed
more accurately the time-dependent relationship between the
sequences compared to the analysis using the C2-V3-C3 region
The phylogenetic analysis of the pol gene confirmed the
relatedness of the two individuals viruses.
The one-amino-acid deletions in the C2-V3-C3 region pro-
vided a very strong epidemiological link between the two pa-
tients, and we were not able to detect this deletion in any of the
sequences from the Los Alamos database that we included in
In conclusion, the results from the phylogenetic analyses, the
sequence distances between the virus from the man and the
virus from the child, and the identification of the unique mo-
lecular fingerprint in the env gene together indicate that the
virus from the man and that from the child were epidemiolog-
We thank Bende Frederiksen for the excellent work she has done
working on this transmission case.
We also thank the Velux Foundation and the AIDS Foundation for
supporting our work.
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