BRIEF REPORT
hormonal contraceptives may affect the complexity of the trans-
A Substantial Transmission Bottleneck
among Newly and Recently
HIV-1–Infected Injection Drug Users
in St Petersburg, Russia
Alexey E. Masharsky,1,2,a Elena N. Dukhovlinova,1,a
Sergei V. Verevochkin,1,2 Olga V. Toussova,1 Roman V. Skochilov,1
Jeffrey A. Anderson,4,6 Irving Hoffman,4,6 Myron S. Cohen,4,6
Ronald Swanstrom,5,6 and Andrei P. Kozlov1,3
1
Biomedical Center, 2State Research Institute of Highly Pure Biopreparations,
and 3St Petersburg State University, St Petersburg, Russia; 4Division
of Infectious Diseases, 5Department of Biochemistry and Biophysics,
and 6Center for AIDS Research, University of North Carolina, Chapel Hill
There are limited data on the genetic complexity of human
immunodeficiency virus type 1 (HIV-1) after transmission
among a cohort of injection drug users (IDUs). We used
single-genome amplification of HIV-1 env to determine the
genotypic characteristics of virus among IDUs with acute
infection in St Petersburg, Russia. Our results indicate that
a single variant was transmitted in a majority of cases (9 of
13 participants), which is analogous to what is observed in
sexual transmission. These data are most consistent with a
genetic bottleneck during transmission by injection drug use
that is due to a small inoculum, which most often results in
the transmission of a low-complexity viral population.
In sexual transmission of human immunodeficiency virus type
1 (HIV-1), a single variant is established in the recipient in
∼80% of cases [1–3]. This bottleneck phenomenon is currently
explained by the low efficiency of virus penetration through
mucosal layers and the potential selective pressure at the sites
of transmission in either the donor or the recipient that could
shape the transmitted viral strains. However, cofactors such as
the presence of a sexually transmitted infection or the use of
Received 20 August 2009; accepted 21 December 2009; electronically published 27 April
2010.
Potential conflicts of interest: none reported.
Financial support: Russian Federal Agency of Science and Innovations (contract
02.512.11.2188); Civilian Research and Development Foundation (award RUB1-7000-ST-08);
Bill and Melinda Gates Foundation (grant 383-0925).
a
A.E.M. and E.N.D. contributed equally to this work.
Reprints or correspondence: Andrei P. Kozlov, 194044, 8 Vyborgskaya St, St Petersburg,
Russia (contact@biomed.spb.ru).
The Journal of Infectious Diseases 2010; 201(11):1697–1702
 2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20111-0013$15.00
DOI: 10.1086/652702
mitted virus [2, 4].
The second most frequent mode of HIV-1 transmission
among adults is associated with needle sharing during injection
drug use. In a rhesus macaque model, intravenous infection
has been associated with more heterogeneous infections than
intravaginal or intrarectal infection, with transmission by the
intravenous route also being much more efficient [5–7]. In
addition, the timing of HIV-1 env diversification and the de-
velopment of neutralizing antibodies appear to be different in
macaques infected intravenously from those in macaques in-
fected mucosally [8]. However, the details of HIV transmission
among cohorts of injection drug users (IDUs) have received
much less attention than the details of sexual transmission. The
absence of a mucosal barrier and the direct introduction of the
virus into the blood could theoretically lead to a higher fre-
quency of transmission of multiple variants, as suggested in
one study [7].
The epidemic of HIV-1 infection among IDUs in St Peters-
burg, Russia, is notable for the recent increase of HIV infection
prevalence and incidence. Several longitudinal studies have
been performed in St Petersburg among IDUs [9]. The HIV
Prevention Trials Network study, conducted from 2002 through
2006, found a baseline HIV-1 infection prevalence of 30%
among IDUs and an incidence of 4.5/100 person-years (95%
confidence interval, 2.7–7.0) [10, 11]. Subsequent studies (con-
ducted from 2004 through 2008) have found an even higher
HIV-1 infection prevalence (∼50%) and incidence rates among
IDUs of 12.5–19.6/100 person-years (C. Latkin and A. P. Koz-
lov, unpublished data, 2008).
We have examined the genetic complexity of the viral pop-
ulation in recently HIV-1–infected IDUs in the St Petersburg
cohort. Multiple viral variants were present in only 4 (30%) of
the 13 recently infected participants in this cohort. Phylogenetic
analysis showed that viral sequences did not represent a trans-
mission cluster, and several chronically infected individuals had
much more complex populations. Thus, the low complexity of
the viral population among recently infected IDUs does not
appear to be the result of an overall local epidemic of low-
complexity virus, but it rather represents the limiting infectious
unit for establishing a new infection.
Methods. Samples stored in a serum bank from 4 studies
were used. All study participants provided informed consent
for storage and further analysis of their blood samples. The
human experimentation guidelines of the US Department of
Health and Human Services and the Biomedical Center in St
BRIEF REPORT • JID 2010:201 (1 June) • 1697
Petersburg, Russia, were followed in the conduct of this re-
search. The participants were screened for sexually transmitted
infections and answered questionnaires regarding drug use and
sexual practices. Demographic, clinical, and behavioral data for
the subset of participants included in this transmission study
are presented in Table 1. The 4 studies that were the source of
the samples used are as follows: the HIV Prevention Trials
Network study, which was conducted in St Petersburg from
2002 through 2006; the Russian IDU Peer Network HIV Pre-
vention Intervention Trial; the Sexual Acquisition and Trans-
mission of HIV Cooperative Agreement Program; and a lon-
gitudinal cohort study that was initiated by the Biomedical
Center in July 2008 (Maintenance of IDU Cohort for HIV
Vaccine Clinical Trials).
Plasma or serum samples from participants with negative
enzyme-linked immunosorbent assay (ELISA) results from all
4 studies were pooled and screened for HIV-1 RNA by means
of the Amplicor HIV-1 Monitor test (Roche). Samples from
individuals who had positive ELISA results and indeterminate
Western blot results were included in the viral genetic analysis.
The duration of each HIV-1 infection was categorized on the
basis of HIV-1 RNA load or evolving antibody profiles, as de-
scribed by Fiebig et al [12]. Detection assays for syphilis, herpes
simplex virus infection, and hepatitis C virus infection were
performed as described elsewhere [13]. Single-genome ampli-
fication procedures were performed by following a protocol
described elsewhere [1, 3], with only minor changes to facilitate
amplification and sequencing of subtype A virus. The sequences
were aligned and then manually edited, and neighbor-joining
trees were generated.
To ensure that sequences reflected single templates from viral
populations in vivo, amplicons with sequence chromatograms
with “double peaks,” indicative of coamplification of 11 template,
were excluded. However, if the number of double peaks did not
exceed 1 and included the consensus nucleotide, then the am-
plicon sequence was included, with the consensus nucleotide
placed at that position. Sequence analyses, including visualization
of Highlighter plots (Highlighter is available at http://www.hiv
.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), cal-
culation of Hamming and pairwise distances, and construction
of neighbor-joining trees, were performed as described elsewhere
[1, 3, 5, 14]. The GenBank accession numbers are GU481107–
GU481683.
Results and discussion. The goal of our work was to ex-
amine the complexity of the transmitted virus among a cohort
of IDUs in St Petersburg, Russia. Samples from a total of 772
participants with negative ELISA results from 4 IDU cohort
studies were tested for the presence of HIV RNA; 2 cases of
acute HIV infection (Fiebig stage I or II) were found (Table
1). One individual had positive ELISA results but negative West-
ern blot results (Fiebig stage III). Among individuals with pos-
1698 • JID 2010:201 (1 June) • BRIEF REPORT
itive ELISA results, 7 had indeterminate Western blot results
and were classified as Fiebig stage IV. Three individuals had
positive ELISA results and positive Western blot results (Fiebig
stage VI) with evidence of recent HIV infection, due to negative
ELISA results !1 year ago. Four additional samples from chron-
ically infected individuals were used as a control [10, 11].
Serum samples from these 13 individuals with acute and
early infection and the 4 chronically infected individuals were
used to generate full-length env amplicons by means of single-
genome amplification (Table 1). The multiplicity of infection
was determined by means of phylogenetic analysis of the env
sequences. A neighbor-joining tree of sequences revealed sub-
stantial diversity in the viral population among the 4 chronically
infected individuals, compared with that among the 13 indi-
viduals with acute and early infection. Among those 13 indi-
viduals, we observed sequence homogeneity in 9 individuals
(Figure 1A). A representative Highlighter plot from participant
R163 (Figure 1B) is consistent with infection by a single viral
variant, with the majority of sequences being identical to the
consensus sequence. In contrast, the other 4 individuals (par-
ticipants H408, H410, R053, and SC1283; 30%) appeared to
be infected with multiple (2 or 3) variants (Figure 1A and 1C).
In the viral population among chronically infected participants,
we observed no sequences that were identical to the consensus
sequence (Figure 1D). All participants but 1 harbored HIV-1
strains of subtype A. The virus in participant SC1283 repre-
sented a mixture of HIV-1 CRF06_cpx strains with its subse-
quent recombinant CRF-06_cpx/subtype A variant. We did not
detect any evidence for superinfection: all viral variants from
participants with 11 variant clustered within each participant
(Figure 1A).
Thus, in this small cohort, a single variant was transmitted
∼70% of the time. The rate of multiple variant transmission
events associated with IDUs that was observed in this study
(30%) is higher than, but not statistically different from, the
∼20% transmission rate reported for 2 large cohorts in which
transmission was predominately by sexual contact [1, 3]. Thus,
the complexity of the transmitted virus in this IDU cohort does
not appear to be substantially higher than that observed for
sexual transmission, a possibility that was raised elsewhere [5].
We used behavioral and clinical data to estimate the possible
route of HIV acquisition (Table 1). All of the participants tested
positive for hepatitis C virus, which indicates the risk associated
with unsafe injection practices. Self-reports of injection prac-
tices revealed a high risk of IDU-related HIV transmission.
However, inconsistent or no condom use, a high number of
sexual partners (⭓3 in the past 6 months), and/or the presence
of a sexually transmitted infection also suggested the possibility
of sexual transmission of HIV. We could define the risk of
probable parenteral transmission for 5 of the 13 participants
with acute and early infection (R163, R497, R575, K08, and
Table 1. Cohort and Demographic Characteristics of Injection Drug Users with Acute and Early Human Immunodeficiency Virus Type 1 (HIV-1) Infection

No. of
History Viral load, No. of sexual
Age, of IDU, log10 Fiebig env variants No. of Maximum partners/period,
a b
Participant ID years/sex years copies/mL stage transmitted env amplicons HD Drug and frequency of use Mode of use STI(s) months Condom use Risk estimation

H408 21/male NA 5.4 IV 2 20 36 Heroin 3–4 times per week 1 HSV 7/6 No IDU and sexual contact

H410 24/male NA 5.8 III 3 27 36 Heroin 1–2 times per week 1 Negative 5/6 No IDU and sexual contact

R053 35/female 10 5.6 IV 12 21 12 Heroin 2–5 times per day, methadone occasionally 2 Syphilis, HSV 7/3 Occasionally IDU and sexual contact
(intravenously)
c
SC1283 29/male 2 4.7 VI 2 24 97 Heroin daily Safe use in 30 days Syphilis 1/6 NA IDU and sexual contact

H386 27/female NA 5.8 IV 1 26 5 Amphetamines 5–6 times per week (intravenously) 1 HSV 30/6 Occasionally IDU and sexual contact

R163 32/female 16 5.5 I–II 1 21 2 Heroin 2–5 times per day 2, 3 Syphilis, HSV 1/3 Yes IDU

R392 41/male 25 5.7 IV 1 29 4 Heroin daily, other opiates and psychostimulants 4 Negative 1/3 No IDU and sexual contact
1–2 times per week (intravenously)

R497 22/male 8 5.6 IV 1 26 10 Heroin 2–5 times per day, psychostimulants 1–2 2 Negative 3/3 Yes IDU
times per week (intravenously), methadone
occasionally (intravenously)

R526 30/male 7 5.7 I–II 1 28 3 Heroin daily 4 Negative 5/3 Occasionally IDU and sexual contact

R575 22/male 3 5.8 IV 1 18 5 Heroin 3–4 times per week 2, 3 Negative 0/3 NA IDU

R589 29/female 9 4.8 VI 1 29 10 Heroin daily 2, 3 HSV 1/3 NA IDU and sexual contact

K08 30/male 16 6.2 VI 1 22 7 Heroin 5–6 times per week 2, 3 Negative 0/3 NA IDU

K84 18/male 4 5.4 IV 1 29 5 Heroin 1–2 times per week Safe use in 3 months Negative 1/3 Yes IDU

NOTE. HD, Hamming distance; HSV, herpes simplex virus; IDU, injection drug use; NA, not available; STI, sexually transmitted infection.
a
1, Regular sharing of needles, syringe rinse water, cooker, and filter and front-load and back-load of drug from used syringe; 2, regular sharing of syringe rinse water, cooker, and filter and front-load and back-load
of drug from used syringe; 3, occasional sharing of needles; 4, occasional sharing of needles, syringe rinse water, cooker, and filter and front-load and back-load of drug from used syringe.
b
Estimation of the route(s) with the most risk of HIV-1 transmission.
c
Participant SC1283 was infected with 2 different HIV-1 genetic variants, CRF06_cpx and CRF06_cpx/subtype A recombinant.
Figure 1. Phylogenetic analysis of a cohort of injection drug users with human immunodeficiency virus type 1 (HIV-1) infection. A, Neighbor-joining
tree of HIV-1 env sequences (blue, individuals with multiple transmitted variants; red, individuals with a single transmitted variant; green, individuals
with chronic infection; black, reference strains). B–D, Highlighter plots revealing a single transmitted virus, transmission of 3 viral variants (V1, V2,
and V3), and a diverse viral population in a chronically infected individual, respectively. Horizontal lines represent full-length env sequences. Vertical
ticks indicate nucleotide mismatches from the consensus (green, adenine; red, thymine; orange, guanine; light blue, cytosine; gray, sequence gap).
Scale bar, genetic distance of 0.01 nucleotide substitutions per nucleotide.
K84) on the basis of unsafe drug use practices, consistent con-
dom use, and, in all but 1 case, an absence of sexually trans-
mitted infections. The remaining 8 participants had a risk of
transmission by injection drug use and sexual contact. How-
ever, even among the 5 participants for whom injection drug
use was the most obvious risk factor, the complexity of the
viral population indicated that a single variant was transmitted.
The epidemic of HIV-1 subtype A infection among IDUs in
the Russian Federation is known to have a recent origin and
has less diversity among isolates [14]. Consistent with a more
recent clonal introduction of HIV-1 subtype A in Russia, the
mean ( standard deviation) pairwise distance between the
consensus sequences of the variants from 12 participants (ex-
cluding SC1283) was 0.046 (0.010), compared with 0.073
(0.008) for 79 single-variant subtype B transmitted viruses
in North America [1] and 0.063 (0.008) for 54 single-variant
subtype C transmitted viruses in sub-Saharan Africa [3]. All of
these subtype A transmitted viruses were estimated to be CCR5-
tropic on the basis of the sequence of the V3 loop (specifically,
the presence of a basic amino acid substitution at position 11
or 25), with 1 exception—the virus from participant R589 ap-
peared to be a CXCR4-tropic virus on the basis of the presence
of lysine at position 25 of the V3 loop. The entry phenotype
of 8 of the presumed CCR5-tropic viruses has been confirmed
using cloned env genes in a pseudotype virus assay (data not
shown).
In this study, we have demonstrated the presence of a genetic
bottleneck during HIV-1 transmission among IDUs. In ∼70%
of the participants examined, HIV infection was initiated by a
single genetic variant. The frequency of the transmission of
multiple variants in this cohort of IDUs (30%) is higher than,
but not statistically different from, the frequency of the trans-
mission of multiple variants in cohorts of individuals with sex-
ually transmitted infection [1, 3]. Specific practices may affect
the probability of infection among IDUs and thus potentially
the complexity of the transmitted virus. For example, the mode
of unsafe drug injection, the frequency of coitus, and the prev-
alence of STDs could affect the complexity of the transmitted
virus within any given cohort. In addition, among a population
with high HIV-1 infection incidence like that in St Petersburg,
donors who are experiencing acute infection could transmit
multiple variants that would appear as if they were derived
from a single variant. Although the potential for the trans-
mission of multiple variants exists in IDU cohorts relative to
sexual transmission, a majority of the infections in our cohort
are being initiated with 1 virus; however, this conclusion is
limited by the relatively small sample size.
Little is known about the viral population during the first
few days after transmission. At one extreme, a single virus
infects a single cell and the successful propagation of this single
infectious event gives rise to the infection. At the other extreme,
multiple viruses are transmitted but the complexity is reduced
after multiple initial infectious events, which gives the appear-
ance of a low-complexity infection, as has been suggested else-
where [15]. In the former case, the relatively low frequency of
transmission would be consistent with exposure to a limiting
infectious dose that leads to a single infectious event. In the
latter case, infections would be expected to be more common,
because each infection could result in the transmission of mul-
tiple viruses that establish an initial infection, but some post-
infection phenomenon would winnow the population down to
a single genotype. However, it is clear that the viral population
is increasingly homogeneous in both humans and macaques at
times closer to the transmission event, at least for virus detected
in the blood [1, 7]. Thus, if there is a posttransmission bot-
tleneck, it must take place before systemic infection has oc-
curred, presumably at the site of transmission. Because trans-
mission by injection drug use does not include a mucosal site
barrier and represents just the systemic infection stage, the
simplest explanation for the low-complexity infection is that it
is the result of a single successful infectious event that gave rise
to the transmission. We conclude that the low complexity of
the virus in transmission by injection drug use may be related
to a small inoculum size, which results in a low probability of
transmission and most often allows for a single successful in-
fectious event. Recently, it has been reported that 6 of 10 IDUs
were infected with multiple variants [16], suggesting that, on
average, IDUs may be infected with multiple variants more
often than during sexual transmission, although due to the
small sample size there is not a statistically significant difference
between these results and those reported here. There is also the
possibility that different cohorts may have different risks due
to differences in the way drug-injecting equipment is used or
shared.
Additional studies among cohorts of IDUs will lead to a
better understanding of the natural history of and virus-host
interactions after parenteral HIV transmission, with important
implications for treatment, prevention, and vaccine strategies.
The Russian IDU cohort, with the low complexity of its trans-
mitted virus and the low diversity of the virus at this stage in
the epidemic, would have considerable advantages for the eval-
uation the efficacy of a prophylactic HIV-1 vaccine.
Acknowledgment
We thank the clinical, counseling, and recruitment staff, as well as the
participants in the HIV infection prevention studies.
References
1. Keele BF, Giorgi EE, Salazar-Gonzalez JF, et al. Identification and char-
acterization of transmitted and early founder virus envelopes in pri-
mary HIV-1 infection. Proc Natl Acad Sci U S A 2008; 105:7552–7557.
2. Haaland RE, Hawkins PA, Salazar-Gonzalez J, et al. Inflammatory gen-
BRIEF REPORT • JID 2010:201 (1 June) • 1701
 ital infections mitigate a severe genetic bottleneck in heterosexual trans-
mission of subtype A and C HIV-1. PLoS Pathog 2009; 5:e1000274.
3. Abrahams MR, Anderson JA, Giorgi EE, et al. Quantitating the mul-
tiplicity of infection with human immunodeficiency virus type 1 sub-
type C reveals a non-Poisson distribution of transmitted variants. J
Virol 2009; 83:3556–3567.
4. Sagar M, Lavreys L, Baeten JM, et al. Identification of modifiable factors
that affect the genetic diversity of the transmitted HIV-1 population.
AIDS 2004; 18:615–619.
5. Sagar M, Kirkegaard E, Long EM, et al. Human immunodeficiency
virus type 1 (HIV-1) diversity at time of infection is not restricted to
certain risk groups or specific HIV-1 subtypes. J Virol 2004; 78:7279–
7283.
6. Greenier JL, Miller CJ, Lu D, et al. Route of simian immunodeficiency
virus inoculation determines the complexity but not the identity of
viral variant populations that infect rhesus macaques. J Virol 2001; 75:
3753–3765.
7. Keele BF, Hui L, Learn GH, et al. Low-dose rectal inoculation of rhesus
macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal
infection by HIV-1. J Exp Med 2009; 206:1117–1134.
8. Rybarczyk BJ, Montefiori D, Johnson PR, et al. Correlation between
env V1/V2 region diversification and neutralizing antibodies during
primary infection by simian immunodeficiency virus sm in rhesus
macaques. J Virol 2004; 78:3561–3571.
1702 • JID 2010:201 (1 June) • BRIEF REPORT
9. Beyrer C, Baral S, Shaboltas A, et al. The feasibility of HIV vaccine efficacy
trials among Russian injection drug users. Vaccine 2007; 25:7014–7016.
10. Shaboltas AV, Toussova OV, Hoffman IF, et al. HIV prevalence, socio-
demographic, and behavioral correlates and recruitment methods among
injection drug users in St. Petersburg, Russia. J Acquir Immune Defic
Syndr 2006; 41:657–663.
11. Kozlov AP, Shaboltas AV, Toussova OV, et al. HIV incidence and factors
associated with HIV acquisition among injection drug users in St. Pe-
tersburg, Russia. AIDS 2006; 20:901–906.
12. Fiebig EW, Wright DJ, Rawal BD, et al. Dynamics of HIV viremia and
antibody seroconversion in plasma donors: implications for diagnosis
and staging of primary HIV infection. AIDS 2003; 17:1871–1879.
13. Abdala N, Krasnoselskikh TV, Durante AJ, et al. Sexually transmitted
infections, sexual risk behaviors and the risk of heterosexual spread of
HIV among and beyond IDUs in St. Petersburg, Russia. Eur Addict Res
2008; 14:19–25.
14. Bobkov AF, Kazennova EV, Selimova LM, et al. Temporal trends in
the HIV-1 epidemic in Russia: predominance of subtype A. J Med Virol
2004; 74:191–196.
15. Learn GH, Muthui D, Brodie SJ, et al. Virus population homogeni-
zation following acute human immunodeficiency virus type 1 infection.
J Virol 2002; 76:11953–11959.
16. Bar KJ, Li H, Chamberland A, et al. Wide variation in the multiplicity
of HIV-1 infection among injection drug users. J Virol (in press).