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Journal of Virology, October 2000, p. 8946-8952, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of Genital Tract Inflammation on Human Immunodeficiency
Virus Type 1 V3 Populations in Blood and Semen
Li-Hua
Ping,1,2
Myron S.
Cohen,1,2
Irving
Hoffman,1,2
Pietro
Vernazza,3
Françoise
Seillier-Moiseiwitsch,1,4
Hrishikesh
Chakraborty,4
Peter
Kazembe,5
Dick
Zimba,5,
Martin
Maida,5
Susan A.
Fiscus,1,6
Joseph J.
Eron,1,2
Ronald
Swanstrom,1,7 and
Julie A. E.
Nelson1,*
UNC Center for AIDS
Research1 and Departments of
Medicine,2
Biostatistics,4 Microbiology and
Immunology,6 and Biochemistry and
Biophysics,7 University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599; Institute
for Clinical Microbiology and Immunology, Kantonsspital, St.
Gallen, Switzerland3; and Lilongwe
Central Hospital, Lilongwe, Malawi5
Received 12 January 2000/Accepted 8 July 2000
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ABSTRACT |
We have examined cell-free viral populations in the blood plasma
and seminal plasma compartments of men infected with subtype C human
immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD)
clinic for genital tract inflammation in the form of urethritis (n = 43) or a dermatology clinic (controls,
n = 14) in Malawi. We have previously shown that the
presence of urethritis is associated with an eightfold increase in
virus load in the seminal plasma compartment (M. S. Cohen et al.,
Lancet 349:1868-1873, 1997). The purpose of this study was to
determine whether genital tract inflammation and its treatment caused
genetic instability in cell-free HIV-1 populations. In a
cross-sectional analysis at study entry, three-fourths of the STD and
control subjects had multiple V3 populations in their blood while 60%
of the STD subjects and 79% of the control subjects had multiple V3
populations in their semen. Overall, one-fourth of all of the subjects
showed discordance between results with blood and semen specimens when
samples were compared for the presence and absence of subpopulations.
When differences in the relative levels of abundance of bands were also
taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in
whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between
STD and control cohorts for these comparisons of the compartments in
this cross-sectional analysis at study entry. Longitudinal analysis of
the viral populations from two separate clinic visits over 1 to 4 weeks
showed that the complexity of each V3 population as measured by Shannon
entropy was different in blood and semen at the two time points,
indicating that the blood and semen constitute different compartments
for HIV-1. The seminal plasma compartment was more dynamic than the
blood plasma compartment for the STD subjects who were treated for
urethritis, with changes being noted in the presence or absence of
V3-HTA bands in the semen of 29% of these subjects but in the blood of
only 9% of these subjects. However, the changes were generally small.
Overall, our results suggest that 40% of male subjects show
discordance between seminal and blood viral populations and that the
complexity of each V3 population was different between the two
compartments. Both of these results point to the partial independence
of the seminal compartment as a viral niche within the body.
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INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) is primarily transmitted by sexual intercourse (23).
The efficiency of transmission of HIV-1 is likely to depend on the
concentration of HIV-1 in genital secretions; increased heterosexual
transmission has been associated with higher viral loads in blood
(17, 21, 22). The genotypic and phenotypic characteristics
of HIV-1 are also important for efficient transmission (12, 13,
31). The male genital tract represents a unique compartment,
since mutations conferring resistance to antiviral drugs, changes in
the viral envelope, and/or other characteristics can differ in HIV-1
sequences recovered from blood and semen (5, 7, 8, 11, 28, 32). The amount of HIV-1 (cell free and cell associated) in semen
can vary widely between different men (4, 7, 25) and can
vary over time in each man (16). While high HIV-1 levels in
semen are correlated with advanced HIV-1 disease, the virus has been
detected and/or cultured from semen collected during all phases of
infection (1, 25, 26, 30).
Classical sexually transmitted infections that cause genital ulcers or
mucosal inflammation facilitate transmission of HIV-1 by increasing the
infectiousness of the index case, the susceptibility of the exposed
sexual partner, or both (14). We have shown that men with
urethritis have higher HIV-1 levels in their semen than does a control
population and that appropriate antibacterial therapy reduces HIV-1
concentration in semen (6).
We have previously used a V3-specific heteroduplex tracking assay
(V3-HTA) to examine populations of HIV-1 variants in blood plasma
(18, 20). In the present study we used this assay to compare
populations of HIV-1 in the cell-free plasma fractions of blood and
semen in different patient cohorts over time and before and after
antibacterial therapy for urethritis. We focused on the virus from
cell-free plasma from blood and semen since cell-free virion pools
contain the actively replicating virus and, in one documented case, the
cell-free virus from semen from a transmitter contained the same
variant as was in the recipient (32). We were particularly
interested in determining the relationship between HIV-1 variants in
blood and semen because of previous work showing differences between
the peripheral blood and seminal cell compartments in men with subtype
B HIV-1 (8). In addition, our clinical study (6)
provided samples appropriate for evaluating the effect(s) of local
inflammation caused by urethritis on populations of HIV-1 in semen and blood.
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MATERIALS AND METHODS |
Source of patient samples.
A subset of the cohort of
HIV-1-infected men from Malawi originally described by Cohen et al.
(6) was chosen for this study. Blood and semen were
collected at 1- and 2-week intervals from two groups of men seen either
at a sexually transmitted disease (STD) clinic or (as controls) at a
dermatology clinic in Lilongwe, Malawi. A subset was chosen because
reverse transcription (RT)-PCR products for V3-HTA could be obtained
from matched blood and semen samples collected on the initial visits of
the subjects to either the STD clinic or the dermatology clinic. Men
from this subset were then selected for a second subset if RT-PCR
products for V3-HTA could be obtained from a second set of matched
blood and semen samples collected at a subsequent visit to one of the
clinics. All participants in the study were given physical exams and
were tested for STDs (6). Subjects were determined to have
urethritis if discharge was found during the physical examination and
there were at least five white blood cells per high-power field on a Gram-stained urethral smear. All of the STD subjects and none of the
controls in the present analysis had urethritis. Two of the control
subjects had syphilis, and three had asymptomatic trichomonas and were
included because they did not have urethritis. All subjects with STDs
were treated with appropriate antibiotics, and controls who did not
have an STD were not treated with any antibiotics.
Viral RNA isolation, RT-PCR, and V3-HTA.
Cell-free plasma
from blood and semen were separated as described previously
(6) and stored frozen. Viral RNA was extracted from the
blood plasma and seminal plasma using a QIAamp viral RNA extraction kit
(Qiagen, Valencia, Calif.). RT-PCR was performed as described
previously (20), with each RT-PCR being repeated at least
once to verify representative sampling of the RNA templates. V3-HTA was
performed with the subtype C probe as described previously (20). The consensus subtype B probe was constructed by
amplifying the same 159-bp V3 sequence as that used for the subtype C
probe from a JR-FL molecular subclone (pUC112-1 from Irvin Chen,
University of California, Los Angeles [15]) by using
the C+V3 and C
V3 primers (20). The PCR product was cloned
using a pT7Blue Perfectly Blunt cloning kit (Novagen, Madison, Wis.).
The subtype B probe was labeled by first digesting the plasmid with
NheI, end-labeling one strand by a fill-in reaction of the
NheI overhangs with 
-35S-dATP and the Klenow
fragment of DNA polymerase I (New England Biolabs, Beverly, Mass.), and
then inactivating the Klenow fragment and digesting with
KpnI. The labeled probe was then purified away from the
unincorporated nucleotides using a MicroSpin G-50 column (Amersham
Pharmacia, Arlington Heights, Ill.). V3-HTA with the subtype B probe
was otherwise performed in the same manner as with the subtype C probe.
For subjects with four samples (two blood plasma and two seminal plasma
samples), multiple V3-HTA bands first were scored for the presence or
absence of bands between samples. The relative abundance of each band
within a sample was then visually estimated for each subject to
generate a data set for statistical analysis.
Statistical methods.
We used Fisher's exact test for
hypotheses of independence between two factors with two levels when
tables contained cells with results from fewer than five observations,
and we used the chi-square test otherwise. To compare two groups with
more than two levels, we used the Mantel-Haenszel test. We used the
Wilcoxon rank sum test to compare medians between the STD and control
groups and used the Student paired t test to examine
subject-specific differences. To study the frequency distribution of
bands in a viral population rather than the total number of bands, we
computed the Shannon entropy, i.e.,
where p(i) is the relative frequency of band i
(i = 1,...n, where n is the total number of
bands) (24). This quantity measures the amount of
uncertainty in the distribution and was used in a similar manner by
Delwart et al. (9). It is 0 when there is a single band
(i.e., n = 1) and reaches its maximum value when the
observed bands are equally frequent [i.e., p(i) = 1/n
for all i]. We monitored the changes in the distribution of
bands in an individual via determining the entropy and looked for
changes in this value. To relate the changes in RNA values to changes in the entropy, we fitted a generalized-estimating-equation model with
an unstructured covariance matrix (for visits 1 and 2 in both STD and
control groups) using PROC GENMOD in SAS version 7.0 (SAS Institute,
Inc., Cary, N.C.).
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RESULTS |
The STD and control groups are well matched.
We have used a
collection of samples previously obtained (6) to examine the
effects of STDs on cell-free HIV-1 populations in blood and seminal
plasma. Paired blood and semen samples had been collected during
initial clinic visits by 43 HIV-1-infected men with urethritis from an
STD clinic cohort (Table 1) and 14 HIV-1-infected men from a dermatology clinic (control) cohort (Table
2). Age, CD4+ T-cell counts,
and viral loads in blood were similar in STD and control subjects.
Median ages were 28 years for the STD group and 29 years for the
control group, respectively (P = 0.94). Median CD4+-T-cell counts were 259/µl for the STD subjects and
268/µl for the control subjects (P = 0.73). Median
viral loads in blood plasma were 1.6 × 105 and
3.4 × 105 copies/ml for the STD and control groups,
respectively (P = 0.52). The median viral load in
seminal plasma was higher in subjects with urethritis (1.8 × 105 copies/ml) than in control subjects (6.2 × 104 copies/ml), but the difference was not statistically
significant (P = 0.31). Men in the larger STD cohort
from the original study (n = 86) had significantly
higher viral loads in semen than did men in the original control cohort
(n = 49) (6); however, only subjects from
whom V3 sequences could be reproducibly amplified from both blood and
semen were studied here.
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TABLE 1.
HIV-1 viral loads in blood and semen, the number of days
between the two clinic visits, CD4+ T-cell counts,
diagnosed STDs, and ages for the 43 subjects enrolled at the
STD clinic
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TABLE 2.
HIV-1 viral loads in blood and semen, the number of days
between the two clinic visits, CD4+ T cell counts, and ages
for the 14 control subjects enrolled at the dermatology clinic
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Gonorrhea was the most common cause of urethritis, being present in a
majority of the members of the small STD cohort studied
(Table
1); 10 subjects had trichomonas, 1 subject had chlamydia,
and 6 subjects had
more than one pathogen. Twelve subjects also
had a genital ulcer, which
we have previously shown is correlated
with increased excretion of
HIV-1 in semen (
10). Two of the
control subjects had
syphilis and three had asymptomatic trichomonas,
but none of these five
subjects had
urethritis.
Blood and seminal plasma samples from a second clinic visit by 34 of
the STD subjects and 12 of the control subjects were
also examined
(Tables
1 and
2). The subjects seen at the STD
clinic were treated for
their STDs with antibiotics, so that the
inflammation had cleared in
most of the men by the second clinic
visit. None of the subjects were
under treatment with antiretroviral
therapy. The median changes in
blood plasma viral loads between
the two visits were decreases of
3.6 × 10
3 copies/ml for the STD group and 2.3 × 10
4 copies/ml for the control group; neither of these
median changes
was statistically significant (
P = 0.78,
P = 0.66), and the medians
were not significantly different
from one another (
P = 0.67).
The median change in
seminal plasma viral load for the control
group was an increase of
2.5 × 10
4 copies/ml, which was also not statistically
significant (
P =
0.47). For the STD group, the median
change in seminal plasma
viral load after therapy was a decrease of
1.0 × 10
5 copies/ml, which was statistically
significant (
P = 0.04) and
was significantly different
from the median change in the control
group (
P = 0.003).
Cross-sectional comparison of the blood and seminal compartments at
the first visit.
HIV-1 populations in the cell-free fractions of
blood and semen samples from the first clinic visit were analyzed using
V3-HTA, and the patterns were scored for the presence of multiple
bands, with each band representing a distinct genotype. All of the
samples were first analyzed using a subtype C probe. However, because many of the subjects had only a single V3 population that was detected
with the subtype C probe, we also used a subtype B probe for V3-HTA
that was more sensitive to minor differences among subtype C sequences
because of the clusters of mismatches between the subtype B probe and
the subtype C sequences (Fig. 1A). By using both probes, a larger number of V3 variants could be identified. V3-HTA with the subtype B probe was done only on the RT-PCR products from those subjects for whom only single bands were seen in both the
blood plasma and seminal plasma compartments using the subtype C probe.
Figure 1B shows the V3-HTA results with the subtype B and subtype C
probes on the same samples from two representative subjects. For both
of these subjects, single bands with the subtype C probe were resolved
as multiple bands using the subtype B probe. Overall, 31 of the 43 STD
subjects (72%) and 10 of the 14 controls (71%) had multiple
populations in their blood. Similar results were obtained for the
seminal compartment with the two cohorts; 26 STD subjects (60%) and 11 control subjects (79%) had multiple genotypic populations in their
semen (P = 0.22).
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FIG. 1.
(A) Comparison of the probes used for V3-HTA.
The subtype C probe was cloned from subject C128 as described
previously (20), while the subtype B probe was derived from
the JR-FL molecular clone (15). (B) Examples of V3-HTA
patterns for two subjects that showed different numbers of bands with
the subtype C and subtype B probes. Only the bottom portions of the
gels are shown. The arrow indicates the position of the probe
homoduplex. Lanes are labeled by sample type at the first visit (blood
[1B] or semen [1S]).
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The V3-HTA patterns in the blood and seminal compartments of each
subject were next compared for differences in the number
of bands.
V3-HTA results illustrating examples of the V3 population
patterns are
shown in Fig.
2. Subjects S159 and C011
had the same
V3-HTA pattern in their blood and semen (Fig.
2A). In
contrast,
subject S171 had two V3 populations in his blood but only one
of them was detected in his semen, while subject C047 had two
V3
populations in his semen and only one was detected in his blood
(Fig.
2B). In all cases, the test was reproduced at least once
with each
sample to document sufficient template sampling to justify
the
comparisons.
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FIG. 2.
Examples of the V3-HTA patterns of subjects
with the same bands in blood and semen (A), subjects with different
bands in blood and semen (B), and subjects with different relative
levels of abundance of the same bands in blood and semen (C). The lanes
are labeled as in Fig. 1, and the arrow indicates the position of the
probe homoduplex. The V3-HTA patterns with the subtype C probe are
shown for subjects S099 and C034; the V3-HTA patterns with the subtype
B probe are shown for the other four subjects.
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The results of the compartment comparisons are shown in Table
3. Nearly every subject (in both the STD
and control groups)
with a single viral population in his blood had
that same population
in his semen. In the STD and control subjects with
multiple viral
populations in their blood, most had the same
populations in their
semen. Overall, less than one-fourth of the
subjects showed differences
between the two compartments. The STD and
control groups had similar
percentages of subjects in each category in
Table
3 (
P = 0.78),
indicating that the higher HIV-1
RNA load in the semen of men
with an STD did not change the likelihood
that their seminal virus
would be different from the virus in blood, at
least as assessed
by a determination of discrete V3 populations.
As a more sensitive measure of the similarity or difference between the
blood and seminal compartments, we rescored the V3-HTA
patterns for the
subjects with multiple bands in their blood for
differences in the
relative levels of abundance of the populations.
Examples of V3-HTA
patterns showing this type of difference are
shown in Fig.
2C. More
subjects showed differences between the
compartments by this analysis,
with half of the subjects with
multiple V3 populations having different
V3-HTA patterns between
blood and semen (16 of 31 STD subjects and 5 of
10 control subjects),
and again there was no difference between the STD
and control
subjects (
P = 0.75). When
relative-abundance differences are taken
into account, two-fifths of
all of the subjects showed discordance
between the
compartments.
Temporal changes in virus populations in blood and semen.
Blood and seminal plasma samples from two separate clinic visits were
analyzed by V3-HTA for 34 STD and 12 control subjects. The median time
between visits was 13 days for the STD group and 14 days for the
control group. Examples of the V3-HTA patterns seen in the comparison
of the two sets of blood and semen samples are shown in Fig.
3. Subject S094 had the same pattern in
all four samples, while subject S029 had the same pattern in his blood over time but a change in his seminal pattern over time. Subject S036
had the same pattern in his blood and semen at both visits, but the
pattern changed over time.
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FIG. 3.
Examples of V3-HTA patterns for subjects from whom
specimens were collected at two time points for each compartment. Lanes
are labeled by visit number (1 or 2) as well as sample type (blood
[B] or semen [S]). The subtype C probe was used for subject S094,
and the subtype B probe was used for subjects S029 and S036. The arrow
indicates the position of the probe homoduplex.
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The V3-HTA patterns were scored for each of the four samples from the
46 subjects first for changes in which bands were present
and then for
changes in the relative abundance of each band. Changes
in the blood
and the seminal compartments were analyzed in parallel
for each subject
for both analyses (Table
4). When only
the presence
or absence of specific bands was taken into account, the
virus
populations were stable in both compartments, since only one
control
subject (8%) showed changes in the seminal plasma and none
showed
changes in the blood plasma. For the STD subjects, the seminal
compartment was more dynamic while their genital inflammation
was being
treated because 10 of the STD subjects (29%) showed
changes in their
semen but only three of these subjects (9%) showed
changes in their
blood (
P = 0.02).
In the second analysis in which the relative abundance of each band was
also considered, similar results were obtained (Table
4). For the
control group, three subjects (25%) showed changes
in the viral
populations in their blood and three other subjects
showed changes in
their semen, which were not statistically different
between the
compartments (
P = 0.51). In the STD group, 3 subjects
(9%) showed changes in their blood while a total of 15 subjects
(44%)
showed changes in their semen, although in this case the
difference
between the compartments did not reach statistical
significance
(
P = 0.08). It should be noted that the subjects
included in these groups were selected based on our ability to
reproducibly amplify the V3 regions from all four samples. The
ability
to amplify the V3 region from extracted RNA tended to
correlate with
higher viral loads; thus, this subset of subjects
is, by necessity, a
biased sampling of the original cohort (
6).
The complexity of the V3 populations in both the numbers of bands
and abundance of each band was calculated as the Shannon
entropy.
This entropy was calculated for the four samples from
each subject.
Statistical modeling was done using the entropy
values and the viral
loads (at the first visit) from each compartment
for the STD and
control subjects together. Higher viral loads
in the blood at the first
visit were significant predictors of
higher V3 complexity at both
visits (
P < 0.0001), but the seminal
viral loads at
the first visit were not predictive (
P = 0.57).
The
complexity of the V3 populations in the blood plasma was different
from
that in the seminal plasma at each time point (first blood
sample
versus first semen sample and second blood sample versus
second semen
sample) and across time points (first blood sample
versus second semen
sample and second blood sample versus first
semen sample), indicating
that these are separate compartments
for HIV-1 (
P = 0.01 for all comparisons). The complexity of each
V3 population
was the same, however, between the two visits within
each compartment
(
P = 0.65 for blood plasma and
P = 0.95
for seminal
plasma). All of these comparisons held true for the STD
group
alone, but the control group sample size was too small for
reliable
parameter
estimation.
In only one of the analyses of these data was there a statistically
significant difference in the frequency of changes between
the two
compartments, which was for the STD group when changes
were scored by
the presence or absence of bands. When relative
abundance was included,
the differences between changes in the
blood and changes in the semen
were no longer statistically significant;
also, when the magnitude of
the differences was included in the
entropy calculation, the
compartments were the same. Therefore,
there are differences between
the seminal samples and the blood
samples at each time point, but the
differences are
small.
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DISCUSSION |
We examined the V3 populations in the cell-free fractions of
matched blood and semen specimens collected from men who were concomitantly infected with HIV-1 and one or more STD pathogens causing
urethritis. Virions in the cell-free fractions of both blood and semen
were studied because they represent the viral variants being produced
at the time of sample collection. At the initial visit, multiple V3
populations were present in the blood and semen from both the STD
subjects and a control population. About two-fifths of all of the
subjects had detectable differences in viral populations between the
blood and semen and the complexity of each V3 population was
significantly different between the compartments, as might be predicted
based on earlier work (5, 7, 8, 11, 28, 32). There were no
differences in these analyses between the STD and control groups at the
initial visit, indicating that the presence of urethritis did not
result in an increased likelihood of having multiple V3 populations and
did not change the relationship between the blood and seminal compartments.
We also compared the blood and seminal compartments over time. Our
results demonstrated that HIV-1 populations in blood remained largely
stable over the full 28 days of the study. However, the viral
populations in semen specimens from 29% of the treated STD subjects
showed changes in the presence or absence of V3-HTA bands over this
time, although the changes were generally small and this difference in
the frequency of changes in the two compartments was not significant
when it was calculated using values for the relative abundance of the
bands or entropy measures. This observation suggests that HIV-1
populations in the seminal compartment are more dynamic than in the
blood compartment, specifically while the viral load is decreasing
during treatment and resolution of genital inflammation.
Inflammation of the genital tract caused by sexually transmitted
diseases causes a significant increase in excretion of HIV-1 (reviewed
in reference 29) and likely facilitates transmission of HIV-1 (21). However, it is not clear whether the increase in viral burden in the genital tract results from HIV-1 replication in
cells already present in the genital tract, HIV-1-infected cells from
blood directed to the genital tract by inflammatory cytokines and
chemokines (2), transudation of cell-free virus from the
blood plasma to the seminal plasma, or combinations of the above.
However, increased recovery of HIV-1 in semen has been associated with
increased numbers of leukocytes in semen (1).
These results do not allow final conclusions about the source of HIV-1
in semen. However, if the influx of cell-free HIV-1 or HIV-1-infected
cells from blood was primarily responsible for the increased level of
HIV-1 in semen from men with urethritis, we would expect a stronger
relationship between HIV-1 in blood and semen from the STD subjects at
the initial visit. The continued change in HIV-1 in semen after
appropriate antibacterial therapy supports the idea of local genital
tract HIV-1 replication, likely in response to continued exposure of
HIV-1 to key inflammatory cytokines (2, 25).
We examined the viral populations in these compartments using V3-HTA in
order to understand better the relationship between HIV-1 in blood and
semen. We adapted the V3-HTA to detect viral populations based on other
features of V3 sequence variability because subtype C HIV-1, unlike
other subtypes, tends to remain R5 throughout the disease (3, 19,
20, 27). By using a probe for V3-HTA from a different subtype as
well as the homologous subtype C probe, we were able to increase the
sensitivity of the assay for other features of genetic variation (Fig.
1).
Limitations of this study should be emphasized. First, while V3-HTA can
identify subpopulations of HIV-1, such populations must represent at
least 3 to 5% of the whole (W. Resch, N. Parkin, E. L. Stuelke,
and R. Swanstrom, submitted for publication), and this method of
population characterization was restricted to the small region encoding
V3. Therefore, we would not be able to detect variations in minor
subpopulations or subpopulations defined by variability in other
regions of the viral genome. Second, our questions would be best served
if we were able to study subjects before, during, and after urethral
inflammation. However, we were able to examine each subject's HIV-1
populations only at the time of inflammation and briefly thereafter.
Our conclusions about the effect of genital inflammation on the
relationship between the blood and seminal compartments were drawn from
cross-sectional comparisons of the STD and control cohorts. Third, our
study cohorts were small subgroups of the original cohorts and were
biased toward higher viral loads in both compartments, since higher RNA
levels are generally correlated with better template sampling during PCR amplification. Finally, antibiotic treatment was a variable in this
study, since most of the control subjects did not receive antibiotics
and each subject who was treated was given different antibiotics
appropriate for his infection.
These limitations notwithstanding, the results of this study offer
further insight into issues critical to our understanding of the
transmission and the prevention of transmission of HIV-1 from men to
their partners. Reduction of viral burden in the semen of men with STDs
is not likely to reduce heterogeneity in viral populations. In
addition, biological interventions to prevent sexual transmission of
HIV-1 from men to their partners, whether through vaccines,
antiretroviral agents, or other means, must include an examination of
HIV-1 in the genital tract, from which the virus is sexually
transmitted. Populations of HIV-1 in blood cannot be expected to
represent fully the populations in the semen, especially since HIV-1
populations in semen can change without similar changes occurring in blood.
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ACKNOWLEDGMENTS |
We thank the AIDSCAP Malawi Research Group (E. Nkata,
E. Kachenje, T. Banda, G. Mughogho, C. Koller, J. Schock, G. Dallabetta, and B. Gilliam); Jerry Russell, Peter Killick, F. L. Musisi, and Terrie Taylor for administrative and logistical support;
John Schmitz, Michelle Fiodisi, Elaine Doherty-Leach, and Lance Nkana for technical and laboratory support; and Carol Porter for assistance with data management.
This work was supported by grants R01-AI44667 (R.S.), R01-DK49381
(M.S.C.), P30-HD37260 (UNC Center for AIDS Research), U01-AI31496 (UNC
Center for STD Research), N01-AI75329 (STD-CTU), and M01-RR00046 (UNC
General Clinical Research Center) from the National Institutes of
Health. This work was also supported by the National Institutes of
Health Office of AIDS Research, the U.S. Agency for International Development (USAID) as part of Family Health International's AIDS Control and Prevention Project (623-02380A-00-4031-00), the World Health Organization (SD1/94/009), and Pfizer Inc. P.V. was supported by
the Swiss National Science Foundation (3233-048902.96/1).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CB 7295, Room
22-062 Lineberger Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: (919) 966-5757. Fax: (919) 966-8212. E-mail: jaen{at}med.unc.edu.
Deceased.
| |
REFERENCES |
| 1.
|
Anderson, D. J.,
T. R. O'Brien,
J. A. Politch,
A. Martinez,
G. R. I. Seage,
N. Padian,
C. R. Horsburgh, Jr., and K. H. Mayer.
1992.
Effects of disease stage and zidovudine therapy on the detection of human immunodeficiency virus type 1 in semen.
JAMA
267:2769-2774[Abstract/Free Full Text].
|
| 2.
|
Anderson, D. J.,
J. A. Politch,
L. D. Tucker,
R. Fichorova,
F. Haimovici,
R. E. Tuomala, and K. H. Mayer.
1998.
Quantitation of mediators of inflammation and immunity in genital tract secretions and their relevance to HIV type 1 transmission.
AIDS Res. Hum. Retroviruses
14:S43-S49.
|
| 3.
|
Bjorndal, A.,
A. Sonnerborg,
C. Tscherning,
J. Albert, and E. M. Fenyo.
1999.
Phenotypic characteristics of human immunodeficiency virus type 1 subtype C isolates of Ethiopian AIDS patients.
AIDS Res. Hum. Retroviruses
15:647-653[CrossRef][Medline].
|
| 4.
|
Byrn, R. A., and A. A. Kiessling.
1998.
Analysis of human immunodeficiency virus in semen: indications of a genetically distinct virus reservoir.
J. Reprod. Immunol.
41:161-176[CrossRef][Medline].
|
| 5.
|
Byrn, R. A.,
D. Zhang,
R. Eyre,
K. McGowan, and A. A. Kiessling.
1997.
HIV-1 in semen: an isolated virus reservoir.
Lancet
350:1141[CrossRef][Medline].
|
| 6.
|
Cohen, M. S.,
I. F. Hoffman,
R. A. Royce,
P. Kazembe,
J. R. Dyer,
C. C. Daly,
D. Zimba,
P. L. Vernazza,
M. Maida,
S. A. Fiscus, and J. J. Eron, Jr.
1997.
Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1.
Lancet
349:1868-1873[CrossRef][Medline].
|
| 7.
|
Coombs, R. W.,
C. E. Speck,
J. P. Hughes,
W. Lee,
R. Sampoleo,
S. O. Ross,
J. Dragavon,
G. Peterson,
T. M. Hooton,
A. C. Collier,
L. Corey,
L. Koutsky, and J. N. Krieger.
1998.
Association between culturable human immunodeficiency virus type 1 (HIV-1) in semen and HIV-1 RNA levels in semen and blood: evidence for compartmentalization of HIV-1 between semen and blood.
J. Infect. Dis.
177:320-330[Medline].
|
| 8.
|
Delwart, E. L.,
J. I. Mullins,
P. Gupta,
G. H. Learn, Jr.,
M. Holodniy,
D. Katzenstein,
B. D. Walker, and M. K. Singh.
1998.
Human immunodeficiency virus type 1 populations in blood and semen.
J. Virol.
72:617-623[Abstract/Free Full Text].
|
| 9.
|
Delwart, E. L.,
H. Pan,
H. W. Sheppard,
D. Wolpert,
A. U. Neumann,
B. Korber, and J. I. Mullins.
1997.
Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.
J. Virol.
71:7498-7508[Abstract].
|
| 10.
|
Dyer, J. R.,
J. J. Eron,
I. F. Hoffman,
P. Kazembe,
P. L. Vernazza,
E. Nkata,
C. Costello Daly,
S. A. Fiscus, and M. S. Cohen.
1998.
Association of CD4 cell depletion and elevated blood and seminal plasma human immunodeficiency virus type 1 (HIV-1) RNA concentrations with genital ulcer disease in HIV-1-infected men in Malawi.
J. Infect. Dis.
177:224-227[Medline].
|
| 11.
|
Eron, J. J.,
P. L. Vernazza,
D. M. Johnston,
F. Seillier-Moiseiwitsch,
T. M. Alcorn,
S. A. Fiscus, and M. S. Cohen.
1998.
Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission.
AIDS
12:F181-F189[Medline].
|
| 12.
|
Fiore, J. R.,
A. Bjorndal,
K. A. Peipke,
M. Di Stefano,
G. Angarano,
G. Pastore,
H. Gaines,
E. M. Fenyo, and J. Albert.
1994.
The biological phenotype of HIV-1 is usually retained during and after sexual transmission.
Virology
204:297-303[CrossRef][Medline].
|
| 13.
|
Fiore, J. R.,
Y. J. Zhang,
A. Bjorndal,
M. Di Stefano,
G. Angarano,
G. Pastore, and E. M. Fenyo.
1997.
Biological correlates of HIV-1 heterosexual transmission.
AIDS
11:1089-1094[CrossRef][Medline].
|
| 14.
|
Fleming, D. T., and J. N. Wasserheit.
1999.
From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection.
Sex. Transm. Infect.
75:3-17[Abstract].
|
| 15.
|
Koyanagi, Y.,
S. Miles,
R. T. Mitsuyasu,
J. E. Merrill,
H. V. Vinters, and I. S. Y. Chen.
1987.
Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms.
Science
236:819-822[Abstract/Free Full Text].
|
| 16.
|
Krieger, J. N.,
R. W. Coombs,
A. C. Collier,
D. D. Ho,
S. O. Ross,
J. E. Zeh, and L. Corey.
1995.
Intermittent shedding of human immunodeficiency virus in semen: implications for sexual transmission.
J. Urol.
154:1035-1040[CrossRef][Medline].
|
| 17.
|
Lee, T. H.,
N. Sakahara,
E. Fiebig,
M. P. Busch,
T. R. O'Brien, and S. A. Herman.
1996.
Correlation of HIV-1 RNA levels in plasma and heterosexual transmission of HIV-1 from infected transfusion recipients.
J. Acquir. Immune Defic. Syndr.
12:427-428.
|
| 18.
|
Nelson, J. A. E.,
S. A. Fiscus, and R. Swanstrom.
1997.
Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay.
J. Virol.
71:8750-8758[Abstract].
|
| 19.
|
Peeters, M.,
R. Vincent,
J. L. Perret,
M. Lasky,
D. Patrel,
F. Liegeois,
V. Courgnaud,
R. Seng,
T. Matton,
S. Molinier, and E. Delaporte.
1999.
Evidence for differences in MT2 cell tropism according to genetic subtypes of HIV-1: syncytium-inducing variants seem rare among subtype C HIV-1 viruses.
J. Acquir. Immune Defic. Syndr.
20:115-121.
|
| 20.
|
Ping, L.-H.,
J. A. E. Nelson,
I. F. Hoffman,
J. Schock,
S. L. Lamers,
M. Goodman,
P. Vernazza,
P. Kazembe,
M. Maida,
D. Zimba,
M. M. Goodenow,
J. J. Eron, Jr.,
S. A. Fiscus,
M. S. Cohen, and R. Swanstrom.
1999.
Characterization of V3 sequence heterogeneity in subtype C HIV-1 isolates from Malawi: underrepresentation of X4 variants.
J. Virol.
73:6271-6281[Abstract/Free Full Text].
|
| 21.
|
Quinn, T. C.,
M. J. Wawer,
N. Sewankambo,
D. Serwadda,
C. Li,
F. Wabwire-Mangen,
M. O. Meehan,
T. Lutalo, and R. H. Gray.
2000.
Viral load and heterosexual transmission of human immunodeficiency virus type 1.
N. Engl. J. Med.
342:921-929[Abstract/Free Full Text].
|
| 22.
|
Ragni, M. V.,
H. Faruki, and L. A. Kingsley.
1998.
Heterosexual HIV-1 transmission and viral load in hemophilic patients.
J. Acquir. Immune Defic. Syndr.
17:42-45.
|
| 23.
|
Royce, R. A.,
A. Sena,
W. Cates, Jr., and M. S. Cohen.
1997.
Sexual transmission of HIV.
N. Engl. J. Med.
336:1072-1078[Free Full Text].
|
| 24.
|
Shannon, C. E., and W. Weaver.
1949.
The mathematical theory of communication.
University of Illinois Press, Champaign, Ill.
|
| 25.
|
Speck, C. E.,
R. W. Coombs,
L. A. Koutsky,
J. Zeh,
S. O. Ross,
T. M. Hooton,
A. C. Collier,
L. Corey,
A. Cent,
J. Dragavon,
W. Lee,
E. J. Johnson,
R. R. Sampoleo, and J. N. Krieger.
1999.
Risk factors for HIV-1 shedding in semen.
Am. J. Epidemiol.
150:622-631[Abstract/Free Full Text].
|
| 26.
|
Tindall, B.,
L. Evans,
P. Cunningham,
P. McQueen,
L. Hurren,
E. Vasak,
J. Mooney, and D. A. Cooper.
1992.
Identification of HIV-1 in semen following primary HIV-1 infection.
AIDS
6:949-952[Medline].
|
| 27.
|
Tscherning, C.,
A. Alaeus,
R. Fredriksson,
A. Bjorndal,
H. Deng,
D. R. Littman,
E. M. Fenyo, and J. Albert.
1998.
Differences in chemokine coreceptor usage between genetic subtypes of HIV-1.
Virology
241:181-188[CrossRef][Medline].
|
| 28.
|
Vernazza, P. L.,
J. J. Eron,
M. S. Cohen,
C. M. van der Horst,
L. Troiani, and S. A. Fiscus.
1994.
Detection and biologic characterization of infectious HIV-1 in semen of seropositive men.
AIDS
8:1325-1329[Medline].
|
| 29.
|
Vernazza, P. L.,
J. J. Eron,
S. A. Fiscus, and M. S. Cohen.
1999.
Sexual transmission of HIV: infectiousness and prevention.
AIDS
13:155-166[CrossRef][Medline].
|
| 30.
|
Vernazza, P. L.,
B. L. Gilliam,
J. Dyer,
S. A. Fiscus,
J. J. Eron,
A. C. Frank, and M. S. Cohen.
1997.
Quantification of HIV in semen: correlation with antiviral treatment and immune status.
AIDS
11:987-993[CrossRef][Medline].
|
| 31.
|
Zhu, T.,
H. Mo,
N. Wang,
D. S. Nam,
Y. Cao,
R. A. Koup, and D. D. Ho.
1993.
Genotypic and phenotypic characterization of HIV-1 patients with primary infection.
Science
261:1179-1181.
|
| 32.
|
Zhu, T.,
N. Wang,
A. Carr,
D. S. Nam,
R. Moor-Jankowski,
D. A. Cooper, and D. D. Ho.
1996.
Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission.
J. Virol.
70:3098-3107[Abstract].
|
Journal of Virology, October 2000, p. 8946-8952, Vol. 74, No. 19
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
