High Viral Fitness during Acute HIV-1 Infection
Alicia Arnott1,2¤a, Darren Jardine1¤b, Kim Wilson1, Paul R. Gorry3,4,5, Kate Merlin6, Patricia Grey6,
Matthew G. Law6, Elizabeth M. Dax1,4, Anthony D. Kelleher6,7, Don E. Smith6¤c, Dale A. McPhee1,2,4* and
the Pulse Study Team"
1National Serology Reference Laboratory, St Vincent’s Institute, Melbourne, Victoria, Australia, 2Department of Microbiology, Monash University, Melbourne, Victoria,
Australia, 3Burnet Institute, Melbourne, Victoria, Australia, 4Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia,
5Department of Medicine, Monash University, Melbourne, Victoria, Australia, 6National Centre in HIV Epidemiology and Clinical Research, University of New South Wales,
Sydney, New South Wales, Australia, 7St. Vincent’s Centre for Applied Medical Research, Sydney, New South Wales, Australia
Several clinical studies have shown that, relative to disease progression, HIV-1 isolates that are less fit are also less
pathogenic. The aim of the present study was to investigate the relationship between viral fitness and control of viral load
(VL) in acute and early HIV-1 infection. Samples were obtained from subjects participating in two clinical studies. In the
PULSE study, antiretroviral therapy (ART) was initiated before, or no later than six months following seroconversion. Subjects
then underwent multiple structured treatment interruptions (STIs). The PHAEDRA study enrolled and monitored a cohort of
individuals with documented evidence of primary infection. The subset chosen were individuals identified no later than 12
months following seroconversion to HIV-1, who were not receiving ART. The relative fitness of primary isolates obtained
from study participants was investigated ex vivo. Viral DNA production was quantified using a novel real time PCR assay.
Following intermittent ART, the fitness of isolates obtained from 5 of 6 PULSE subjects decreased over time. In contrast, in
the absence of ART the fitness of paired isolates obtained from 7 of 9 PHAEDRA subjects increased over time. However, viral
fitness did not correlate with plasma VL. Most unexpected was the high relative fitness of isolates obtained at Baseline from
PULSE subjects, before initiating ART. It is widely thought that the fitness of strains present during the acute phase is low
relative to strains present during chronic HIV-1 infection, due to the bottleneck imposed upon transmission. The results of
this study provide evidence that the relative fitness of strains present during acute HIV-1 infection may be higher than
previously thought. Furthermore, that viral fitness may represent an important clinical parameter to be considered when
deciding whether to initiate ART during early HIV-1 infection.
Citation: Arnott A, Jardine D, Wilson K, Gorry PR, Merlin K, et al. (2010) High Viral Fitness during Acute HIV-1 Infection. PLoS ONE 5(9): e12631. doi:10.1371/
Editor: Esper Georges Kallas, University of Sao Paulo, Brazil
Received May 14, 2010; Accepted August 7, 2010; Published September 9, 2010
Copyright: ? 2010 Arnott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Alicia Arnott, Monash University Post-graduate Scholarship; Australian Centre for HIV and Hepatitis Research Grant; the Foundation for AIDS Research
(amfAR) Grant (106669) and the Australian National Health and Medical Research Council Project Grant (502617). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Institut Pasteur, Phnom Penh, Cambodia
¤b Current address: Department of Microbiology, St Vincent’s Hospital, Melbourne, Victoria, Australia
¤c Current address: Head of Research Development, Albion Street Centre, Sydney, New South Wales, Australia
" Members of the Pulse Study Team are listed in the Acknowledgments.
HIV-1 exists within the host as a swarm of genetically related
strains, termed quasispecies . The heterogeneity of the
quasispecies occurs largely as a result of the highly erroneous
reverse transcription process . Combined with the rapid rate of
virion production (between 108and 109virions per day) and the
large number of infected cells (107to 108), the result is a highly
diverse HIV-1 population [3,4,5]. Additionally, recombination
between distinct strains within a host can also occur, further
increasing diversity within the virus population [1,6].
The inherent genetic diversity of HIV-1 facilitates rapid
evolution and adaptation to a given or changing environment
within the infected host, referred to as viral fitness [6,7].
Adaptation of HIV-1 involves migration and dissemination
throughout the host, escape from adaptive and innate immune
responses, and from antiretroviral drug pressure . Fitness
therefore is dependent upon viral and host factors, and has been
associated with HIV-1 disease progression in individuals with
chronic HIV-1 infection [6,8,9]. It is thought that individuals
harbouring virus isolates that are attenuated or replicate poorly
are able to control virus replication and delay disease progression
compared with individuals infected with rapidly replicating virus
isolates. A correlation between poor ex vivo replication and VL
suppression was observed following analysis of individuals infected
with a nef/LTR attenuated strain [9,10,11,12,13,14]. In the
findings of Trkola et al. (2003), viral fitness of isolates obtained
prior to initiation of ART strongly correlated with the degree of
VL rebound following treatment cessation in a group of 20
individuals with chronic HIV-1 infection . A strong correlation
between ex vivo viral fitness and disease progression was
demonstrated following analysis of virus isolates obtained from
three well characterised long term survivors (LTS) of HIV-1
infection, and three individuals with chronic, progressive HIV-1
PLoS ONE | www.plosone.org1September 2010 | Volume 5 | Issue 9 | e12631
infection . Similarly, Campbell et al. (2003) reported a strong
linear relationship between HIV-1 replication ex vivo and plasma
VL for 12 individuals with chronic HIV-1 infection .
Collectively, these observations suggest a correlation between
ex vivo viral fitness and clinical outcome in chronic HIV-1 disease
Little is known regarding viral fitness during the acute phase of
infection. From what is known, the fitness of isolates present
during acute HIV-1 infection is thought to be low relative to
isolates present at later stages of infection, due to the significant
genetic bottleneck imposed upon transmission [1,18]. Indeed,
findings from two studies investigating founder viruses and viral
diversification in acute HIV-1 infection revealed that in the
majority of individuals investigated, infection occurred as a result
of transmission or expansion of a single founder virus [19,20]. The
genetic properties required for efficient transmission may differ
from those required for effective establishment and dissemination
of HIV-1 infection throughout the new host. As a result, the
adaptive potential of transmitted strains may be reduced .
To examine the relationship between ex vivo viral fitness and
control of VL in the acute or early chronic stage of HIV-1
infection in this study, viral strains obtained from participants of
two clinical cohorts were investigated [21,22]. Relative viral fitness
was assessed using a highly sensitive, quantitative real time PCR
(QPCR) assay to measure production of total HIV-1 DNA. Total
HIV-1 DNA production can be detected as early as 3 h post-
infection ex vivo, preceding production of integrated and circular
forms . Hence, total HIV-1 DNA production was thought to
represent a sensitive, early and reliable marker to assess the
relative viral fitness of isolates investigated in this study. We found
that ex vivo viral replicative fitness did not correlate with coincident
plasma VL from individuals in the acute and early chronic stages
of HIV-1 infection. Surprisingly, the fitness of isolates obtained
from individuals prior to, or immediately following seroconversion
to HIV-1 was equal to or greater than that of isolates obtained
from ART naı ¨ve individuals with early, chronic HIV-1 infection.
The results of this study suggest that despite the genetic bottleneck
occurring upon transmission of HIV-1, the replication capacity of
transmitted strains is not necessarily reduced. As viral pathoge-
nicity has been linked to fitness, the findings of this study also
suggest that the pathogenicity of isolates present during acute
HIV-1 infection may be higher than previously thought, perhaps
providing further evidence for the initiation of ART during this
phase of HIV-1 infection.
Plasma samples were obtained from 20 of 60 participants of the
PULSE study  (Table S1). The PULSE study was designed to
investigate whether individuals with acute HIV-1 infection could
suppress HIV-1 replication following multiple structured inter-
ruptions (STIs) to ART. Briefly, the PULSE study consisted of four
phases: A, B, C and D. Baseline plasma samples were collected
from subjects upon enrolment into the study, prior to initiation of
ART (Phase A). Subjects received ART [stavudine, lamivudine,
ritonavir-boosted indinavir with randomisation to hydroxyurea
(HU) or not] until plasma VL decreased to ,50 RNA copies/ml
for three consecutive months. Patients selected were stratified to
ensure a balance of acute or early primary HIV-1 infection (PHI)
with or without HU . Once VL was contained below detection
in Phase A, subjects underwent carefully monitored STI in Phase
B. Subjects remained off ART if the VL remained below 5 000
RNA copies/ml. Once the VL increased above 5000 RNA
copies/ml, ART was reinitiated as Phase C. Treatment interrup-
tion (Phase B) followed by reinitiation of ART (Phase C), occurred
a maximum of three times for each subject, prior to entry into
Phase D. Phase D was a follow-up phase, a period of clinical
monitoring following the completion of the mandated treatment
interruptions study .
Seventeen participants of the PHAEDRA study were investi-
gated in parallel with PULSE study subjects (Table S2). The
PHAEDRA study was a natural history cohort study, patients
could elect to be treated or not. It was established to monitor
immunological and virological characteristics of individuals with
acute and early HIV-1 infection. Documentation of acquiring
HIV within the past 12 months was the criteria for entry. This
particular substudy was restricted to a cohort of patients who had
elected not to receive ART. All these participants had serocon-
verted to HIV-1 at enrollment. Samples were collected at baseline
and 24, 36 and 52 weeks subsequently. Seroconversion for both
cohorts was defined according to stages described by Fiebig and
collegues  (Tables S1 and S2). For the subjects from whom
virus was successfully isolated and further study performed, at
baseline the PULSE subjects had a median Fiebig stage of 4 with a
mean VL and CD4 T cell count of 1 383 342 RNA copies/mL
and 533.5 cells/ml, respectively. At baseline, the median Fiebig
stage was 6 for the PHAEDRA subjects, with a mean VL and CD4
cell count of 159 286 RNA copies/mL and 720.7 cells/ml,
respectively (Tables S1 and S2).
Plasma samples were stored at 280uC, and patient PBMCs in
liquid nitrogen, until required. Research ethics approval was given
by St Vincent’s Hospital, Sydney, St Vincent’s Health, Melbourne
and the University of New South Wales Research Ethics
Committees. All participants signed an informed consent form
before study entry.
Peripheral blood mononuclear cells (PBMCs) were isolated by
density gradient centrifugation from buffy packs collected from
healthy, HIV-1 seronegative individuals, obtained from the
Australian Red Cross Blood Service (ARCBS, Melbourne,
Australia), as described . Cells were maintained in RF-10
medium (RPMI-1640 medium supplemented with 10% [v/v]
heat-inactivated foetal bovine serum, 0.03 mg/ml L–glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin), and activated
with 10 mg/ml of phytohemagglutinin (PHA) for 3 days prior to
infection with primary HIV-1 strains.
Replication of primary isolates can vary considerably in PBMCs
from different donors . To minimise the impact of donor
variability, all donor PBMCs used for virus isolation and viral
fitness experiments were screened against a diverse panel of
primary HIV-1 isolates to determine permissiveness to infection
with HIV-1, prior to use. Cells were selected for use in the fitness
assay based on the ability to support replication of a genetically
diverse panel of primary HIV-1 isolates . The level of CD4
expression on the surface of PBMCs capable of supporting
replication of genetically diverse primary HIV-1 strains was
significantly higher than on PBMCs that could not (Pate and
McPhee, unpublished). To further minimise the effects of donor
variability, pooled preferred PHA-PBMCs from two separate
HIV-1 negative donors were used for all experiments.
The reference isolate HIV-1MBC925was isolated from PBMCs
collected from an AIDS patient, and characterised as described
. This highly pathogenic, clade B, CCR5-using primary isolate
was selected as it had been observed to replicate efficiently and
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reproducibly in PHA-PBMCs (McPhee, D.A., unpublished) .
The use of HIV-1MBC925 also enabled a direct comparison
between the fitness of isolates present during acute infection
relative with that of an isolate obtained from an individual with
advanced disease. Virus isolation was attempted from 36 and 34
plasma samples collected from PULSE and PHAEDRA subjects,
respectively, by centrifugation over a 20% (w/v) sucrose cushion at
45 0006g for 1 h. Plasma was preferred as it best represents the
circulating quasispecies. The pelleted virus was resuspended in IL-
2 medium (RF-10 medium containing 10 U/ml IL-2 and 12 mM
HEPES) containing 16107PHA-PBMCs and cultured for 14 days
. Virus production was analysed by measurement of cell-free
reverse transcriptase (RT) activity or p24 antigen production.
Virus isolation was attempted from plasma collected at Baseline
prior to the initiation of ART, from all PULSE subjects, and from
any additional, available Phase B (STI) plasma sample with a VL
$5 000 RNA copies/ml. Coincident plasma VL measurements
ranged from 260 to 7 500 000 RNA copies/ml (Table S1). Isolates
were successfully obtained from 19 of the 36 plasma samples: 15
from Baseline and 4 from plasma collected subsequent to Baseline.
A strong correlation between plasma virus isolation from PULSE
subjects, and high coincident VL, was observed. Virus isolation
was unsuccessful from plasma samples with a VL ,153 000 RNA
copies/ml (Table S1).
Virus isolation was attempted from two plasma samples
obtained from each PHAEDRA subject: a sample collected at
Baseline and a sample collected at either week 24, 36 or 52
subsequent to Baseline. Two sequential isolates were successfully
obtained from 12 of the 17 PHAEDRA subjects (Table S2). Only
one isolate, obtained from plasma collected at Baseline, was
obtained from an additional PHAEDRA subject (data not shown).
Successful virus isolation from plasma obtained from PHAEDRA
subjects did not correlate with plasma VL (Table S2). A total of 25
viruses were isolated from PHAEDRA cohort members (Table
S2). The relative fitness of 18 of the 25 isolates was subsequently
investigated in this study.
Virus isolation was attempted from cryopreserved PBMCs
available from a subset of PULSE subjects, where plasma was
unavailable, or when virus isolation from plasma was unsuccessful,
using co-culture with preferred PBMCs as described above .
After recovery from storage in liquid nitrogen, the viability of all
PBMCs collected from PULSE subjects and subsequently used for
co-culture was $70% (data not shown). PBMCs were available
from two post-Baseline time-points from six of 10 PULSE subjects,
and one post-Baseline time point from a further four subjects, a
total of 16 samples. Following co-culture, eight additional isolates
were successfully obtained from eight PULSE subjects. There was
no correlation between VL and successful virus isolation from
PBMCs (Table S1). A total of 28 isolates were obtained from 16 of
20 PULSE subjects; the relative fitness of 24 of the 28 isolates was
Parallel infection assays
A standardised input of 600 pg of p24 antigen of each primary or
referenceisolatewasincubatedwith 26105PHA-PBMCsfor2 h,in
triplicate. Isolates were minimally passaged in an attempt to ensure
isolates reflected the replication competent virus present in vivo
[28,29]. Where 600 pg of p24 could not be achieved, undiluted
infection supernatant was added. Cells were washed in IL-2
medium and transferred to 96 well plates at 26105cells/well in a
final volume of 200 ml, achieved using IL-2 medium. Cells were
harvested at various time-points between 0 and 158 h post-
infection. Following harvest, cells were washed and resuspended
in200 ml of TEbuffer.To lyse infected cells,300 ml ofMagNA Pure
lysis buffer (Roche, Castle Hills, NSW, Australia) was added and the
cells incubated (15 minutes, room temperature). Lysed cells were
stored at 280uC until required for DNA extraction. Harvested
supernatant was stored at 220uC and virus production analysed by
measurement of cell-free RT activity or p24 antigen production.
DNA was extracted from HIV-1 infected PHA-PBMCs using the
Invitrogen Easy DNA kit as per the manufacturer’s instructions,
with the exception of the initial cell lysis step.
Measurement of total HIV-1 DNA production to estimate
relative viral fitness
To evaluate relative viral fitness, a quantitative real time PCR
(QPCR) assay was developed to measure production of total HIV-
1 DNA (extrachromosomal, integrated and 2-LTR circular forms)
for a period of between 96 and 158 h post-infection. Published
primer and probe sequences targeting a highly conserved region of
the 59-LTR and the human Albumin gene, were used (Figure 1)
[30,31]. Prior to use assay sensitivity and intra- and inter-assay
variation were extensively tested (Text S1; Tables S3 and S4). To
detect total HIV-1 DNA, the real time PCR reaction mix
contained 5 ml DNA in a final volume of 20 ml. The mix contained
QPCR Probe Mastermix (Integrated Sciences, Australia), 100 nM
dual labelled probe, 300 nM of HIV-1 LTR forward and reverse
primers, and nuclease free water (NFW). To detect Albumin DNA,
the real time PCR reaction mix contained 5 ml DNA in a final
volume of 20 ml. The reaction mix contained QPCR Probe
Mastermix, 100 nM dual labelled probe, 300 nM Albumin
forward and reverse primers and NFW. All DNA amplifications
were performed using a Stratagene MX3000P real time PCR
machine (Integrated Sciences, Australia) with the following
Figure 1. Primers and probes used in this study. The original sources and sequences of the primers and probes used in this study are
summarised. The fluorophores used were FAM and HEX for the HIV-1 LTR and albumin probes, respectively. The quencher used for both probes was
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conditions: 1 cycle at 95uC for 10 mins; 40 cycles at 95uC for 30
sec and 60uC for 1 min.
Quantification and calculation of relative viral fitness
As each strain was tested in triplicate, the mean Ct for each time
point was calculated following QPCR analysis. Copies of target
DNA were quantified by converting the mean Ct value generated
for each sample to DNA copies using the standard curve generated
by MX3000P software from the quantified DNA standards
included in each run. Copies of total HIV-1 cDNA were
calculated per 200 000 cells, the number of PHA-PBMCs in each
sample. Using the copies of HIV-1 DNA measured for each strain,
a relative viral fitness score was calculated for each isolate. Total
HIV-1 DNA production at 96 h post-infection was measured for
all isolates tested; hence calculation of viral fitness scores at this
time-point enabled direct comparison between the relative fitness
of all PULSE and PHAEDRA isolates tested. A second score at the
final time-point tested (either 110 or 158 h post-infection) enabled
analysis of DNA production for those isolates not detected at 96 h
post-infection. As the viral fitness measured in this study was
relative to that of the pathogenic reference strain HIV-1MBC925,
fitness scores for test strains were calculated relative to HIV-1
DNA production by HIV-1MBC925from coincident time points.
To calculate viral fitness scores, copies of DNA produced by test
strains were divided by copies of DNA produced by the reference
strain at a coincident time point post-infection [Fitness score =
(HIV-1 DNAT/HIV-1 DNAR)], where HIV-1 DNATand HIV-1
DNARcorrespond to copies of HIV-1 DNA produced by the test
and reference strains, respectively. Fitness scores throughout the
text and figures are represented as a fraction of 1. Isolates with a
relative fitness score of $0.1 were classified as fit; isolates with a
relative viral fitness score of 0.1 to 0.01 were classified as
moderately fit; the relative fitness of isolates with a score of ,0.01
was classified as low.
Reduced relative fitness of a nef/LTR attenuated virus
compared with a primary wild type HIV-1 strain,
Whether primary HIV-1 isolates of variable replicative fitness
could be distinguished on the basis of total HIV-1 DNA
production was investigated using HIV-1MBC925and a nef/LTR
attenuated isolate, HIV-1D36III. The HIV-1D36IIIisolate, obtained
from a long term non-progressor (LTNP), replicates poorly, as a
result of deletions/mutations in the nef/LTR region [9,11,32].
Production of viral DNA by both isolates was detected at four h
post-infection, however a significant difference in replicative fitness
over time was observed (Figure 2). The difference between total
HIV-1 DNA produced by HIV-1MBC925and HIV-1D36IIIat 96 h
post-infection was 38.7-fold (Figure 2). It has been observed by
studies in our laboratory, and those by Kim and collegues, that a
single replication cycle takes between 20 and 24 h [33,34]. Hence,
several rounds of infection were required to demonstrate
differences in the kinetics of total HIV-1 DNA production. A
slow/low replication phenotype was observed for the attenuated
virus strain compared with a fast/rapid DNA production profile
for the reference virus. Increased DNA production at all time-
points tested by HIV-1MBC925relative to HIV-1D36IIIindicated
that the replicative fitness of the reference strain was greater than
that of the attenuated isolate. For both virus infections there was
an increase followed by a modest decrease between 4 and 12 h
post infection as observed previously in a study of one step growth
kinetics of HIV-1 . Furthermore, these results indicated that
using the QPCR assay, primary HIV-1 isolates with variable
replicative fitness could be readily distinguished on the basis of
viral DNA production over several rounds of replication.
Decreased replicative fitness from acute to early chronic
HIV-1 infection, following treatment with ART (PULSE
The replicative fitness of isolates obtained from 14 PULSE
subjects was investigated using the QPCR assay. The reference
isolate HIV-1MBC925was cultured in parallel with test isolates in
each viral fitness experiment, enabling calculation of a relative
fitness score and to monitor any potential inter assay variation.
Two isolates from different time-points obtained from 6 PULSE
subjects, and 8 single isolates obtained from 8 PULSE subjects,
were tested (Figure 3). From the viral fitness scores calculated using
Figure 2. Production of HIV-1 DNA by a reference and a known
attenuated virus, quantified using the QPCR assay. PHA-PBMCs
were infected with 6 000 pg of p24 of the reference strain HIV-1MBC925
and known attenuated isolate HIV-1D36III, and cultured for 110 h.
Infected cells were harvested at 4, 8, 12, 24, 48, 72, 96 and 110 h post-
infection. DNA was extracted and the HIV-1 and albumin DNA
quantified using QPCR. Copies of HIV-1 DNA per 200 000 cells,
determined for each isolate, are plotted on a logarithmic scale against
time (A). In (B), relative fitness of HIV-1D36III was determined by
calculating the amount of HIV-1 DNA produced at 96 h post-infection,
expressed as a percentage of HIV-1MBC925DNA production at the same
time-point. The results are representative of three experiments.
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total HIV-1 DNA production at 158 h post-infection, isolates
obtained from PULSE subjects were categorised into three groups:
high fitness, moderate fitness and low fitness (Figure 3). The seven
isolates classified as highly fit were all obtained from plasma
collected at Baseline, during acute HIV-1 infection, and prior to
initiation of ART. For three BL isolates replication was near
equivalent to the reference strain used (Figure 3).
Four of the 8 isolates classified as moderately fit were obtained
from plasma collected at Baseline, four were obtained from plasma
collected during STI, subsequent to Baseline. The relative fitness
of eight isolates was classified as low, indicating that total HIV-1
DNA production by these isolates was less than 1% of total HIV-1
DNA production by the reference isolate at a coincident time-
point post-infection ex vivo (Figure 3). Six of the 8 isolates with low
fitness were obtained from plasma collected during STI. Viral
DNA was only detected after 96 h post-infection for these 8
isolates. The rapid kinetics of HIV-1 DNA production after 96 h
post-infection lead to selection of 158 h post-infection as the final
timepoint for analysis of relative fitness in subsequent experiments
(data not shown).
Over time, following the initiation of ART, the fitness of isolates
obtained from 6 PULSE subjects decreased (Figure 4). Due to the
small number of subjects analysed, the decrease observed was not
significant (p=0.14). Furthermore, although decreasing viral
fitness coincided with decreasing plasma VL for 4 of 6 subjects
from whom multiple isolates were obtained, overall, viral fitness
did not correlate with plasma VL for the 14 PULSE subjects
investigated (Borderline statistical significance p=0.051; Figure 5).
There was no correlation between CD4+ T cell counts and relative
viral fitness for the PULSE subjects investigated (data not shown).
Increasing replicative fitness during chronic HIV-1
infection (PHAEDRA subjects)
Sequential isolates from 9 of 12 PHAEDRA subjects were
analysed using the QPCR assay (Figure 6). Based on relative viral
fitness scores calculated using total HIV-1 DNA production at
Figure 3. Viral fitness scores and clinical data for PULSE subjects. Shown are clinical and experimental data obtained for PULSE subjects from
which virus was successfully isolated and subsequently tested using the real time PCR assay. Indicated by the column headings are the subject
identification code and seroconversion status at Baseline (‘+’ indicates subject had seroconverted, ‘2’ indicates subject was seronegative, ‘w+’
indicates that a weak antibody response was detected). Also shown are the number of STIs experienced by the subject, whether VL was suppressed
below 5 000 RNA copies/ml upon STI (indicated by ‘controller’ or ‘non-controller’), and the phase of the PULSE study during which the relevant
sample was collected. The time (in weeks) post Baseline that the sample was collected, coincident VL and CD4+T cell counts and the sample type
from which virus was successfully isolated, are also shown. Finally, viral fitness scores calculated using DNA production measured at 96 h post-
infection ex vivo, and at the final time-point analysed (158 h post-infection), are shown for each isolate. The fitness scores generated for the isolate
obtained from subject 3.13 were calculated from total HIV-1 DNA produced at 60 and 72 h post-infection. ‘ND’ indicates that the specified
measurement was not done.
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110 h post-infection, isolates were classified according to the 3
groups used above. Four isolates were categorised as highly fit, 3 of
which were obtained from plasma collected 36 weeks subsequent
to Baseline (Figure 6). In contrast, all of the isolates obtained from
PULSE subjects that were classified as fit were obtained from
plasma collected at Baseline, prior to the initiation of ART
Of the 5 PHAEDRA isolates classified as moderately fit, 3
isolates were obtained from plasma collected 52 weeks subsequent
to Baseline, and 2 isolates were obtained from plasma collected at
Baseline (Figure 6). The relative fitness of 9 isolates obtained from
PHAEDRA subjects was classified as low. Interestingly, 6 of the 9
isolates with low relative fitness were obtained from plasma
collected at Baseline, in contrast to results obtained for the PULSE
Over time, the relative fitness of isolates obtained from seven
PHAEDRA subjects increased significantly (p=0.03; Figure 7).
However, viral fitness was not found to correlate with plasma VL
following analysis of the 18 isolates obtained from PHAEDRA
subjects (Figure 8). In addition, relative viral fitness was not found
to correlate with CD4+ T cell counts for the PHAEDRA subjects
investigated (data not shown).
High relative fitness of isolates from acute infection
(PULSE) compared with early chronic HIV-1 infection
It is widely believed that, due to a genetic bottleneck occurring
upon transmission, the fitness of isolates present during acute
infection is low relative to isolates obtained later in infection
[6,35,36,37,38,39]. To investigate this, we compared the viral
fitness of isolates obtained from Baseline plasma from PULSE
subjects, to those collected 36 to 52 weeks post-baseline from
PHAEDRA subjects. The isolate groups were selected to enable
the relative fitness of viruses present during acute HIV-1 infection,
naı ¨ve to any selection pressures exerted by ART (PULSE), and
those found during untreated, early chronic infection (PHAE-
DRA), to be compared.
Relative to isolates obtained from PHAEDRA subjects,
replication of PULSE Baseline isolates was considerably slower,
with replication of 57% of isolates not detected by 96 h post-
infection. However, between 96 and 110 or 158 h post-infection,
total HIV-1 DNA production increased substantially, with
replication of 84% of PULSE viruses detected (Figure 3). By
comparison, replication of 39% of PHAEDRA post-baseline
isolates was not detected by 110 h post-infection ex vivo (Figure 6).
In addition, the increase in total HIV-1 DNA production between
96 and 110 h post-infection for isolates obtained from PHAEDRA
subjects was not substantive relative to wild type or the isolates
obtained from PULSE subjects (data not shown). We observed
that overall, PULSE Baseline isolates were slower to establish a
productive infection relative to the PHAEDRA post-baseline
isolates (Figure 9). From this we suggest that the genetic diversity of
isolates obtained post-Baseline from the PHAEDRA subjects was
greater than that of the PULSE isolates obtained at Baseline,
evidenced by greater relative adaptive ability. However, once
infection was established, the amount of HIV-1 DNA produced by
the PULSE Baseline isolates was comparable to, or higher than,
the amount of viral DNA produced by PHAEDRA post-Baseline
isolates (Figure 9). These findings provide evidence that the
relative fitness of isolates present during acute HIV-1 infection
may be higher than previously thought. When viral fitness scores
were plotted relative to the stage of seroconversion the results are
even more striking. The most fit viruses were observed during the
earliest stage of seroconversion monitored (Figure 10). Conceiv-
ably, in vivo viral fitness is compromised as HIV-1 infection
progresses, in response to selective immunological pressure on
Figure 4. Decreasing fitness over time observed following
analysis of paired isolates obtained from acute HIV-1 infection
subjects, measured using QPCR. Relative viral fitness scores were
calculated for isolates obtained from PULSE subjects and represented
on a box-plot. Only subjects from whom a Baseline isolate and at least
one additional isolate (Week 27 to 106) were obtained were included in
the analysis (n=6). Where multiple isolates from additional time-points
were obtained, the average of the combined viral fitness scores was
used. Shown are viral fitness scores calculated at the final time-point
tested (158 h; exception was 3.13 which was at 72 h) ex vivo for paired
isolates obtained from 6 PULSE subjects. The box represents the middle
50% of values for the data set, the solid line indicates the median value.
The vertical ‘whiskers’ extending from the box respectively indicate the
lowest and highest observed values. The open circle represents an
outlier; the asterisk represents an extreme outlier. The significance of
the observed changes in viral fitness over time is shown (p=0.14),
calculated using a signed rank test.
Figure 5. Viral fitness did not correlate with VL following
analysis of isolates obtained from acute HIV-1 infection
subjects. Coincident plasma VL measurements (log10RNA copies/ml)
were plotted against relative viral fitness scores (log10) for 16 isolates,
obtained from plasma, from PULSE subjects. The Pearson correlation
was rho=0.496, p=0.051.
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In this study we investigated the relative viral fitness of isolates
obtained from individuals with acute and early HIV-1 infection.
Temporal changes in relative viral fitness were observed for 6 and
10 subjects participating respectively in the PULSE (acute HIV-1
infection) and PHAEDRA (early HIV-1 infection) studies
(Figures 3, 4, 6 and 7). Consistent with the findings of previous
studies investigating viral fitness during untreated HIV-1
infection [6,15,36], the relative fitness of paired isolates obtained
from 7 PHAEDRA subjects increased significantly over time
(p=0.03; Figure 7). Viral fitness decreased over time following
intermittent ART for 5 of the 6 PULSE subjects analysed
(Figure 4), an observation that might be expected due to the
potential bottleneck imposed by suppressive ART. Most unex-
pected was the high relative fitness of isolates obtained from
PULSE subjects during acute HIV-1 infection, prior to the
initiation of ART, compared to isolates obtained from individuals
with early chronic HIV-1 infection. Furthermore, total HIV-1
DNA production by several PULSE Baseline isolates was
comparable to, or greater than that of the highly pathogenic,
primary reference isolate HIV-1MBC925 obtained from an
individual with AIDS (Figure 3) . These findings provide
evidence that despite the bottleneck occurring upon transmission,
the relative fitness of isolates present during acute HIV-1
infection may indeed be high.
To investigate relative viral fitness, a ‘parallel infection assay’
was used . Parallel infection assays have been successfully used
in other studies to examine replication of primary HIV-1 isolates
in primary cell types [35,40,41]. Alternatively, viral fitness can be
investigated using a growth competition assay, whereby replication
of test and reference strains is compared in the same culture,
primarily performed using recombinant viruses [6,15,17,42,
43,44,45]. The use of recombinant strains, as in recent studies
by Miura et al.,  and Kong et al.,  to investigate the
contribution of specific genes to the fitness of viruses during acute
infection, does not permit investigation of the fitness of the
circulating viral quasispecies. We used a parallel infection assay to
enable investigation of the replicative fitness of strains isolated
directly from patient plasma, to maximise the clinical relevance of
results obtained .
It is widely accepted that regardless of the route of HIV-1
infection, the virus encounters an extreme genetic bottleneck
upon transmission, resulting in a highly homogenous virus
population in the recipient [19,37,38,39,48]. Decreased genetic
diversity is thought to activate Muller’s ratchet , therefore,
the fitness of strains present during acute infection is thought to
be low. As 10 of the 20 PULSE individuals investigated had not
fully seroconverted to HIV-1 (Table S1), we anticipated that the
fitness of viruses isolated from coincident plasma samples would
be low. A virus population with highly constrained genetic
diversity would not be expected to readily adapt to an
Figure 6. Clinical and experimental data obtained for PHAEDRA subjects. Shown are clinical and experimental data obtained for PHAEDRA
subjects from which virus was successfully isolated and subsequently tested using the real time PCR assay. Indicated by the column headings are the
subject identification code, seroconversion status at Baseline (‘+’ indicates subject had seroconverted, ‘-’ indicates subject was seronegative), and the
phase of the PHAEDRA study at which the relevant sample was collected. The time (in weeks) post Baseline that the sample was collected, coincident
VL and CD4+T cell counts, and the sample type from which virus was successfully isolated are shown. The viral fitness scores calculated using DNA
production measured at 96 h post-infection ex vivo, and at the final time-point analysed (110 h post-infection), are shown for each isolate.
HIV-1 Viral Fitness
PLoS ONE | www.plosone.org7 September 2010 | Volume 5 | Issue 9 | e12631
environment distinct to that found within the host, such as the
ex vivo system used in this study to measure relative viral fitness
However, 7 of the 13 isolates obtained from plasma collected at
Baseline from PULSE subjects were classified as highly fit
(Figures 3 and 9). Indeed, analogous to the findings of this study,
rapidly replicating variants have been identified in similar,
smaller studies investigating the fitness of isolates present during
acute and early HIV-1 infection [6,36,40]. In the findings by
Ferbas et al. (1996) for one individual, high viral fitness was
observed following analysis of the ex vivo fitness of isolates
obtained at the time of peak viremia, but prior to seroconversion
. Kong et al. (2008) recently reported that strains with higher
Figure 7. Increasing viral fitness over time observed following
analysis of paired isolates obtained from early chronic HIV-1
infection subjects, measured using QPCR. Relative viral fitness
scores were calculated for isolates obtained from PHAEDRA subjects
and represented on a box-plot. Only subjects from whom a Baseline
isolate and at least one additional isolate (Week 36 to 52) were obtained
were included in the analysis (n=8). Shown are viral fitness scores
calculated at the final time-point tested (110 h) ex vivo for paired
isolates obtained from 8 PHAEDRA subjects. The box represents the
middle 50% of values for the data set, the solid line indicates the
median value. The vertical ‘whiskers’ extending from the box
respectively indicate the lowest and highest observed values. The
asterisk represents an extreme outlier. The significance of the observed
changes in viral fitness over time is shown (p=0.03), calculated using a
signed rank test.
Figure 8. Viral fitness did not correlate with VL following
analysis of isolates obtained from early chronic HIV-1 infection
subjects. Coincident plasma VL measurements (log10RNA copies/ml)
were plotted against relative viral fitness scores (log10) for 16 isolates,
obtained from plasma, from PHAEDRA subjects. The Pearson correlation
was rho=0.133, p=0.697.
Figure 9. Increased fitness of Baseline isolates obtained from
acute HIV-1 infection subjects relative to isolates obtained
subsequent to Baseline from early chronic HIV-1 infection
subjects. Relative viral fitness scores were calculated for isolates
obtained from PULSE and PHAEDRA subjects. Shown on a box-plot are
the viral fitness scores generated for the Baseline isolates obtained from
13 PULSE subjects compared with the viral fitness scores of the ‘Late’
isolates obtained from eight PHAEDRA subjects, at 96 h PI (A) and at
the final time-point tested (158 h PI for PULSE and 110 h PI for
PHAEDRA isolates; B). The box represents the middle 50% of values for
the data set; the solid line indicates the median value. The vertical
‘whiskers’ extending from the box respectively indicate the lowest and
highest observed values. Outliers are represented by an open circle;
extreme outliers are represented by an asterisk. The significance of
difference in viral fitness between the two groups at 96 h PI (A; p=0.12)
and the final time-point tested (B; p=0.45) is shown.
HIV-1 Viral Fitness
PLoS ONE | www.plosone.org8 September 2010 | Volume 5 | Issue 9 | e12631
replicative fitness with respect to the env gene were vertically
transmitted by mothers with chronic HIV-1 infection .
Combined with the observation of highly fit strains present
during acute HIV-1 infection in this study, these results suggest
the bottleneck that occurs upon initial transmission of HIV-1
does not necessarily result in loss of fitness.
The level of relative viral fitness has been linked to the genetic
diversity of the viral quasispecies. Kong et al. (2008) reported
transmission of multiple virus strains; Borderia et al. (2010)
recently demonstrated a direct correlation between increasing
genetic diversity and increasing in vivo viral fitness of clonal
populations [47,50]. Troyer et al. (2005) reported strong
correlation between genetic diversity of the viral quasispecies,
and ex vivo viral fitness . In our study, with subjects that were
therapy naive, viral fitness increased over time for 7 of the 9
PHAEDRA subjects investigated. Observations that genetic
diversity correlates with viral fitness are certainly not novel; fitness
of an RNA virus population increasing with genetic diversity is
described by the Red Queen hypothesis . This has been
applied extensively in the field of HIV-1 research [7,40], and is
highly relevant given the level of genetic diversity of the viral
quasispecies present in infected individuals. Cloning of the env
sequences of isolates obtained from PULSE subjects is currently
underway, to investigate whether the observed high level of fitness
correlated with genetic diversity of the quasispecies present at
baseline, during acute infection.
Following commencement and subsequent interruption of
suppressive ART, viral fitness decreased for 5 of 6 PULSE
subjects investigated (Figure 3). Analogous to the findings of this
study, reduced viral fitness was also observed for individuals
experiencing STI following initiation of ART during acute
infection by Wang et al. (2007) . Suppressive antiretroviral
therapy can result in the development of drug resistant mutations
in the viral quasispecies to evade inhibition, which has been
shown to reduce viral fitness [6,21]. Development of drug
resistance mutations in this study was not suspected as VL
suppression was observed upon resumption of ART in all PULSE
subjects investigated . Instead, analogous to the findings of
Wang et al., (2007)  and Borderia et al. (2010) ,
decreasing relative viral fitness over time was thought to be a
direct result of a genetic bottleneck created by suppressive ART,
activating Muller’s ratchet . Muller proposed that when
genetically diverse populations are randomly reduced, such as
during treatment with ART, or the development of potent
immune responses, the overall fitness of the population also
decreases [6,40]. The fitness of Baseline isolates obtained from 6
of the 9 PHAEDRA subjects was also classified as low (Figure 6).
At Baseline, all PHAEDRA subjects had clearly seroconverted to
HIV-1 (Table S2). The observed low relative fitness may have
resulted from mutation of the viral quasispecies as a direct result
of the development of potent immune responses following
seroconversion. Indeed, escape from targeted immune responses
has been observed in similar studies investigating anti-HIV-1
immune responses during early HIV-1 infection [17,53].
The accumulation of escape mutations can incur a high fitness
cost to the virus, depending on the genomic location of the
mutation [54,55,56]. Indeed, Goonetilleke and colleagues (2009)
reported that selection of viral escape mutants, following
development of adaptive T-cell responses, occurred rapidly
following containment of peak viremia in 4 individuals with acute
HIV-1 infection confirming earlier studies [57,58,59]. However,
there was no obvious fitness cost to the viruses studied .
Similarly, as relative viral fitness increased subsequent to Baseline
for 7 of 9 PHAEDRA subjects investigated in this study,
accumulation of deleterious mutations seems unlikely. Not as
restrictive as suppressive ART, development of potent immune
responses upon seroconversion may have created a ‘‘wider’’
bottleneck, limiting but not preventing the expansion and
diversification of the viral quasispecies . Consequently, we
propose that increasing fitness subsequent to seroconversion
observed for 7 of 9 PHAEDRA subjects occurred as a result of
virus evolution and diversification within the host to evade
Figure 10. Viral fitness of baseline isolates obtained from acute HIV-1 infection subjects (PULSE) and from early chronic HIV-1
infection subjects (PHAEDRA) relative to the stage of seroconversion as detailed by Fiebig and collegues (24). Baseline viral fitness
scores for both PULSE (orange) and PHAEDRA (mauve) subjects from final timepoints (110 h or 158 h) have been grouped according to the stage of
seroconversion (Fiebig stages IV, V or VI; reference 24) for direct comparison.
HIV-1 Viral Fitness
PLoS ONE | www.plosone.org9 September 2010 | Volume 5 | Issue 9 | e12631
adaptive immune responses [6,7,40,51]. Although contribution of
cellular immune responses to containment of virus replication has
not been investigated we are currently assessing neutralising
antibody responses for both the PULSE and the PHAEDRA
There were several limitations to the present study. The use of
an ex vivo system, such as that used in this and other studies, does
not reflect the sensitivity of the virus to antiretroviral drugs,
chemokines or additional inhibitory agents that may affect fitness
in vivo. Furthermore, for 6 of the 14 PULSE subjects from whom
plasma virus could not be isolated, virus was isolated from PBMC
(Figure 3). In addition to PBMC-derived isolates, for 5 of these 6
subjects, virus was obtained from plasma collected at distinct time-
points throughout the study. The fitness of both PBMC and
plasma derived viruses was subsequently investigated (Figure 3). It
has long been understood that HIV-1 can evolve separately in
distinct physiological compartments [60,61]. In addition, it is a
widely held belief that the current, circulating viral quasispecies
are present in the plasma and that cellular reservoirs of HIV-1
contain archived strains. However, the findings of recent studies
suggest otherwise [62,63]. Indeed, we observed that the kinetics of
HIV-1 DNA production by the PBMC-derived isolates tested in
this study were distinct relative to plasma derived isolates obtained
at different time-points from the same PULSE subject (data not
Combined, observations of the relative fitness of PULSE and
PHAEDRA isolates suggest selection of the fittest virus, or viruses,
upon transmission which progressively become less fit upon
development of adaptive immune pressure and/or commence-
ment of antiviral therapy. Further studies to investigate the long-
term impact of viral fitness on disease progression are warranted.
Muira and collegues recently reported the attenuated replication
capacity of isolates obtained from individuals who became HIV-1
controllers during early infection . In this study, none of the
PULSE subjects from whom Baseline isolates with high replicative
fitness were obtained controlled HIV-1 replication in the absence
of therapy (data not shown). Although the role of viral fitness in
disease progression remains unclear, what is clear from the
findings of this study is that the fitness of strains present during
acute/early HIV-1 infection can be high.
In conclusion, the findings of this study suggest that despite the
bottleneck transmission of a strain or strains with high relative
fitness does occur. Furthermore, these results suggest that viral
fitness decreases subsequent to the development of adaptive
immune pressure and/or commencement of antiviral therapy.
The findings of this study make a substantial contribution towards
understanding that the selection process during transmission of
HIV-1 from donor to recipient can be for a very fit virus.
Found at: doi:10.1371/journal.pone.0012631.s001 (0.04 MB
Detail of viral fitness QPCR assay validation.
Shown are the clinical results and the results of attempted virus
isolation from plasma or PBMCs obtained from PULSE subjects;
‘‘triangle’’ indicates that virus isolation was attempted from the
sample indicated. A single asterisk indicates the sample used for
virus isolation was plasma; a double asterisk indicates that virus
isolation was attempted from PBMCs when either plasma was not
available or virus isolation from plasma was unsuccessful. Shown is
the subject identification number followed by the phase of the
PULSE study during which the sample was collected. ‘‘A’’, ‘‘B’’
Clinical and virus isolation data for PULSE subjects.
and ‘‘C’’ indicate PULSE study Phases A, B and C. The
subsequent number indicates during which of up to three B or C
phases sample collection occurred; prefaced by ‘‘W’’ (weeks), the
following number indicates duration of the specified phase at
sample collection. Seroconversion status according to the Fiebig et
al  stages, coincident CD4+ T cell counts and plasma VL at
the time of sample collection, are shown: ‘‘.log10 5.88’’ indicates
VL was above the upper limit of detection, and was not quantified.
Whether subjects received HU in addition to ART is indicated.
Reverse transcriptase and p24 antigen EIA assay results,
performed following virus isolation, are also shown: ‘‘ND’’
indicates culture supernatant was not tested using the RT assay;
‘‘NQ’’ indicates that the relevant result for the isolate was above or
below the limit of detection for the assay and was not quantified;
‘‘2’’ indicates that virus isolation was attempted but RT activity or
p24 antigen were not detected.
Found at: doi:10.1371/journal.pone.0012631.s002 (0.32 MB
subjects. Shown are the clinical results and the results of attempted
virus isolation from plasma obtained from PHAEDRA subjects. A
single asterisk indicates the plasma sample from which virus
isolation was attempted. Indicated by the column headings are the
subject identification code, the phase of the PHAEDRA study at
which the relevant sample was collected, and seroconversion status
according to Fiebig et al . Coincident CD4+ T cell counts and
plasma VL at the time of sample collection are shown: ‘‘.log10
5.88’’ indicates VL was above the upper limit of detection, and
was not quantified. Reverse transcriptase and p24 antigen EIA
assay results, performed following virus isolation, are also shown:
‘‘ND’’ indicates culture supernatant was not tested using the RT
assay; ‘‘NQ’’ indicates that the relevant result for the isolate was
above or below the limit of detection for the assay and was not
quantified; ‘‘2’’ indicates that virus isolation was attempted but
RT activity or production of p24 antigen was not detected
Found at: doi:10.1371/journal.pone.0012631.s003 (0.17 MB
Clinical and virus isolation data for PHAEDRA
examine intra-assay variation, 20 replicates of each HIV-1 (A) and
albumin (B) DNA standard were tested in the same run. Data
represent the mean Ct value (Mean), standard deviation (SD) and
coefficient of variation (COV, expressed as a percentage) for each
standard. ‘‘N’’ indicates the number of replicates detected for each
Found at: doi:10.1371/journal.pone.0012631.s004 (0.04 MB
Intra-assay variation analysis for the QPCR assay. To
variation, five consecutive runs with the HIV-1 (A) and albumin
(B) DNA standards were performed. Standards were tested in
triplicate within each run. Shown are the mean Ct values obtained
for each standard following each of the five independent runs.
Data represent the total number of replicates detected (N), mean
Ct value (Mean), standard deviation (SD) and coefficient of
variation (COV, expressed as a percentage) for each standard.
Mean, SD and COV values were calculated using Ct values
obtained for each replicate detected of the specified standard. A
HIV-1 negative non-amplification control (NAC) was included,
consisting of cellular DNA. ND indicates that the specified sample
was not detected.
Found at: doi:10.1371/journal.pone.0012631.s005 (0.06 MB
Inter-assay variation analysis. To examine inter-assay
HIV-1 Viral Fitness
PLoS ONE | www.plosone.org10 September 2010 | Volume 5 | Issue 9 | e12631
Members of the Pulse Study Team comprised:
National Centre in HIV Epidemiology and Clinical Research, UNSW:
D Smith, K Petoumenous, K Irvine, P Grey, R Munro, M Law, J
Kaldor, and D Cooper.
St Vincent’s Hospital, Darlinghurst, Sydney:
A Carr, R Feilden, M Lacey, S Pett and DA Cooper.
407 Doctors, Darlinghurst, Sydney:
R Macfarlane, D Baker, W Genn, H McLeod, R Vale.
Holdsworth House General Practice, Darlinghurst, Sydney:
M Bloch, D Quan, D Austin, S Miller.
Taylor Square Private Clinic, Darlinghurst, Sydney:
R Finlayson, R Richardson, J Price.
Burwood Road Clinic, Burwood, Sydney:
Centre for Immunology, St. Vincent’s Hospital:
A Kelleher, J Zaunders, P Cunningham, C Satchell, M-L Munier, K
Prahan Market Clinic, Prahan, Melbourne:
N Roth, H Wood.
Victorian Infectious Disease Reference Laboratory, Melbourne:
C Birch, T Middleton.
Miami Sexual Health Clinic, Gold Coast:
Conceived and designed the experiments: AA DJ PRG PG ADK DES
DAM. Performed the experiments: AA KW. Analyzed the data: AA DJ
KW ML EMD ADK DES DAM. Contributed reagents/materials/analysis
tools: KW PRG KM PG ML EMD ADK DES. Wrote the paper: AA PRG
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HIV-1 Viral Fitness
PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12631