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 (, , and ). Consistent with the findings of previous studies investigating viral fitness during untreated HIV-1 infection 
, the relative fitness of paired isolates obtained from 7 PHAEDRA subjects increased significantly over time (p
0.03; ). Viral fitness decreased over time following intermittent ART for 5 of the 6 PULSE subjects analysed (), an observation that might be expected due to the potential bottleneck imposed by suppressive ART. Most unexpected 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 () 
. 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 
. 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 
. 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 
. 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 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 ( and ). 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 
. 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 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 
. 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 
, 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 (). 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 
. 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 
. The fitness of Baseline isolates obtained from 6 of the 9 PHAEDRA subjects was also classified as low (). 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 
The accumulation of escape mutations can incur a high fitness cost to the virus, depending on the genomic location of the mutation 
. 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 
. 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 adaptive immune responses 
. 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 subjects.
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 (). 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 (). It has long been understood that HIV-1 can evolve separately in distinct physiological compartments 
. 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 
. 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 shown).
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 commencement 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.