Co-existence of recent and ancestral nucleotide sequences in viral quasispecies of human immunodeficiency virus type 1 patients

Gonzalo Bello1, Concepción Casado1, Soledad García2, Carmen Rodríguez2, Jorge del Romero2 and Cecilio López-Galíndez1

1 Centro Nacional de Microbiología (CNM), Instituto de Salud Carlos III, Majadahonda, Madrid 28220, Spain
2 Centro Sanitario Sandoval (CSS), Comunidad Autónoma de Madrid, Madrid 28010, Spain

Correspondence
Cecilio López-Galíndez
clopez{at}isciii.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In human immunodeficiency virus type 1 (HIV-1) infection, the presence of divergent nucleotide sequences within a quasispecies has been associated with double infections or samples from different times or from different tissue compartments. The authors analysed HIV-1 proviral quasispecies from PBMC of three untreated Spanish patients displaying highly divergent nucleotide sequences without evidence of double infection. The origin of these nucleotide sequences was determined by phylogenetic analysis and by dating of the different groups using a genetic divergence versus sampling year plot from a set of Spanish samples. By their short genetic distance to the node of the patient's HIV-1 phylogenetic tree and by their early date of origin, close to the seroconversion time, some groups of sequences were considered ancestral. The presence within HIV-1 quasispecies of ancestral sequences, dated up to 10 years earlier than present ones, has important consequences for in vivo viral evolution, in the pathogenesis and treatment of HIV-1 infection.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human immunodeficiency virus type 1 (HIV-1) replication is inevitably associated with the accumulation of mutations due to the error-prone enzymic machinery of the virus. The variation in the in vivo evolution of HIV-1 is the result of the continuous virus replication and of host factors such as immune response, genetic susceptibility, or activation state (Fauci, 1996). As a consequence, HIV-1-infected individuals show heterogeneous viral populations of related genomes best described as viral quasispecies (Domingo & Holland, 1988; Eigen & Biebricher, 1988). Genetic analysis of patient viral populations has been extensively used to gain information on the pathogenesis of the virus as well as to clarify in vivo evolution of HIV-1 (Delwart et al., 1997; Lukashov et al., 1995; Plikat et al., 1997; Shankarappa et al., 1999; Wolinsky et al., 1996).

In HIV-1 quasispecies, the presence of highly divergent nucleotide sequences within quasispecies has been detected in sequential samples from the same patient (Delwart et al., 1997; Halapi et al., 1997; Wolinsky et al., 1996), and also from different tissue compartments of the same patient (Wong et al., 1997b; Delwart et al., 1998; Itescu et al., 1994; Poss et al., 1998). Highly divergent variants have been reported in single blood samples generally associated with double infections (Diaz et al., 1995; Sala et al., 1994; Zhu et al., 1995). In this study, we analysed HIV-1 quasispecies from three Spanish patients infected for more than 9 years and displaying divergent nucleotide sequences without evidence of double infection. We determined the origin of these nucleotide sequences by phylogenetic analysis and by dating the origin of the different clades forming the quasispecies. Nucleotide sequence groups within individual quasispecies had dates of origin up to 10 years apart. Some clades were considered ancestral because they dated close to the seroconversion time and they displayed a short genetic distance to the node of the patient's HIV-1 phylogenetic tree.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Study subjects.
Three asymptomatic and untreated HIV-1-infected individuals (identified as 30, 45 and 10) were selected from a group of 16 patients because of the presence of highly divergent nucleotide sequences (up to 12 %) between individual sequences and because the mean values between clades of the quasispecies were up to 9 % different. Samples were obtained from an outpatient clinic (Centro Sanitario Sandoval, CAM, Madrid) from two homosexuals and one intravenous drug user. The patients had been infected for 9–11 years and the seroconversion time was dated as the mean time between the last negative and the first positive serological test separated by a maximum of 1 year. Table 1 summarizes the clinical data of the patients. Patient 30 was followed yearly for 4 years. C1 and C2 were two asymptomatic HIV-1 patients used as controls because of a tight control of virus replication the 5 years for which they were followed. Patient C1, infected in 1997, showed an RNA viral load between 300 and 2500 copies (ml plasma)-1 and a genetic divergence of 0·3 % per year. Patient C2, infected in 1989, showed an RNA viral load between <50 and 2500 copies (ml plasma)-1 and a divergence of 0·1 % per year.


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Table 1. Clinical and virological data of patients

 
Separation of viral nucleic acids.
PBMC and plasma samples were obtained as described by Casado et al. (2001). Proviral PBMC-associated DNA was obtained from 1x107 cells by standard phenol extraction method. Viral RNA was isolated from 200 µl plasma according to Boom et al. (1990).

Amplification and quantification of viral nucleic acids.
PCR and RT-PCR amplification conditions were as described by Casado et al. (2001). Proviral load was carefully determined as previously described (Rodrigo et al., 1997) in the same nested PCR as used to amplify the entire gp120. To avoid genetic bottlenecking, the first PCR included at least 20 copies of proviral DNA and primers 91ECU (5'-CTTAGGCATCTCCTATGGC-3', 5956–5974 HXB2 position) and 22ED (described by Casado et al., 2001) were used. The second PCR included 1 µl of the first PCR products and primers 27EU (described by Casado et al., 2001) and 96ED (5'-AGACAATAATTGTCTGGCCTGTACCGT-3', 7862–7836 HXB2 position). Plasma HIV-1 RNA was quantified with the Amplicor HIV Monitor test kit with a detection limit of 50 copies ml-1 (Roche Diagnostics Systems), following the manufacturer's instructions. Nested RT-PCR was used to amplify the V3 env region. The first RT-PCR included at least 100 copies of viral RNA and primers 169ECU (5'-AATGGCAGCACAGTACAATGTACAC-3', 6945–6969 HXB2 position) and 96ED were used. The second PCR included 1 µl of the first RT-PCR products and primers 27EU and 28ED (5'-ATGAATTCTGGGTCCCCTCCTGAGGA-3', 7314–7339 HXB2 position).

Cloning and sequencing.
Two microlitres of the nested PCR products were ligated into plasmid pCR 2.1 and cloned according to the TA Cloning Kit instructions (Invitrogen). Eighteen to twenty clones per sample were sequenced with primer 27EU using the ABI PRISM Dye Terminator reaction kit (Perkin-Elmer) according to manufacturer's instructions in an ABI PRISM 377 automated sequencer (Perkin-Elmer).

Phylogenetic analysis.
All nucleotide sequence analyses were carried out on a 614 bp fragment of the env gene spanning from the distal portion of C2 to the proximal portion of C5 (nucleotides 7068–7682 in the HXB2 clone). Nucleotide sequences were aligned using CLUSTAL W (Thompson et al., 1994) and later hand edited. All positions with an alignment gap in at least one nucleotide sequence were excluded from the analysis, giving a 551 nucleotide fragment. All tree reconstructions in the study were performed by two independent methods: the neighbour-joining (NJ) method (with Kimura two-parameter model and a transition/transversion ratio of 2·0) in 1000 bootstrapped datasets as implemented in the MEGA version 2.1 program (Kumar et al., 2001); and by the maximum-likelihood (ML) method with the DNAML program (random input order, empirically found base frequencies and transition/transversion ratios and single rate of substitution) as implemented in PHYLIP version 3.6 (Felsenstein, 1993). The likelihood of the DNAML tree was significantly higher than that of the DNAMK tree (which assumes that all sequences are equidistant to the node of the tree) with a log likelihood ratio test (P<0·001).

Nucleotide distance analysis.
Intrasample (heterogeneity) and intersample (divergence) genetic distances were estimated by the Kimura two-parameter model (as indicated above) using MEGA version 2.1 (Kumar et al., 2001) in 100 bootstrapped datasets and by the F84 model (random input order, empirically found base frequencies and transition/transversion ratios and single rate of substitution) using the DNADIST program as implemented in PHYLIP version 3.6. Heterogeneity for patients 10, 30 and 45, and divergence for patient 30 clade a, were expressed as the mean distance for all pairwise comparisons between nucleotide sequences within a sample or from two different samples, respectively. Divergence for control patients C1 and C2 was calculated as the genetic distance between the global nucleotide sequences in two samples. The most recent common ancestor (MRCA) for each patient was inferred with the DNAML program (PHYLIP version 3.6) (Felsenstein, 1993). This sequence represents the most distal node of the tree originating all HIV-1 nucleotide sequences from the patient. Mean genetic distances between nucleotide sequence clusters and each patient MRCA were estimated by the F84 model (as indicated above) using DNADIST (PHYLIP version 3.6) (Felsenstein, 1993).

Dating of viral nucleotide sequences within quasispecies.
HIV-1 subtype B V3 env nucleotide sequences from 96 Spanish samples, collected from 1993 to 2002, were generated by us from homosexual and injecting drug users from the Centro Sanitario Sandoval (CAM, Madrid) and from the Hospital General de Navarra (Casado et al., 2000). A consensus nucleotide sequence (Spanish consensus), which included the most frequent nucleotide in each position, was generated as described by Casado et al. (2000). The MRCA for this nucleotide sequence set (Spanish MRCA) was also inferred by the DNAML program as explained above. Samples were grouped by the collection year, and the genetic distance of each sample to the Spanish consensus or to the Spanish MRCA was estimated by the Kimura two-parameter model and by the ML method, respectively, with the DNADIST program using the conditions described above. For dating of the patient's clades, the mean genetic distance of all pairwise comparisons between nucleotide sequences from each cluster to the Spanish consensus or to the Spanish MRCA was used (see Fig. 2).



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Fig. 2. Genetic divergence versus sampling time plot from a set of Spanish isolates. Nucleotide distance (D) to a Spanish consensus was calculated by the Kimura two-parameter model in 96 Spanish HIV-1 subtype B V3 nucleotide sequences and plotted against the sequence sampling year. Samples were taken from 1993 to 2002. Each data point represents a single patient sequence. The regression line and 95 % confidence intervals are shown. The calculated x intercept is 1981·5 (marked with an arrow on the plot).

 
Statistical analysis.
Means and standard errors (SE) for distances to MRCA were calculated with the Statgraphics Plus 5.0 program (Statistical Graphics Corp.). Linear regression analysis was done using GraphPad Prism version 2.01 (GraphPad Software).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of divergent sequences within HIV-1 proviral quasispecies
Phylogenetic analysis by the NJ method of the proviral quasispecies in the C2–C5 env region in three HIV-1-infected patients showed highly divergent nucleotide sequences (Fig. 1). All the nucleotide sequences of each patient formed monophyletic groups and were separated from nucleotide sequences of the other patients with 100 % bootstrap values, excluding the possibility of double infections. A similar analysis with the inclusion of 13 additional contemporaneous patient sequences from the local area supported the conclusion that the sequences of the patients studied are monophyletic (data not shown). In patients 30 and 45 three clusters, a, b and c, were observed and in patient 10 two clusters, a and b, were present. The extent of the groups was arbitrarily defined but statistically supported by bootstrap values higher than 70 % (see Fig. 1). Mean intergroup nucleotide distances ranged from 3·4 % to 9·3 % (see Table 2). The rooting and the tree topology were also estimated by the ML method with identical results (data not shown). The clinical characteristics of the patients are shown in Table 1. Patients were all asymptomatic, with 9–11 years since primary infection, but with differences up to 1 logarithm in viral and proviral loads.



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Fig. 1. Phylogenetic tree constructed by the NJ method of HIV-1 PBMC-derived C2–C5 gp120 env proviral DNA nucleotide sequences. Samples from patients 10 ({blacktriangleup}), 30 ({blacksquare}), and 45 ({bullet}) were taken 9 years after seroconversion for patient 10, and 11 years after serovonversion for patient 30 (1999 sample) and patient 45 (see Table 1 and Methods). Numbers at branch nodes refer to the number of booststrap repetitions (out of 1000) in which nucleotide sequences grouped together. Only frequencies greater than 70 % are shown. Nucleotide sequence clusters within each patient are grouped by vertical lines and denominated by lower-case letters a, b and c. The boxes identify recombinant genomes between sequences from different clusters. ‘?’ indicates unclassified sequences. Horizontal branch lengths are scaled and correspond to the nucleotide differences between nucleotide sequence and nodes.

 

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Table 2. Mean nucleotide distances between proviral sequences clusters within each patient

 
Characterization of the clusters within each HIV-1 proviral quasispecies
To characterize the clusters found in the viral quasispecies we performed two different analyses. First, we calculated by the Kimura and F84 model the genetic distance of each clade to the patient's MRCA. The genetic distance from each patient's MRCA to clusters 10 b, 30 c, and 45 b and c was significantly shorter than the distance to the other clusters (t-test; P<0·0001; see Table 3). The short distance of these sequence clusters to the patient's MRCA was suggestive of ancestral variants.


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Table 3. Genetic distances of the different clusters to MRCA

The MRCA for each patient pool of sequences was reconstructed as explained in Methods. Mean genetic distances to MRCA from sequence clusters were estimated by the Kimura two-parameter model (see Methods). Genetic distances estimated with the F84 model gave similar results (see Methods).

 
Second, we dated the different clusters in each quasispecies, using the correlation between the V3 nucleotide sequence divergence and sampling time previously obtained with a Spanish isolate set by our group (Casado et al., 2000). A significant correlation between the sampling year of the nucleotide sequence and the genetic distance to the Spanish consensus was found using a linear regression analysis (P<0·0001; Fig. 2). The accuracy of this plot for dating individual nucleotide sequences was tested with different types of data. First, the x intercept of this plot identified the date of the beginning of the HIV-1 epidemic in Spain to year 1981·5 (±2 years), which corresponds to the actual epidemiological data (http://www.msc.es/sida/). Second, we dated the viral sequences of control patients C1 and C2 which had the seroconversion time documented and showed very limited virus replication (see Methods). Sequences of patients C1 and C2 analysed 3 and 10 years, respectively, after seroconversion time dated very close to the patient's seroconversion year (see Table 4). For a 1999 sample of patient C1, who seroconverted in 1997, the distance value to the Spanish consensus was 0·091. Using this value in the correlation equation we dated the viral population to 1996. Similarly, a 1999 viral sample of patient C2, who seroconverted in 1989, was dated to 1987. The coincidence of the dates calculated with the divergence plot with the authentic ones proved the usefulness of the correlation plot for the dating of nucleotide sequences of Spanish isolates.


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Table 4. Dating of patient nucleotide sequences

 
The results of the dating of the different clusters within each viral quasispecies in the three patients are shown in Table 4. In a 1998 sample of patient 10, cluster a was dated to 2001 but the b population was dated to 1992. In a 1999 sample of patient 30, populations a and b were dated to 2000, but cluster c was dated to 1990. Finally, in a 1998 sample of patient 45, clade a dated to 2000, cluster b to 1993 and cluster c to 1991. A correlation between the sampling year of the nucleotide sequence and the genetic distance to the Spanish MRCA was also used for dating. No significant difference between the two regressions lines was observed (P=0·84), and very similar results were obtained with differences of less than 2 years (see Table 4). The dating of the different clusters confirms the co-existence in the same quasispecies of nucleotide sequences with very different dates with up to 10 years difference. By their earlier date and short distance to the tree node of each infecting virus, sequences of clade b in patient 10, clade c in patient 30 and clades b and c in patient 45 were considered ancestral.

Temporal evolution of proviral quasispecies
To further analyse the presence and temporal evolution of the different nucleotide sequence clades in the proviral quasispecies, the same viral population analysis was carried in consecutive samples of the two low viral load patients; the results are shown for patient 30 in Fig. 3. In the 1998 sample the quasispecies was dominated by the ancestral clade c and the modern clade b sequences, representing 52 % and 35 % respectively. In the following years there was a rapid turnover of the proviral population and the quasispecies was dominated by sequences of the recent clade a, although the recent b population showed a slower turnover than the ancestral c clade. Patient 45 showed a similar pattern of temporal evolution, with the detection of ancestral nucleotide sequences in the first two samples and their replacement by the recent ones in later samples (data not shown).



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Fig. 3. Temporal evolution of proviral DNA nucleotide sequence clusters of patient 30 over a 5-year period. (A) Phylogenetic tree by the NJ method of HIV-1 PBMC-derived gp120 C2–C5 env nucleotide sequences taken 10 ({bullet}), 11 ({blacklozenge}), 13 ({blacktriangleup}) and 14 ({blacksquare}) years after seroconversion, along with reference strains (italic) as outgroups. Conditions and symbols as described in Fig. 1. The box indicates a 2002 recombinant clone between a and c clusters. Arrows identify possible parental clones. (B) Histogram representing the proportion of the different sequence clades in the proviral population with time. ‘?’ indicates unclassified sequences.

 
Detection and analysis of recombinant genomes in the quasispecies
We detected recombinant genomes between sequences of different clades in the three patients analysed, as can be seen for patients 10 and 45 in Fig. 1 and for patient 30 in Fig. 3. These recombinants usually correspond to nucleotide sequences intermediate between recent and ancestral groups in the phylogenetic trees. The recombinant nucleotide sequences in patients 10 and 45 were confirmed by phylogenetic analysis (data not shown). However, the observed recombination could be the result either of an in vivo event or of a PCR recombination (Meyerhans et al., 1990) because of the co-existence in the same PCR of both parental groups. In patient 30, it was possible to detect an intermediate nucleotide sequence in the phylogenetic tree (boxed a/c sequence in Fig. 3). The nucleotide sequences showed that clone a/c was in fact the product of recombination between genomes of clusters a and c with a breaking point 18–19 amino acids downstream of V3 as shown in Fig. 4(A). The nucleotide sequence was split accordingly into a 5' region (from C2 to C3, Fig. 4B) and a 3' region (from C3 to C5, Fig. 4C). NJ analysis of the 5' region indicates that clone a/c was most closely related to recent cluster a sequence 12.01; conversely, analysis of the 3' portion indicates that clone a/c was most closely related to the ancestral cluster c sequence 7.98. Boostrap analysis indicated that these findings were robust. Because the recombinant clone a/c appeared in the 2002 sample where no ancestral nucleotide sequences were detected, it could only be the result of an in vivo recombination event.



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Fig. 4. Recombination analysis of the a/c recombinant clone in the 2002 sample of patient 30. (A) Predicted amino acid sequences in the gp120 of the recombinant clone a/c and of the potential parental clones 7.98 (cluster c, 1998 sample) and 12.01 (cluster a, 2001sample). Sequences are shown with the following symbols with reference to the baseline clone 7.98: dots (.), identity with 7.98 clone; hyphens (-), gap inserted to maintain alignment of sequence; replacements are indicated by the appropriate code letter. The arrow indicates the possible crossing-over point. (B, C) Phylogenetic analysis by the NJ method of the PBMC-derived sequences from patient 30 in the two gp120 segments C2–C3 (positions 7068–7281, B) and C3–C5 (positions 7281–7682, crossing-over defined by the crossing point, C) along with reference strains (italic). Conditions and symbols are as described in Fig. 1. Only bootstrap frequencies at the main nodes are shown.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work we report the co-existence of ancestral and recent nucleotide sequences in viral quasispecies of HIV-1-infected individuals. These highly divergent groups of sequences are each monophyletic groups, excluding the possibility of double infections.

The ancestral nucleotide sequences were defined by two criteria: first, by their short distance to the MRCA of each patient; and second, by the early date of the sequences reflected in the short distance to the Spanish consensus or Spanish MRCA sequence. The correlation between genetic divergence and time was used by our group (Casado et al., 2000) and by Lukashov & Goudsmit (2002) for the dating of the HIV-1 epidemic in Spain and the USA and the Netherlands respectively. Furthermore, a correlation between genetic divergence and time was also used for the dating of an individual sequence from an old HIV-1 sample (Zhu et al., 1998). Genetic distance to patient MRCA was also used to characterize the presence of ancestral sequences in treated patients (Gunthard et al., 1999). These analyses were based upon the existence of a molecular clock in in vivo HIV-1 evolution, which was demonstrated by Leitner & Albert (1999), using the same V3 region, and by Lukashov & Goudsmit (1997). There are controversies regarding the use of genetic distances for the dating of individual samples because of the wide dispersion of the genetic distance with time due to the heterogeneous nature of the HIV-1 infection (Korber et al., 1998; Wolinsky et al., 1996; Zhu et al., 1998). However, the genetic distance dispersion is bigger when comparisons are made between subtypes (Korber et al., 1999), but our analysis was performed in the context of an epidemic caused by the introduction and circulation of a single variant of subtype B in Spain (Casado et al., 2000). Finally, although every dating approach has an error and it is conditioned by the estimation used, the differences in the dates of the clades between 7 and 11 years were greater than any calculation error.

The old date and small genetic distance to patient's MRCA of the ancestral nucleotide sequences is incompatible with continuous virus replication for 9–10 years because divergence values for HIV-1 have been estimated at around 1 % per year and then could only be the result of viral latency (Shankarappa et al., 1999). In addition, the detection of these ancestral sequences at high proportion 10 years after their date of origin suggest a recent activation and/or a clonal expansion of the memory or naïve CD4+ T cell latent reservoir (Cheynier et al., 1998), but not the long-term persistence of ancestral sequences (Simmonds et al., 1991) because of the lack of intermediate sequences. The best evidence that the ancestral nucleotide sequences in the proviral population resulted in active replicating virus was the discovery, in the 2002 sample of patient 30, of one recombinant clone displaying ancestral and recent sequences. In our patients, however, the presence of the ancestral nucleotide sequences in the proviral viral populations was not correlated with their presence in the plasma viral populations. Plasma RNA viral quasispecies of patients 10 and 30 were analysed, but no RNA ancestral nucleotide sequences were detected, following the procedure described in Methods. This could be explained by the faster turnover of the viral RNA populations compared with the proviral ones (Simmonds et al., 1991), and/or by the lower fitness of early variants in comparison to late ones because of the continuous optimization associated with virus replication (Nijhuis et al., 1999). A similar result was observed in a study by Bagnarelli et al. (1999), where 12 out of 36 proviral clones were ancestral in one patient, while only one archival clone out of 44 was detected in the plasma.

The persistence of ancestral sequences could have important implications for the understanding of the in vivo evolution of HIV-1. First, the follow-up of patient 30 showed that changes in the proportion of the ancestral and recent clusters in the quasispecies have profound effects in the mean values of divergence, diversity and dN/dS (data not shown) and could result in discontinuities in the pattern of viral evolution as observed in other studies (Ostrowski et al., 1998; Simmonds et al., 1991). Second, the activation of HIV-1 variants resident in memory or naïve T-cells illustrates the importance of stochastic processes in HIV-1 population dynamics (Cheynier et al., 1994, 1998), although it does not rule out the impact of immune selective forces (Delwart et al., 1997; Lukashov et al., 1995; Wolinsky et al., 1996). In fact, while immune selection seems to drive evolution of clusters a and b, a stochastic activation probably conditioned the appearance of cluster c.

It is of great interest to know if the persistence of ancestral nucleotide sequences is a general phenomenon in HIV-1 infection. In the present study, the three patients analysed should be considered as slow progressors (SP) (see Table 2). A cluster of archival nucleotide sequences was also detected in another SP patient (Ostrowski et al., 1998) and in three Italian SP patients (Bagnarelli et al., 1999). Nevertheless, the detection of the persistence and activation of wild-type viruses after many years under antiretroviral therapy in HIV-1-infected patients (Finzi et al., 1997; Wong et al., 1997a), and the detection of ancestral nucleotide sequences in SIV infection (Ryzhova et al., 2002), suggest the generality of this phenomenon.

This work has shown the co-existence of recent and ancestral sequences within viral quasispecies in HIV-1-infected patients, and confirmed that a population of latently infected cells harbouring ancestral nucleotide sequences could persist despite many years (up to 10) of ongoing virus replication in untreated HIV-1 patients. The analysis of the causes and the frequency of ancestral viral variants will be helpful not only for the understanding of the evolutionary dynamics of HIV-1 in vivo but also for the pathogenesis and treatment of HIV-1 infection.


   ACKNOWLEDGEMENTS
 
Work in the CNM was supported by project SAF 2002/00626 and in part by the Red Tematica Cooperativa de Investigación en SIDA (Red de grupos 173) del FISss. Specific work at CNM and CSS was supported by FIS grant 00/0268.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 22 May 2003; accepted 26 September 2003.