Characterization of a new circulating recombinant form comprising HIV-1 subtypes C and B in southern Brazil
Santos, André Fa; Sousa, Thatiana Ma; Soares, Esmeralda AJMa; Sanabani, Sabrib; Martinez, Ana MBc; Sprinz, Eduardod; Silveira, Jussarac; Sabino, Ester Cb; Tanuri, Amílcara; Soares, Marcelo Aa
From the aLaboratório de Virologia Molecular, Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
bFundação Pró-Sangue, Hemocentro, São Paulo, SP, Brazil
cFundação Universidade do Rio Grande, Rio Grande, RS, Brazil
dHospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.
Received 10 March, 2006
Revised 4 July, 2006
Accepted 3 August, 2006
Correspondence and requests for reprints to Marcelo A. Soares, CCS Bloco A, sala A2-120, Cidade Universitária, Ilha do Fundão, 21949-570 Rio de Janeiro, RJ, Brazil. Tel: +55 21 2562 6383; fax: +55 21 2562 6396; e-mail: email@example.com
Objective: To identify a new circulating recombinant form (CRF) of HIV-1 comprising two circulating subtypes in the southern region in Brazil, subtypes B and C.
Methods: A total of 152 HIV-positive patients followed at two hospitals in southern Brazil had their viral pol genes isolated by reverse transcriptase–polymerase chain reaction (PCR) from plasma. PCR products were sequenced and phylogenetically analysed using HIV-1 subtype reference sequences. Six full-length subtype C viruses from Brazil previously described as ‘pure’ strains were included in the analysis. Sequences suggestive of recombination were analysed by bootscanning and phylogenetic analyses of separate fragments. The common ancestry of recombinant strains was evaluated by similarity plot and informative site analyses.
Results: HIV-1 subtypes commonly found in Brazil (B, C and F1) were observed. Sixty-two viruses were initially assigned as subtype C, but 15 viruses clustered in a separate internal clade. Pol from two full-length genomes of subtype C viruses grouped together with those samples. Bootscanning analysis showed that all 17 viruses had the same recombinant structure, with a 240 base pair fragment of subtype B in the middle of the reverse transcriptase pol region. Subtype B assignment of this fragment was confirmed by phylogenetic analyses using different methods of tree inference and cluster robustness tests. Mosaics were shown to have a common ancestry.
Conclusion: As CRF_BC represents 11% of the HIV-1 viruses circulating in the southern region of the country, which borders several south American countries, the assessment of its spread is of pivotal importance to the HIV/AIDS epidemic in Brazil and Latin America.
High genetic diversity is one of the major hallmarks of HIV-1. Phylogenetic analysis of numerous strains showed that HIV-1 can be divided into groups, subtypes, sub-subtypes, circulating recombinant forms (CRF) and unique recombinant forms . HIV-1 groups refer to the three very distinct lineages M, N and O. The vast majority of strains found worldwide are classified as group M. This group is the major group responsible for the AIDS pandemic. Group O, on the other hand, is endemic to Cameroon and neighbouring countries , whereas group N is represented by a limited number of isolates from Cameroon . Within group M nine subtypes have been characterized, A–D, F–H, J and K . Subtypes form roughly equidistant clusters in phylogenetic trees, being separated by 25–35% distance between env sequences and by 10–15% between pol sequences at the nucleotide level. Within some subtypes further phylogenetic structure can be identified, leading to a classification into sub-subtypes (A1 and A2, F1 and F2) [4,5]. This diversity of HIV-1 is the result of high rates of mutation of its reverse transcriptase  and rapid viral turnover in patients [7,8].
The phylogenetic analysis of the HIV-1 full-length genome sequences demonstrated that certain isolates clustered with two or more subtypes depending on the genomic region analysed [9,10]. These mosaic forms appear through HIV-1 recombination during reverse transcription [11,12], and this phenomenon promotes an additional source of HIV-1 variation. Some of the recombinant forms may achieve an epidemic level of dissemination, and currently there are at least 16 published different CRF (http://www.hiv.lanl.gov/content/hiv-db/CRFs/CRFs.html). More recently, CRF18_cpx  and CRF19_cpx  have been described.
In Brazil, the prevailing subtype is B, but other subtypes, such as F1, C, D, A and B/F co-circulate [15–18]. Analysis of protease (PR) and reverse transcriptase (RT) genomic regions have shown that subtype B was responsible for approximately 65% of infections in 2001, followed by subtype C (approximately 30%) . The same study showed that approximately 15% of samples were recombinants. Recent studies conducted by our group showed a high prevalence of subtype C in the southern region, including the states of Rio Grande do Sul and Paraná (prevalence of 45 and 30%, respectively) [19–21], and more recently six full-length genomes of local subtype C viruses were characterized .
A few studies of Brazilian B/F recombinants have been published [23–26], but CRF28_BF and CRF29_BF were described only recently . Given the high and similar frequencies of subtypes B and C in southern Brazil, we decided to investigate the potential generation of a CRF_CB in that part of the country.
Samples of 152 HIV-positive patients from two HIV/AIDS reference centres in Rio Grande do Sul, Hospital de Clínicas de Porto Alegre (in the capital of the state) and University Hospital of Rio Grande (in the city of the same name, further south in the state) were included this study. Subjects signed a written consent form for their inclusion in the study, and upon acceptance a single sample of peripheral blood was drawn. The date of HIV diagnosis was extracted from patients' medical records. This study was approved by the local hospitals' internal review boards.
RNA isolation, polymerase chain reaction and sequencing
HIV viral RNA was isolated as previously described . Complementary DNA synthesis and genomic amplification by polymerase chain reaction (PCR) of the HIV-1 pol fragment spanning the entire protease gene and approximately two-thirds of the RT gene (285 codons; nucleotides 2201–3353 relative to the HXB2 strain) were conducted in two steps with specific nested primers . A 1152 base pair (bp) fragment was obtained and purified using Microcon PCR cartridges (Millipore Corp., Billerica, Massachusetts, USA). Primers used in the PCR reactions were described elsewhere . Purified products were sequenced in an automated ABI Prism ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, California, USA). All sequencing chromatograms obtained were assembled with PC/Windows computers using the software SeqMan (DNAStar, Madison, Wisconsin, USA) and manually edited. For five strains out of the 15 generated in this study that grouped in a cluster suggestive of recombination (see below), a larger pol fragment (nucleotides 2157–5220 relative to the HXB2 strain) was PCR-amplified, sequenced and further analysed.
All sequences generated in this study were submitted to the GenBank database and were assigned the accession numbers AY275717–AY275802, AY390076, AY390179–AY390194, DQ190951–DQ191039 and DQ343964–DQ344021.
HIV-1 subtype determination
For HIV-1 genetic subtype determination and to discard sample mix-ups or contaminations, all sequences in FASTA format were aligned with reference sequences representative of all HIV-1 subtypes obtained from the Los Alamos database (http://hiv-web.lanl.gov) in ClustalW . In this analysis, we also included homologous pol fragments from six samples of Brazilian subtype C isolates recently described by Sanabani et al.  (GenBank accession nos. AY727522–AY727527). Aligned sequences were subjected to phylogenetic inference through the neighbour-joining method and Kimura two-parameter model implemented in the MEGA 2.1 package . One thousand bootstrap replicates were used to assess the phylogenetic robustness of the clusters. The sequence of SIVCPZGAB (GenBank accession no. X52154) was used as an outgroup to root the trees. In addition, maximum parsimony and maximum likelihood analyses were also conducted for the same dataset in MEGA 2.1 and in PHYML 2.4.4  to corroborate the neighbour-joining findings. In the maximum likelihood analysis, the nucleotide substitution model was estimated using Modeltest 3.7 . In addition to bootstrap analysis, the internal branch length test was used to evaluate the robustness of sequence clusters, implemented in MEGA 2.1.
Recombinant HIV-1 identification
HIV-1 subtype C sequences forming a high bootstrap-supported cluster inside the C clade were further analysed by bootscanning analysis implemented in Simplot 3.5.1 for Windows  using representatives of all HIV-1 subtypes. A sliding window of 300 bp, an increment step of 20 bp and the Kimura two-parameter model were used in the analysis. Bootstrap support was calculated based on 100 data replicates. Fragments of sequences assigned to specific HIV-1 subtypes were further confirmed by separate phylogenetic analyses conducted as described above. As two out of the six full-length subtype C genomes recently described in Brazil  grouped within the above-mentioned cluster, we re-analysed them by bootscanning in Simplot 3.5.1 with a sliding window of 400 bp and an increment step of 50 bp.
To characterize the recombination breakpoints suggested in the previous analyses more precisely, the putative recombinants were subjected to informative site analysis by generating consensus DNA sequences of CB recombinants, and of local subtype B and C viruses. Consensus sequences were generated with 60% of threshold frequency and were aligned in ClustalW and manually compared for sites that indicated identities between the recombinants and subtypes B or C. Statistical analyses were conducted for nucleotide frequencies around each recombination breakpoint in the datasets used for the generation of subtypes B and C consensus using Fisher's exact test.
In order to assess the common origin of the recombinant isolates, their sequences were subjected to similarity plot analysis using Simplot . Each CB recombinant sequence was compared with consensus sequences of the remaining CB recombinants, and with consensus of local subtype C and B sequences generated in this study.
HIV-1 subtype distribution
Viral RNA samples isolated from plasma of 152 patients were sequenced in the protease and RT genomic regions and subtyped through phylogenetic analysis. Sixty-two samples were initially assigned to subtype C and 70 to subtype B. Another six samples were identified as subtype F1, one as subtype D, and 13 represented mosaic viruses that had the protease gene of one subtype and the RT gene of another. These included the genotypes PRD/RTB, PRF1/RTB, PRB/RTC and PRC/RTB.
Neighbour-joining distance-based phylogenetic analysis has shown that the Brazilian subtype C viruses grouped in a cluster (with a bootstrap of 77%) when compared with subtype C viruses from other countries (Fig. 1). The interior branch test of phylogeny analysis rendered a probability of 93% for that clade, whereas maximum parsimony analysis showed a bootstrap of 65% (data not shown), also corroborating previous observations of monophyly by our group . Samples of subtypes B and F1 characterized in this study clustered into their respective groups with high bootstrap values in all trees (Fig. 1 and data not shown).
HIV-1 CB recombinant identification
Seventeen samples belonging to the Brazilian subtype C clade (15 characterized herein and two described by Sanabani et al. ) clustered in a group supported by a high bootstrap value (97%) external to the remaining isolates (Fig. 1). This cluster was also supported by maximum parsimony (bootstrap of 90%), by maximum likelihood (bootstrap of 96%) and by the interior branch test of phylogeny (99%) (data not shown). As this tree topology is suggestive of common ancestry, we decided to characterize those samples in further detail. Five representative isolates of the CB recombinant clade had a larger fragment of genomic cDNA amplified, encompassing the whole protease, RT and the 5′ half of integrase. Each of these isolates was subjected to bootscanning analysis. We included isolates 04BR137 and 04BR142 characterized in Sanabani et al.  that had clustered together with those samples, and the results are depicted in Fig. 2. All isolates had the same pattern of subtype recombination, with an internal pol fragment spanning approximately 240 nucleotides assigned to subtype B. All three fragments (both outer subtype C and inner subtype B) were separately subjected to phylogenetic analysis to corroborate the bootscanning results. The internal 240 bp assigned to subtype B from all isolates clustered with a bootstrap value of 64% (Fig. 3b), and an internal branch length test of 91% (data not shown), whereas the remaining fragments grouped with subtype C, particularly with the Brazilian reference isolate C.BR.92.U52953 (Fig. 3a and c). The phylogenetic support has undoubtedly enabled us to confirm the recombinant nature of those isolates. In addition, the bootscanning re-analysis with full-length genome of isolates 04BR137 and 04BR142 (previously described as pure subtype C in Sanabani et al. ) was performed. To our surprise, this analysis also confirmed the same recombinant breakpoints in the pol region (data not shown).
Informative site analysis of recombination breakpoints
To characterize the recombination breakpoints of our CRF more precisely, we performed an informative site analysis comparing consensus sequences of the CB recombinants with those of subtypes B and C. That analysis is depicted in Fig. 4. By analysing signature patterns among the three consensus sequences, we were able to narrow the recombination breakpoints down to nucleotides 712–731 and to 925–938 of our fragment (2965–2984 and 3190–3203 of HXB2, respectively). At positions 711, 732 and 924, 100% of the subtype C strains used for the consensus generation presented an adenosine, whereas all subtype B presented a guanine (P = 0.001082 for all three cases). At position 939, the frequency of cytosine was 100% in subtype C and 16.7% in subtype B (P = 0.004079). In external borders the mosaic consensus showed the same signatures of subtype C, whereas in internal borders it showed the same signatures of subtype B.
Common ancestry of CB recombinant viruses
We wanted to confirm further the epidemically circulating property of the CB recombinant strains. For this, we generated three sets of local sequences, one representing subtype B, another subtype C, and a third one with the CB recombinant strains (with the exception of the one queried). Each recombinant strain was compared against those three consensus sequences through similarity plots. The results of these analyses can be seen in Fig. 5. They revealed that all recombinant strains analysed were closer to their CB consensus throughout the fragment analysed, even when they belonged to subtypes B or C and were compared with subtype B and C strains from the same geographical region (Fig. 5 and data not shown). We used only the initial 950 bp fragment because we only had that information from the ‘pure’ local subtypes, but on the other hand that allowed us to test all 17 putative recombinant sequences available. All of them showed the same pattern of similarity (data not shown), indicating a common ancestry of all recombinant strains described.
Equation (Uncited)Image Tools
Earlier studies of HIV-1 subtype distribution in Brazil have shown the prevalence of approximately 3% of subtype C , but we and others have revealed the southern region as an endemic site for subtype C [19,20,33–35], notably in the state of Rio Grande do Sul. The molecular profile of HIV-1 subtypes observed in this study corroborates those previous observations. We found 62 samples belonging to subtype C, which represented 41% of the total.
Equation (Uncited)Image Tools
Phylogenetic analyses have shown that Brazilian HIV subtype C viruses clustered in a well-defined clade (Fig. 1), in agreement with previous studies [22,33]. Altogether, these data show that the introduction of subtype C viruses in Brazil was probably a single event. The maximum parsimony and interior branch test of phylogeny with the neighbour-joining distance method tree and Kimura two-parameter model confirmed such an hypothesis (data not shown).
We have observed a subcluster within the Brazilian subtype C clade in this study comprising 17 samples with high bootstrap value, including two samples, 04BR137 and 04BR142, which had previously been described as pure subtype C strains . A more detailed analysis revealed that all samples were CB recombinants and shared the same recombination breakpoint, suggesting that mosaic viruses shared a single ancestor. A 2242 bp fragment within the pol gene was confirmed by bootscanning analysis and further phylogenetic inference.
The probability of generating a CRF of HIV requires and is directly related to the co-circulation of distinct subtypes in a population. The high co-prevalence of subtypes B and C, which together made up 87% of the HIV viruses in the state of Rio Grande do Sul, is thus likely to generate CB recombinants. The presence of CB recombinants is also common in countries where these subtypes predominate, such as India [36,37], China [38–40], and in south American countries where subtype C has recently been introduced [41–43]. An informative site analysis that compared the CB recombinants with local subtype B and C viruses has also shown that the former shared signatures at the DNA level with the latter, suggesting a local origin (Fig. 4).
The subtype B genomic fragment incorporated into the recombinant form described here comprises codons 138–217 of RT, and therefore harbours several positions associated with resistance to nucleoside (codons 151, 184, 210 and 215) and non-nucleoside (codons 181, 188 and 190) RT inhibitors. None of the 15 mosaic isolates found here harboured drug resistance-associated mutations at those positions, and they were all drug naive at the time of sample collection (data not shown). The nature of the selective advantage in incorporating such an RT region into the CRF remains to be determined. One explanation could be that such a region is more prone to develop drug resistance in subtype B than in C, but there is no current evidence for this bias. Previous work from our group has failed to show any differences in genetic barrier for those two subtypes in developing RT drug resistance .
Many unique recombinant forms have been described in Brazil, mostly BF mosaics [19,20,23,25,26,45]. Previous studies by our group also identified unique CB recombinant forms in Brazil [19–21], but all have failed to detect CRF. More recently, two CRF_BF viruses were isolated in southeastern Brazil . In the present study, we gathered two full-length genomes previously sequenced in another study  and five partial genomes of different samples with the same mosaic CB structure, which formally characterizes the required evidence for describing a new CRF . Our bootscanning re-analyses of the isolates 04BR137 and 04BR142 showed that these are mosaics (data not shown) and not pure subtype C as previously characterized . One possible explanation for the discrepancy in the results obtained by us and in Sanabani et al.  is the size of the sliding window used in the bootscanning analyses. We used here a sliding window of 400 bp and an increment step of 50 bp, whereas Sanabani et al.  used a sliding window of 500 bp and the same increment step. A small region of approximately 200 bp could not be noticed with this size window, but only a peak for subtype B bootstrap. Although both analyses also differed in the substitution model used (Kimura two-parameter versus F84), this difference did not influence the results (data not shown).
Our samples presented the same breakpoint structure, a common origin and nucleotide signatures that indicated a local origin from subtype B and C isolates. To the best of our knowledge, this is the first concrete evidence of the existence of a Brazilian HIV-1 CRF comprising subtypes B and C.
Despite the fact that subtype C is responsible for more than 56% of HIV-1 infections worldwide , until now only three out of 29 CRF comprising subtype C had been described, CRF07_BC , CRF08_BC  and CRF10_CD . Our study may provide a new exemplar of CRF comprising subtype C in its structure.
It is noteworthy that among all samples from Rio Grande do Sul analysed in this study, 24% of the initially assigned subtype C samples now represent a CRF_BC. An independent study recently published  found mosaic samples with this same breakpoint structure in 43% of samples previously described as subtype C, corresponding approximately to 25% of the total viruses circulating in newly infected individuals. Interestingly, as we have data on the time of diagnosis for our patients, we can trace this CRF back to at least 1990, which means that it has been circulating in southern Brazil for over 15 years. We are currently estimating the more precise time of generation of this CRF in the region using coalescence techniques. Additional studies are necessary to obtain a complete understanding of the role of this CRF in the Brazilian and ultimately in the Latin American HIV/AIDS epidemic.
The authors would like to thank the staff from both clinical sites at Rio Grande do Sul, the Hospital de Clínicas de Porto Alegre and University Hospital of Rio Grande (Drs C.S. Moss, D.R. Mendoza and M. Gonzaga), for their help with patient care and sample collection, as well as to all patients who agreed to participate in this study. The authors are deeply indebted to Professor Rodrigo Brindeiro, Universidade Federal do Rio de Janeiro (UFRJ) for his support for this study and to Ana Flávia Pires and Renan B. Lengruber (UFRJ) for their technical support. Finally, they would like to thank Dr Carlos G. Schrago for helping with the phylogenetic analyses. This study is part of the dissertation of A.F.A.S. for his masters degree from the Graduate Programme in Genetics, UFRJ.
Sponsorship: Conselho Nacional de Desenvolvimento Científico e Tecnológico (A.F.S. scholarship and grant no. 403589/2004–5) and Fundação de Amparo Pesquisas do Estado do Rio de Janeiro (grant no. E-26/170.545/2004), Brazil.
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Circulating recombinant form; HIV-1; southern Brazil; subtype C; recombinant virus
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