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Infection, Genetics and Evolution 6 (2006) 368–377

www.elsevier.com/locate/meegid

Analysis of the near full length genomes of HIV-1 subtypes


B, F and BF recombinant from a cohort of 14
patients in São Paulo, Brazil
Sabri Sanabani a,b, Walter Kleine Neto a, Erika M.N. Kalmar c,d,
Ricardo Sobhie Diaz b, Luiz Mario Janini b, Ester Cerdeira Sabino a,*
a
Fundação Pro-Sangue, Hemocentro, São Paulo, Brazil
b
Retrovirology Laboratory, Federal University of São Paulo, Brazil
c
Department of Parasitic and Infectious Disease, Faculty of Medicine, University of São Paulo, São Paulo, Brazil
d
STD/AIDS Reference and Training Center, São Paulo, Brazil
Received 11 August 2005; received in revised form 4 January 2006; accepted 6 January 2006
Available online 7 March 2006

Abstract
The human immune deficiency virus (HIV) exhibits strikingly tremendous amount of genetic variability. Such feature is critically
important for the virus to adapt to environmental changes by escaping the host immune system and by escaping candidate vaccine. Therefore,
understanding of such diversity is fundamental for the design of successful drugs or vaccine, which is urgently needed to bring the HIV/AIDS
epidemic under control. In this study, we investigated the magnitude of diversity of the HIV-1 near full-length genomes from
patients previously assigned as infected with non-recombinant HIV-1 subtypes B and F1 variants based on small portion of viral genome.
HIV-1 proviral DNA was extracted from 14 samples previously classified in our laboratory as six subtypes B and eight subtypes F on the basis
of small amplicon sequencing. Reamplifications of DNA from these samples were carried out by an overlapping PCR followed by
direct sequencing. The data were phylogenetically inferred. Sequence analysis revealed that two out of six partially identified subtype B and
six out of eight partially identified subtype F were in fact BF recombinants throughout their full genomes. Two pairs BF recombinants had
identical genomic recombination structure and distinct from the Argentinean CRF 12_BF strains, probably represents a novel circulating
recombinant forms in Brazil. Our data provided new genetic material of some of the HIV-1 subtypes currently circulating in the country
and points to the widespread of BF recombinants which are expected to change the epidemic nature by approaching the level of subtype B
in Brazil.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Full length genome; HIV-1 subtypes; HIV-1 recombination

1. Introduction genetic variations and pattern observed in phylogenetic


reconstruction, researchers have classified the virus into
Human immunodeficiency virus-1 (HIV-1) is the causative groups, subtypes and sub-subtypes (Robertson et al., 2000).
agent of AIDS and can persist in individuals for years before Currently, three groups (M, main; O, outlier; N, neither) have
causing disease. The HIV-1 genome is approximately 9.5 kb so far been recognized. HIV-1 group M viruses are
and displays significant sequence variations as a result of responsible for the current global epidemic and are further
constant mutation and evolutionary pressure. Based on these classified into nine (A–D, F–H, J and K) subtypes,
approximately equidistant, although subtypes B and D seem
to be more closely related. Moreover, early sequencing
studies have provided evidence of interstrand crossovers
* Corresponding author at: Head of the Molecular Biology Laboratory, between genomes of different HIV subtypes (Sabino et al.,
Fundação Pró-Sangue, Hemocentro de São Paulo, Av. Dr. Eneas de Carvalho
Aguiar, 155 18 andar, São Paulo 05403 000, Brazil. Tel.: +55 11 30615544x221;
1994; Robertson et al., 1995). Such interclade recombinant
fax: +55 11 30888317. strains are consistently reported from regions where two or
E-mail address: sabinoec@uol.com.br (E.C. Sabino). more clades are predominant. Recombinant strains from
1567-1348/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2006.01.003
S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377 369

unlinked epidemiological sources that exhibit identical 2. Materials and methods


patterns of mosaicism have been classified separately as
circulating recombinant forms (CRFs) (Carr et al., 1998). 2.1. Samples
There are currently 16 defined CRFs, namely CRF01–CRF16
and are epidemiologically important as subtypes. HIV-1 A total of 14 HIV-1 strains isolated from adult Brazilian
subtypes and CRFs show considerably different patterns of patients attending HIV/AIDS treatment centre were selected as
distribution in different geographical regions. representative of pure subtype B or F1 variants based on small
In South America, HIV epidemic is concentrated among portion of their genome. These isolates were genotyped in our
people at increased risk of infection like injecting drug users, laboratory by sequencing part of pol region to determine their
and men who have sex with men. The most prevalent HIV genotypic resistance to antiretroviral therapy. Sequence
genetic subtypes are subtypes B, BF recombinants and F informations from the V3–V5 env genome regions were
(Hierholzer et al., 2002). CRF12_BF and its related recombi- available from some samples. Partial genome sequencing
nant forms are widely circulating in Argentina (Carr et al., revealed eight isolates as subtype F1 and six as subtype B
2001; Thomson et al., 2002). variants. Based on these results, the isolates were submitted for
Brazil is the Latin American country that has been hardest complete genome characterizations. All patients were from São
hit by the HIV epidemic and has the second highest number Paulo (located at the south-east region of Brazil) and reportedly
of HIV-1 cases in the Americas, after the USA with an to had acquired their infection through sexual contact. Other
estimated number of 610,000 HIV-1/AIDS cases at the end of characteristics of the subjects are given in Table 1.
2001 (Global AIDS program, 2004). HIV-1 subtype B is a
major genetic form circulating in the country, however, 2.2. Methods
existence of small proportion of other subtypes such as F, C,
B/C and B/F have been consistently reported (Sabino et al., DNA was extracted from peripheral blood mononuclear cells
1994; Cornelissen et al., 1996; Bongertz et al., 2000; Caride (PBMCs) using the QIAamp blood kit (Qiagen GmbH, Hilden,
et al., 2000; Brindeiro et al., 1999). Typing strategies of these Germany) following the manufacturer’s instructions. Proviral
strains are based on sequencing of one or multiple short DNA was amplified from the purified genomic DNA by PCR
fragments of the viral genome, which may not accurately using primers and methods described by Thomson et al. (2002)
define the true HIV-1 genetic structure. Efforts to search for with little modifications. Briefly, five overlapping subgenomic
CRFs in Brazil in two recent studies did not find a common fragments were amplified in 50-ml volume reaction mixture
ancestry of Brazilian recombinants or their relationship to containing 0.5–1 mg of template DNA, 10 nmol of each dNTP,
CRF12_BF (Thomson et al., 2004; Sa Filho et al., 2005). The 20 pmol each of primers, 2.0 mM MgCl2, 10 mM Tris–HCl (pH
current study was undertaken to characterize HIV-1 near full- 8.3) and 0.50 U of Taq DNA polymerase (Invitrogen). Thermal
length genomes from patients previously assigned as infected cycling was essentially hot-started (Mastercycler Gradient
with non-recombinant HIV-1 subtypes B and F1 variants thermocycler; Eppendorf Scientific) at 94 8C for 5 min,
based on small portion of viral genome. followed by 35 cycles of 94 8C for 30 s, 58.5 8C for 30 s,

Table 1
Characteristics of patient samples included in this study
Sample Year of collection Sex Age Mode of transmission Subtype identification
Partial genome subtype Complete genome
region
01BR042 2001 M 38 Heterosexual F1 rt a BF1
01BR047 2001 M 38 Heterosexual F1 Protb, rt BF1
01BR087 2001 M 49 Bisexual F1 prot, rt and envc F1
01BR125 2001 F 39 Heterosexual F1 prot, rt and env F1
01BR226 2001 F 43 Heterosexual F1 prot, rt BF1
01BR323 2001 F 44 Heterosexual F1 prot, rt and env BF1
02BR002 2002 F 29 Heterosexual B prot, rt and env B
02BR005 2002 F 29 Heterosexual B prot, rt BF1
02BR006 2002 F 30 Heterosexual B prot, rt BF1
02BR008 2002 M 37 Homosexual B prot, rt B
02BR011 2002 M 27 Bisexual B prot, rt B
02BR013 2002 F 35 Heterosexual B prot, rt B
02BR033 2002 M 36 Bisexual F1 prot, rt BF1
02BR034 2002 M 31 Bisexual F1 prot, rt BF1
a
Reverse transcriptase.
b
Protease.
c
Envelope (V3–V5).
370 S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377

and 72 8C for 3 min; and a final extension step at 72 8C for weight matrix. Aligned sequences were manually edited and
10 min. The first round PCR products (2 ml aliquots) was then trimmed to the minimal shared length in the BioEdit Sequence
amplified with the nested primers under the same conditions. Alignment Editor Program (Hall, 1999).
Efficient PCR amplification was optimized in advance by The gap-stripped aligned sequences were screened for the
manipulating the concentration of MgCl2 and annealing presence of recombination patterns by the similarity plot and
temperatures using an Eppendorf gradient PCR cycler. The bootscan methods as implemented in the SIMPLOT program v
expected sizes of the amplified products were verified using 3.2 beta (Lole et al., 1999; Salminen et al., 1995). These
ethidium-bromide staining after agarose gel electrophoresis. methods identify putative positions of recombination by
Both DNA complementary strands were sequenced directly visually plotting percentage of permuted trees along the length
from purified PCR products in an overlapping fragment of 400 of each sequence. Sequences with suggestive recombination
nucleotides by using variety of internal sequence specific against reference panel by similarity plot were subsequently
primers, fluorescent-dye terminators, and Taq polymerase on an analyzed by bootscan. For both methods, nucleotide distances
automated sequencer (ABI 3100, Applied Biosystems Inc., were calculated in a sliding window of 420 bp moving in steps
Foster City, CA). The data from the sequenced fragments were of 20 bp by using the F84 model of evolution (maximum
edited, assembled into contiguous sequences, and a consensus likelihood) and transition/transversion ratio of 2.0. Bootstrap
of the both strands was formed by the Sequencher program values of 70% were considered definitive for assignment of
(Gene Code Corp., Ann Arbor, MI). All the sequences were parenthood. Exploratory trees of DNA segments between
checked for contamination by BLAST search against HIV-1 breakpoints were constructed.
sequence database and among themselves (Korber et al., 1995). Phylogenetic relationships were established by maximum
Full genome sequences were aligned with reference likelihood and neighbor joining methods with PAUP*4.0b10
sequences representing subtypes A–D, F–H, J and K obtained (Rogers and Swofford, 1998). Model-test (Posada and Crandall,
from the Los Alamos database (http://hiv-web.lanl.gov) using 1998) was used to determine the optimal nucleotide substitution
the CLUSTAL X program (Thompson et al., 1997) with the model for each sequence. The substitution model GTR + I + G
‘‘slow-accurate’’ default alignment parameters and IUB DNA was chosen as the best fitting evolutionary model for likelihood

Fig. 1. Phylogenetic relationship generated from nearly full-length HIV-1 sequences of six non-recombinant Brazilian isolates from the current study (Marked with
black circles and rhombus). Nucleotide sequences were compared with reference sequences of subtype A–D, F–H, J and K (http://hiv-web.lanl.gov). The tree was
constructed by maximum likelihood method under the GTR + I + G substitution model. Values at branches indicate bootstrap support based on 1000 iterations under
NJ method using ML distances and NNI swapping. The scale bar represents 0.1 nucleotide substitution per site.
S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377 371

analysis based on the Akaike information criterion parameters. Maximum likelihood trees were constructed of isolates that
After the starting tree was obtained by neighbor joining, did not show evidence of recombination by similarity plot
heuristic searches for likelihood was performed by using the analysis. Fig. 1 depicts the phylogenetic tree of HIV-1 of 44
NNI branch-swapping algorithm. Phylogenies were boot- sequences, including six isolates from the current study and 38
strapped with 1000 (neighbor-joining) replicates. A Kimura reference strains (GenBank and Los Alamos database)
2P model in MEGA version 2.1 was used to calculate the representing subtypes A–D, F–H, J and K. The computed
genetic distances between and within isolates. overall distance among these six isolates was 5.5–15.7% based
The nucleotide sequences from the 14 HIV-1 isolates have on 8374 nucleotides. Four isolates clustered with subtype B
been submitted to GenBank (accession no. DQ358799- reference sequences (100%; bootstrap), showing nucleotides
DQ358812). distances from 6.7% to 8.2%. Isolates 01BR087 and 01BR125
fell into subtype F1 reference group in 100% of the bootstrap
3. Results trees with distances of 6.0–8.7%.
Isolates were then grouped according to their references and
Reanalysis of HIV-1 subtypes were determined for 14 compared on the base of their genetic distances. The maximum
samples by near full-length genome sequencing. No evidence genetic variability among subtypes B and F1 viruses, including
of sample contamination was observed during the BLAST the F1 Brazilian reference strains were 8.4% and 6.0%,
search. The majority of open reading frames were intact and respectively. The mean variation between both subtypes ranged
opened in all isolates. All samples were previously character- from 14.3% to 15.6%. To gain detailed insight about clustering
ized by partial sequencing and showed six sequences to cluster pattern of our isolates and to define their genetic relation with a
with subtype B, eight with subtype F as shown in Table 1. Full corresponding reference sequences from various countries,
genome similarity plot of all isolates revealed that four of the other phylogenetic trees were constructed independently for
previously identified subtypes B were pure and two were BF each subtype.
recombinants (data not shown). Of the eight isolates previously Evolutionary related subtype B isolates and one sequence
identified as subtype F only two were pure strains throughout from Argentina formed a separate cluster supported by a weak
their genomes and the other six were BF recombinants. bootstrap value of 53% (Fig. 2A). Subtype B sequences from

Fig. 2. Phylogenetic relationship generated from non-recombinant B and F1 subtypes in a nearly full-length tree. (A) Rooted tree showing the geographic cluster of
the four Brazilian subtype B sequences and representative of subtype B. The SIV CPZGAB was used to root the tree. (B) Rooted tree showing the geographic cluster
of the two Brazilian subtype F1 isolates and representative of clade F. The HIV HXB2 was used as an outgroup. References sequences used in A and B, labelled
according to their sources, were obtained from the database (http://hiv-web.lanl.gov). The trees were constructed by maximum likelihood method under the
GTR + I + G substitution model. Values at branches indicate bootstrap support based on 1000 replications under NJ method using ML distances and NNI swapping.
The scale bar represents 0.1 nucleotide substitution per site.
372 S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377

Argentina and Colombia showed the highest inter-isolate between our isolates and the strains from Finland, Belgium and
distances with maximum values of 10.8%. Maximum like- France were 7.2%, 7.5% and 8.9%, respectively.
lihood tree of the near full length sequences of subtype F1 The stability of the cluster of our subtypes B and F1 isolates
identified in this study showed strong clustering (100% were verified by maximum likelihood trees, independently
bootstrap values) with the Brazilian subtype F1 reference made from the HIV-1 gag, pol and env regions using the same
strains. Genetic distances were calculated despite the existence set of reference strains. The results showed that the four subtype
of few number of full-length subtype F1 sequences in the B defined isolates did not form a single cluster in any regions as
databases. The results showed that the mean genetic distances they did in the full genome analysis (data not shown). Unlike

Fig. 3. Phylogenetic relationship of subtype F1 full-length gag (A), pol (B) and env (C) reading frames. The trees were constructed by maximum likelihood method
under the GTR + I + G substitution model. Values at branches indicate bootstrap support based on 1000 replications under NJ method using ML distances and NNI
swapping. Only bootstraps 70% are shown. Trees were rooted using HIV HXB2. The two Brazilian isolates are identified by circle. Branch lengths are drawn to
scale.
S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377 373

clade B, the ability of subtype F1 isolates to form a distinct against a panel of reference sequences of known subtypes by
cluster remained unaffected in all three regions and showed bootscan analysis as shown in Fig. 4. Two isolates, 02BR005
results consistent with those obtained by the full-length tree and 02BR006 were subtype B throughout the majority of their
(Fig. 3). genomes. Isolate 02BR005 had small fragment of subtype F
Data from similarity plot analysis have suggested that at the 30 end of gp 120 and beginning of the 50 end of gp 41
eight isolates, previously assigned either as subtype B or F1, while 02BR006 showed two small fragments of subtype B
were recombinant forms between subtypes B and F1. To fully (Fig. 5). All recombinant isolates, except 02BR005 and
explore this mosaic structure and to accurately identify the 01BR042, shared a common crossover site located between
positions of crossover sites, all sequences were scanned positions 506–551 nt downstream the gag ATG initiation

Fig. 4. Bootscan analysis of the near full length genomes of the eight BF recombinant viruses compared to representatives of HIV-1 subtype reference sequences.
Distances were computed after stripping gaps using F84 (ML) model of substitution. Sliding window of 420 bp moving in steps of 20 nt and 100 replicates of the
dataset were used.
374 S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377

Fig. 5. Schematic representation of the near full length genomic structure and breakpoints profile of eight BF1 recombinant isolates identified in this study. The region
of subtypes B and F1 are indicated at the bottom. Positions of breakpoints are marked with grey arrowhead and numbered according to the HXB2 sequence.

codon which corresponds to the N terminal end of p24gag As depicted in Figs. 4 and 5, the two isolates were comprised
protein. The same isolates, except 02BR033 and 02BR006, of subtype F1 throughout the majority of their genome with
shared other crossover points at positions 330 relative to the three dispersed stretches of subtype B. The exploratory tree
nef start codon. Strains 01BR047 and 01BR226 showed analysis of the seven fragments, delimited by crossover sites,
substantial homology in their mosaic patterns and shared four in isolates 01BR323 and 02BR034 showed them to cluster
crossover points at nearly identical positions as shown in closer to each other with statistical values of more than 70%
Figs. 4 and 5. Maximum likelihood trees of the individual in most of the fragments (Fig. 7). Weak clustering of both
fragments between breakpoints of the later isolates showed isolates was observed in the second fragment (592–1022;
them to cluster with significant bootstrap support to the alignment positions), probably due to few informative sites in
relevant subtypes and positioned closer to each other in some this region.
of the fragments but with low bootstrap values (<70%) In attempt to find sequences with recombinant structure
(Fig. 6). Based on the similarities of the apparent similar to any of our isolates, multiple sequence alignments
recombination pattern and phylogenetic results of subge- including all BF1 published isolates and CRF12_BF were built.
nomic regions, we believed that isolates 01BR047 and The alignment was subjected to bootscan analysis to define and
01BR226 are descendents of the same recombinant strain. compare the breakpoints for each sequence. Using this
Other striking similarity in the recombination profile was approach, we have not found any sequences to share identical
observed between strain 01BR323 and 02BR034, which recombination structure or breakpoints to any of our
exhibited three out of four breakpoints at identical positions. recombinant strains.
S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377 375

Fig. 6. Exploratory tree analysis based on fragments between breakpoints of isolates 01BR047 and 01BR226. ML trees were constructed from regions between
recombination breakpoints as indicated by bootscan plot. The best-fitted evolutionary model was determined for each fragment. Values at the respective nodes
indicate bootstrap support based on 1000 replications under NJ method using ML distances and NNI swapping. The O.BE.87.ANT70 isolate was used to root the tree
but not shown for simplicity. The scale bar represents 0.1 nucleotide substitution per site.

4. Discussion strain from Argentina, albeit with weak statistical support


(bootstrap value of 59%). Further subgenomic trees from gag,
Subtypes B and F viruses and their recombination products pol and env regions showed them to adopt a more dispersed
are prevalent in South American HIV-1 epidemics. Although it distribution in all regions. The diffused clustering of subtype B
became apparent that CRF12_BF and its related sequences play in different parts of the genome indicates different evolutionary
an important role in HIV-1 dissemination in countries as histories in different genomic regions probably caused by
Argentina and Uruguay, a multitude of the BF unique intrasubtype recombination events. Other possibility is that
recombinant forms are being described in places like Brazil. subtype B viruses were the first to be introduced since the
Albeit being one of the most HIV-1 affected South American beginning of AIDS epidemic in Brazil and therefore were able
countries, viral full-length genome data is still very limited to to accumulate genetic changes over time.
allow for a sounding evaluation of the epidemic in Brazil. Brazilian subtype F1 sequences formed a very tight cluster
In this report, we have focused to build a Brazilian panel of with other Brazilian F1 genomes with 100% Bootstrap values.
14 HIV-1 full-length genomes previously identified in our This was not only observed on full-length analysis but also
laboratory as non-recombinant subtypes B and F1 on the base when gag, pol and env regions were analyzed separately. The
of partial genome sequencing. After full-length analysis it was small number of full-length F1 sequences available in the
demonstrated that eight (57%) out of 14 genomes were indeed GenBank and the presence of Brazilian F1 sequences in the
BF1 recombinant forms. Furthermore, six out of eight subtype analysis may have helped to form the observed tight grouping
F1 viruses turned out to be BF1 recombinants. of F1 sequences. Alternatively, this can be explained by recent
Maximum likelihood phylogenetic analysis of non-recom- introduction of subtype F1 into Brazil. Our results were in line
binant viruses classified four strains as subtype B and two as with previous findings (Carr et al., 2001), which suggested that
subtype F1 with 100% bootstrap values (Fig. 1). Subtype B most of the viruses thought to be subtype F1 in South America
viruses were compared with the set of 28 B sequences from are actually BF recombinants. In this study and despite the
different countries and found to form a single cluster with one small size of samples, we proved that subtype F1 is still
376 S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377

Fig. 7. Exploratory tree analysis based on fragments between breakpoints of isolates 01BR323 and 02BR034. ML trees were constructed from regions between
recombination breakpoints as indicated by bootscan plot. The best-fitted evolutionary model was determined for each fragment. Values at the respective nodes
indicate bootstrap support based on 1000 replications under NJ method using ML distances and NNI swapping. The O.BE.87.ANT70 isolate was used to root the tree
but not shown for simplicity. The scale bar represents 0.1 nucleotide substitution per site.

circulating in Brazil, but in low proportion. This may be genomes. Analysis of individual fragments from these isolates
attributed to the replacement of genuine subtype F1 by showed them to cluster to each other in different branches
emerging BF1 recombinants as a result of unknown selective regardless of the non-significant bootstrap support, suggesting
advantage. that they are derived from common recombinant progenitor.
All sequenced recombinant genomes were BF1 mosaics. The other pair includes isolates 01BR323 and 02BR034, which
Neither of them resembled CRF12_BF suggesting that they exhibited almost identical recombination profile and strong
arose either from unrelated ancestors or independent recombi- monophyletic clustering of all but one distinct regions
nation events and indicating that the Brazilian epidemic may suggesting that they might have arisen from a common
harbor its own recombinant forms. ancestor. Moreover, the background history of infection of all
Although only eight recombinant forms were fully four patients did not show any direct epidemiological link.
sequenced, we were able to identify a full range of BF1 Although genomes in both pairs had a very similar genome
mosaics from genomes composed mostly by subtype B to structure, some coincidental breakpoints did not fall exactly at
others in which subtype F1 genetic material was predominant. the same place. We believe that this is a result of punctual
This large sequence diversity present in a limited sample size mutations removing subtype specific signature nucleotides and
suggests that HIV-1 intersubtype recombination is happening thus causing a dislocation in breakpoint positions in the
very frequently in Brazil probably due to HIV-1 transmission bootscan analysis. Other factor that affects the precise
networks operating in the country. However, we were still able localization of breakpoints is the post-recombination evolution
to identify shared breakpoints in these structures, which may in the recombinant strains.
either reflect them as hot spots for recombination or that viruses In this report, we provided evidences to indicate that the two
shared indeed in some extent a common ancestry. pairs of 01BR047/02BR226 and 01BR034/02BR034 are
Analysis of recombinant strains showed particular mosaic representative of two novel CRFs, however, because they did
pattern in two pairs of samples. The first pair consists of not meet one of the formal criteria required for assigning them
isolates 01BR047 and 01BR226, which demonstrated sig- as new CRFs, their formal classification will have to wait
nificant similarity to one another on their virtual complete sequencing of another virus with similar genomic structure.
S. Sanabani et al. / Infection, Genetics and Evolution 6 (2006) 368–377 377

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