Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in São Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/50096-0

Maximum likelihood tree of sequences identified in this study (indicated by black circles) and reference strains inferred from full-length <i>gagpol</i> (A) and <i>env</i> (B) nucleotide sequences.

<p>For clarity purposes, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of≥90% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.</p

Schematic representation of the NFLG structure and breakpoint profiles of the BF1 sequences identified in this study and other BF1 URF and CRF published sequences.

<p>Sequences marked with the symbol (*) were originally classified as pure subtype but were characterized in the current analysis as pure B subtype, thus suggesting that a revised classification of these isolates in the GenBank and the HIV databases is appropriate. The region of subclade F1 and subtypes B are indicated at the bottom.</p

HIV-1 strain subtyping by partial sequences.

<p>HIV-1 strain subtyping by partial sequences.</p

Characteristics and main findings of the Brazilian HIV-1 recently infected individuals.

<p>Characteristics and main findings of the Brazilian HIV-1 recently infected individuals.</p

Phylogenetic tree constructed using a maximum-likelihood method from the NFLG sequences of 64 patient samples and 37 HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

<p>Annotation of samples is as follows: symbol-green circle (subtype B), symbol-blue square (subtype C), symbol-red triangle (subclade F1), symbol-brown square-M indicates heterosexual male, symbol-brown square-F indicates heterosexual female, and symbol-black square indicates homosexual male. The approximate likelihood ratio test (aLRT) values of≥90% are indicated. The scale bar represents 0.05 nucleotide substitutions per site.</p

Phylogenetic relationships among subtype B HIV-1 NFLG sequences sampled from different countries.

<p>(a) Midpoint-rooted maximum-likelihood tree of 435 HIV-1 non recombinant subtype B NFLG sequences sampled from various global locations (colored branch). In addtion to previously published Brazilian subtype B NFLGs and other South American sequences from neighboring countries. The tree also contains 62 isolates sampled in São Paulo from HIV-1 recently infected patients over a 4- year period between 2002–2006 (the present datasets). (b) For clarity, the Brazilian isolates branched as a monophyletic cluster was displayed. Annotation of samples is as follows: symbol-brown square-F indicates heterosexual female, and symbol-black square indicates homosexual male. The approximate likelihood ratio test (aLRT) values of≥70% are indicated. The scale bar represents 0.01 nucleotide substitutions per site.</p

Drug-resistance mutations detected in antiretroviral (ARV) naïve patients.

<p>*HET ^M; heterosexual man, MSM; men who have sex with men.</p

Exploratory tree analysis based on fragments between breakpoints as indicated by bootscan plot of isolate 06BR 1114.

<p>Annotation of samples is as follows: symbol-green circle (subtype B), symbol-blue square (subtype C). The approximate likelihood ratio test (aLRT) values of≥90% are indicated at nodes. The scale bar represents 0.01 nucleotide substitutions per site. The region of subtype C, subtypes B, and subclade F1 are indicated at the bottom.</p