ML trees of sequences of HIV-1 isolates from databases (all from Portugal) or obtained by us (from Spain) clustering with the BG recombinant virus 9196_01 in (a) PR-RT and/or the (b) V3 region.

<p>Sequences obtained in this study are in bold type. HXB2 positions delimiting the analyzed segments are in parentheses. Countries of collection of database viruses of subtype G in PR-RT and of subtype B in V3 are indicated with the two-letter ISO code. Only bootstrap values ≥70% are shown.</p

Identification of an HIV-1 BG Intersubtype Recombinant Form (CRF73_BG), Partially Related to CRF14_BG, Which Is Circulating in Portugal and Spain

<div><p>HIV-1 exhibits a characteristically high genetic diversity, with the M group, responsible for the pandemic, being classified into nine subtypes, 72 circulating recombinant forms (CRFs) and numerous unique recombinant forms (URFs). Here we characterize the near full-length genome sequence of an HIV-1 BG intersubtype recombinant virus (X3208) collected in Galicia (Northwest Spain) which exhibits a mosaic structure coincident with that of a previously characterized BG recombinant virus (9601_01), collected in Germany and epidemiologically linked to Portugal, and different from currently defined CRFs. Similar recombination patterns were found in partial genome sequences from three other BG recombinant viruses, one newly derived, from a virus collected in Spain, and two retrieved from databases, collected in France and Portugal, respectively. Breakpoint coincidence and clustering in phylogenetic trees of these epidemiologically-unlinked viruses allow to define a new HIV-1 CRF (CRF73_BG). CRF73_BG shares one breakpoint in the envelope with CRF14_BG, which circulates in Portugal and Spain, and groups with it in a subtype B envelope fragment, but the greatest part of its genome does not appear to derive from CRF14_BG, although both CRFs share as parental strain the subtype G variant circulating in the Iberian Peninsula. Phylogenetic clustering of partial <i>pol</i> and <i>env</i> segments from viruses collected in Portugal and Spain with X3208 and 9691_01 indicates that CRF73_BG is circulating in both countries, with proportions of around 2–3% Portuguese database HIV-1 isolates clustering with CRF73_BG. The fact that an HIV-1 recombinant virus characterized ten years ago as a URF has been shown to represent a CRF suggests that the number of HIV-1 CRFs may be much greater than currently known.</p></div

Intersubtype breakpoint locations in HIV-1 BG recombinant viruses analyzed in this study, including all available CRF14_BG viruses sequenced in near full-length genomes, as determined with jpHMM.

<p>Intersubtype breakpoint locations in HIV-1 BG recombinant viruses analyzed in this study, including all available CRF14_BG viruses sequenced in near full-length genomes, as determined with jpHMM.</p

Analyses of partial genome sequences of BG recombinant viruses related to X3208 and 9196_01.

<p>(a) Bootscan analyses of <i>pol</i> fragments of X3121, from Spain, and 753_G_0_Rennes, from France. (b) ML tree of the integrase subtype B fragment of X3121 and 753_G_0_Rennes, showing clustering with 9196_01 and X3208. HXB2 positions delimiting the analyzed segment are in parentheses. Only bootstrap values ≥70% are shown. (c) Bootscan analysis of the envelope gene of VLGC_PT_BG3, from Portugal. In the bootscanning graphs, the position in the horizontal axis represents the midpoint of the sliding window in the proviral HXB2 genome.</p

Analysis of the relationship of CRF73_BG with CRF14_BG.

<p>(a) ML tree of concatenated subtype G fragments of X3208 and 9196_01, analyzed with G_Ib and CRF14_BG viruses. Countries of collection of subtype G viruses are indicated with the two-letter ISO code. (b) ML tree of the 5’ <i>env</i> fragment. (c) ML tree of the subtype B 3’ <i>env</i> fragment. HXB2 positions delimiting the analyzed segments in (b) and (c) are in parentheses. CRF73_BG viruses are in bold type. Countries of collection of database viruses of subtype G viruses in (a) and of subtype B viruses in (b) and (c) are indicated with the two-letter ISO code. Only bootstrap values ≥70% are shown.</p

Novel splice junctions identified in this study.

<p>Consensus sequences at both sides of splice junctions are shown. Splice sites involved in splice junctions are indicated, with HXB2 positions in parentheses. Nearby splice sites used in the corresponding samples are also indicated.</p

Schematic depiction of HIV-1 splicing and locations of PCR primers.

<p>Open reading frames are shown as open boxes and exons as black bars. Exons are named as previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158525#pone.0158525.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158525#pone.0158525.ref006" target="_blank">6</a>]. All spliced transcripts incorporate exon 1 and all DS transcripts incorporate exon 7. Optionally, noncoding exons 2 or 3 or both can be incorporated into <i>nef</i>, <i>rev</i>, <i>tat</i>, or <i>env-vpu</i> transcripts, and exon 2 into <i>vpr</i> transcripts. A minority of <i>nef</i> RNAs are generated by splicing from exon 1 to exon 7. Proteins encoded in spliced RNAs are indicated on the right of the middle exon in DS RNAs and of the 3’-terminal exon in SS RNAs. Locations of sequences recognized by primers used for RT-PCR and nested PCR for DS and SS RNA amplification are indicated with arrows, with HXB2 positions of the primers’ 3’ ends in parentheses. 5’RU5-S and 3’nef3 were used for RT-PCR and US22 and TRN-AS for nested PCR for DS RNA amplification; 5’RU5-S and SSD2c were used for RT-PCR, and US22 and SSD1 for nested PCR amplification of SS RNAs.</p

Mosaic structure of CRF73_BG.

<p>Breakpoint positions, according to HXB2 genome numeration, are indicated.</p

Clinical data and subtypes of samples used in this study.

<p>Clinical data and subtypes of samples used in this study.</p

Phylogenetic tree of DS HIV-1 RNA sequences.

<p>The analysis was done with the fragment comprising exons 5 and 7 (HXB2 positions 5977–6045; 8379–8533), common to all DS transcripts, using 20 randomly chosen sequences per sample. Clades comprising sequences from each sample are compressed in triangles. SH-like node support values for sample clades and for subtype clades are shown.</p