Identification of CRF66_BF, a New HIV-1 Circulating Recombinant Form of South American Origin

Abstract

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 110 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n = 4), Spain (n = 3), Paraguay (n = 1), Brazil (n = 1), and Italy (n = 1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17 ^gag , integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF "family" clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.

Keywords: HIV-1; circulating recombinant form; molecular epidemiology; phylodynamics; phylogeny.

FIGURE 1

Maximum likelihood tree of Pr-RT sequences of BF9 cluster constructed with IQ-Tree. Sequences used in this analysis were those from the BF9 cluster shown in the tree of Supplementary Figure 1, constructed with FastTree, plus sequences of F1 subsubtype from different countries and of CRF_BFs that are of nonrecombinant F1 subsubtype in Pr-RT, plus two F2 sequences used as outgroups. Names of sequences obtained by us, all collected in Spain, are in bold type. In database sequences, the country of sample collection is indicated before the virus name with the 2-letter ISO country code. After the names of viruses of the BF9 cluster, the 2-letter ISO code of country of origin of the patient and/or the transmission route, when known, are shown in parentheses. Only ultrafast bootstrap values ≥80% are shown. PY: Paraguay; AR: Argentina; ES: Spain; BR: Brazil; IT: Italy; GQ: Equatorial Guinea; Lat Am: Latin America (unknown country); MSM: man who has sex with men; HT: heterosexual; SX-M: male with unspecified sexual acquisition of HIV-1.

FIGURE 2

Bootscan analyses of 6 NFLG sequences of viruses of the BF9 cluster obtained by us and of two BF1 database NFLG sequences from Paraguay, 02PY_PSP0093 and 02PY_PSP0094. The horizontal axis represents the position in the HXB2 genome of the midpoint of a 400 nt window moving in 20 nt increments and the vertical axis represents bootstrap values supporting clustering with subtype reference sequences.

FIGURE 3

Phylogenetic trees of interbreakpoint genome segments of the BF recombinant viruses analyzed by bootscanning, constructed with IQ-Tree. HXB2 positions delimiting the analyzed segments are indicated on top of the trees. Sequence names of BF viruses are in bold type. Names of subtype references are preceded by the corresponding subtype name. Only ultrafast bootstrap values ≥80% are shown.

FIGURE 4

Maximum likelihood tree of NFLG sequences of viruses of the BF9 cluster and PY02_PSP0094, constructed with IQ-Tree. References of all published CRF_BFs and of HIV-1 subtypes are also included in the analysis. The tree is rooted with SIVcpz virus MB66. Names of sequences obtained by us are in bold type. In reference sequences, the subtype or CRF is indicated before the virus name. Only ultrafast bootstrap values ≥90% are shown.

FIGURE 5

Mosaic structure of CRF66_BF. Breakpoint positions are numbered as in the HXB2 genome. The drawing was made using the Recombinant HIV-1 Drawing Tool https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html.

FIGURE 6

Maximum clade credibility tree of CRF66_BF Pr-RT sequences. Branch colors indicate, for terminal branches, country of sample collection or, for South American individuals residing in Spain, of origin of the individual, which was used as location trait (see Methods), and for internal branches, the most probable location country of the subtending node, according to the legend on the upper left. For database sequence 524026, from a sample collected in Spain, location was assigned to Paraguay as the most probably country of origin, although the only available information in the GenBank entry is that the individual was from Latin America, because 15 (88.2%) of 17 Latin Americans with CRF66_BF sampled in Spain were from Paraguay. Nodes supported by PP ≥ 0.95 and PP 0.9–0.949 are indicated with filled and unfilled circles, respectively. The two most probable countries at the root of the tree are indicated, together with the PPs supporting each location and the time of the MRCA (mean value, with 95% HPD interval in brackets).