The extraordinary genetic variability of human immunodeficiency virus type 1 (HIV-1) group M has led to the identification of 10 subtypes, 102 circulating recombinant forms (CRFs) and numerous unique recombinant forms. Among CRFs, 11 derived from subtypes B and C have been identified in China, Brazil, and Italy. Here we identify a new HIV-1 CRF_BC in Northern Spain. Originally, a phylogenetic cluster of 15 viruses of subtype C in protease-reverse transcriptase was identified in an HIV-1 molecular surveillance study in Spain, most of them from individuals from the Basque Country and heterosexually transmitted. Analyses of near full-length genome sequences from six viruses from three cities revealed that they were BC recombinant with coincident mosaic structures different from known CRFs. This allowed the definition of a new HIV-1 CRF designated CRF108_BC, whose genome is predominantly of subtype C, with four short subtype B fragments. Phylogenetic analyses with database sequences supported a Brazilian ancestry of the parental subtype C strain. Coalescent Bayesian analyses estimated the most recent common ancestor of CRF108_BC in the city of Vitoria, Basque Country, around 2000. CRF108_BC is the first CRF_BC identified in Spain and the second in Europe, after CRF60_BC, both phylogenetically related to Brazilian subtype C strains.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Previous studies have reported the propagation of diverse HIV-1 CRFs of South American origin in Europe. Here, through protease-reverse transcriptase (Pr-RT) and integrase sequence analyses, we identify a Spanish cluster (BC3) branching with a Brazilian virus (10BR_RJ009) previously classified as CRF146_BC. In Pr-RT, BC3 comprised 14 viruses and was nested within a larger cluster, comprising 22 Brazilian viruses and 1 Spanish virus branching outside of BC3. Near full-length genome analyses of five BC3 viruses revealed mosaic structures identical to 10BR_RJ009, with two breakpoints delimiting a ~0.3 kb subtype B fragment within an otherwise subtype C genome. Two other Brazilian viruses previously classified as CRF146_BC (10BR_RJ039 and 01_BR_RGS69) had one and two additional short subtype B fragments, respectively, and failed to group with the 10BR_RJ009/BC3 cluster in subtype C fragments. Based on these results, we contend that 10BR_RJ009 and BC3 viruses, but not 10BR_RJ039 and 01_BR_RGS69, should be classified as CRF146_BC. Bayesian analyses estimated the CRF146_BC emergence in Brazil to be around 1999 and its introduction in Europe around 2011. CRF146_BC is the 10th CRF of South American origin reported to circulate in Europe, reflecting the relationship between South American and European HIV-1 epidemics.

Viruses of HIV-1-infected individuals whose transmission is related group phylogenetically in transmission clusters (TCs). The study of the phylogenetic relations of these viruses and the factors associated with these individuals is essential to analyze the HIV-1 epidemic. In this study, we examine the role of TCs in the epidemiology of HIV-1 infection in Galicia and the Basque County, two regions of Northern Spain. A total of 1158 newly HIV-1-diagnosed patients (NDs) from both regions diagnosed in 2013-2018 were included in the study. Partial HIV-1 pol sequences were analyzed phylogenetically by approximately-maximum-likelihood with FastTree 2. In this analysis, 10,687 additional sequences from samples from HIV-1-infected individuals collected in Spain in 1999-2019 were also included to assign and determine the TCs’ sizes. TCs were defined as those which included viruses from ≥4 individuals, at least 50% of them Spaniards, and with ≥0.95 Shimodaira-Hasegawa-like node support in the phylogenetic tree. Factors associated to TCs were evaluated using odds ratios (OR) and their 95% confidence intervals (CI). Fifty one percent of NDs grouped in 162 TCs. Male patients (OR: 2.6; 95% CI: 1.5-4.7) and men having sex with men (MSM) (OR: 2.1; 95% CI: 1.4-3.2) had higher odds of belonging to a TC compared to female and heterosexual patients, respectively. Individuals from Latin America (OR: 0.3; 95% CI: 0.2-0.4), North Africa (OR: 0.4; 95% CI: 0.2-1.0), and especially Sub-Saharan Africa (OR: 0.02; 95% CI: 0.003-0.2) were inversely associated to belonging to TCs compared to native Spaniards. Differences in distribution and sizes of local TCs were observed in both regions reflecting a different epidemic pattern. Our results show that TCs play an important role in the spread of HIV-1 infection in the two Spanish regions studied, where transmission between MSM is predominant. The majority of migrants were infected with viruses not belonging to TCs that expand in Spain. Molecular epidemiology is essential to identify local peculiarities of HIV-1 propagation. The early detection of TCs and prevention of their expansion, implementing effective control measures, could reduce HIV-1 infections.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Those derived from recombination between subtype B and subsubtype F1, with 18 reported, most of them of South American origin, are among the most diverse. Here we identify a HIV-1 BF1 recombinant cluster that is expanding in Spain, transmitted mainly via heterosexual contact, which, analyzed in near full-length genomes in 4 viruses, exhibited a coincident BF1 mosaic structure, with 12 breakpoints, that fully coincided with that of two viruses from Brazil (10BR_MG003 and 10BR_MG005), previously classified as CRF72_BF1. The three remaining Brazilian viruses previously identified as CRF72_BF1 (10BR_MG002, 10BR_MG004, and 10BR_MG008), exhibited mosaic structures highly similar, but not identical, to that of the Spanish viruses and to 10BR_MG003 and 10BR_MG005, with discrepant subtypes in two short genome segments, located in pol and gp120 ^env . Based on these results, we propose that the 5 viruses from Brazil previously identified as CRF72_BF1 actually belong to two closely related CRFs, one comprising 10BR_MG002, 10BR_MG004, and 10BR_MG008, which keep their CRF72_BF1 designation, and the other, designated CRF122_BF1, comprising 10BR_MG003, 10BR_MG005 and the viruses of the identified Spanish cluster. Three other BF1 recombinant genomes, 2 from Brazil and 1 from Italy, previously identified as unique recombinant forms, were classified as CRF72_BF1. CRF122_BF1, but not CRF72_BF1, was associated with protease L89M substitution, reported to contribute to antiretroviral drug resistance. Phylodynamic analyses estimate the emergence of CRF122_BF1 in Brazil around 1987. Given their close phylogenetic relationship and similar structures, the grouping of CRF72_BF1 and CRF122_BF1 in a CRF family is proposed.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 108 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.

CRF47_BF is a circulating recombinant form (CRF) of the human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS. CRF47_BF represents one of 19 CRFx_BFs and has a geographic focus in Spain, where it was first identified in 2010. Since its discovery, CRF47_BF has expanded considerably in Spain, predominantly through heterosexual contact (∼56% of the infections). Little is known, however, about the origin and diversity of this CRF or its epidemiological correlates, as very few samples have been available so far. This study conducts a phylogenetic analysis with representatives of all CRFx_BF sequence types along with HIV-1 M Group subtypes to place the CRF47_BF sequences in a definitive phylogenetic context. The CRFx_BF sequences cluster into a single, not well supported, clade that includes their dominant parent subtypes (subtype B and subtype F). This clade also includes subtype D and excludes subsubtype F2. The CRF47_BF sequences all share a most recent common ancestor. Further analysis of this clade couples CRF47_BF protease-reverse transcriptase sequences and epidemiological data from an additional 87 samples collected throughout Spain, coupled with additional CRF47_BF database sequences from Brazil and Spain to investigate the origin and phylodynamics of CRF47_BF. The Spanish region with the highest proportion of CRF47_BF samples in the data set was the Basque Country (43.7%) with Navarre next highest at 19.5%. We include in our analysis epidemiological data on host sex, mode of transmission, time of collection, and geographic region. The phylodynamic analysis indicates that CRF47_BF originated in Brazil around 1993-1994 and spread to Spain from Brazil in approximately 1999-2000. The virus spread rapidly throughout Spain with increasing population sizes prior to 2010 and again between 2010 and 2017 with population declines to 2019 and a steady state through 2020. Three strongly supported clusters associated with Spanish regions (Basque Country, Navarre, and Aragon), together comprising 60.8% of the Spanish samples, were identified, one of which was also associated with transmission among men who have sex with men. The expansion in Spain of CRF47_BF, together with that of other CRFs and subtype variants of South American origin, previously reported, reflects the increasing relationship between the South American and European HIV-1 epidemics.