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.

IntroductionThe high recombinogenic potential of HIV-1 has resulted in the generation of countless unique recombinant forms (URFs) and around 120 reported circulating recombinant forms (CRFs). Here we identify through analyses of near full-length genomes (NFLG) a new HIV-1 CRF derived from subtypes B and F1.MethodsHIV-1 protease-reverse transcriptase (Pr-RT) sequences were obtained by RT-PCR amplification from plasma RNA. Near full-length genome sequences were obtained after amplification by RT-PCR in 5 overlapping fragments. Phylogenetic sequence analyses were performed via maximum likelihood. Mosaic structures were analyzed by bootscanning and phylogenetic analyses of genome segments. Temporal and geographical estimations of clade emergence were performed with a Bayesian coalescent method.ResultsThrough phylogenetic analyses of HIV-1 Pr-RT sequences obtained by us from samples collected in Spain and downloaded from databases, we identified a BF1 recombinant cluster segregating from previously reported CRFs comprising 52 viruses, most from Brazil (n = 26), Spain (n = 11), and Italy (n = 9). The analyses of NFLG genomes of 4 viruses of the cluster, 2 from Spain and 2 from Italy, allowed to identify a new CRF, designated CRF75_BF1, which exhibits a complex mosaic structure with 20 breakpoints. All 4 patients harboring CRF75_BF1 viruses studied by us had CD4+ T-cell lymphocyte counts below 220/mm3 less than one year after diagnosis, a proportion significantly higher (p = 0.0074) than the 29% found in other patients studied in Spain by us during the same period. The origin of the clade comprising CRF75_BF1 and related viruses was estimated around 1984 in Brazil, with subsequent introduction of CRF75_BF1 in Italy around 1992, and migration from Italy to Spain around 1999.ConclusionA new HIV-1 CRF, designated CRF75_BF1, has been identified. CRF75_BF1 is the 6th CRF of South American origin initially identified in Western Europe, reflecting the increasing relationship of South American and European HIV-1 epidemics. The finding of low CD4+ T-cell lymphocyte counts early after diagnosis in patients harboring CRF75_BF1 viruses warrants further investigation on the virulence of this variant.

EDITORIAL article Front. Microbiol., 22 August 2022Sec. Virology Volume 13 - 2022 | https://doi.org/10.3389/fmicb.2022.1007037

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 validate that the CRF47_BF sequences share a unique evolutionary history. The CRFx_BF sequences cluster into a single, not well supported, clade that includes their dominant parent subtypes (B and F). This clade also includes subtype D and excludes sub-subtype F2. However, 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, as well as 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 1999–2000 and spread to Spain from Brazil in 2002–2003. The virus spread rapidly throughout Spain with an increase in population size from 2011 to 2015 and leveling off more recently. 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.

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. In this study, we identified a HIV-1 BF1 recombinant cluster that is expanding in Spain, transmitted mainly via heterosexual contact, which, analyzed in near full-length genomes in four viruses, exhibited a coincident BF1 mosaic structure, with 12 breakpoints, that fully coincided with that of two viruses (10BR_MG003 and 10BR_MG005) from Brazil, previously classified as CRF72_BF1. The three remaining Brazilian viruses (10BR_MG002, 10BR_MG004, and 10BR_MG008) previously identified as CRF72_BF1 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 gp120env. Based on these results, we propose that the five 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, two from Brazil and one 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, which was 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.

Integrase strand transfer inhibitor (INSTI)-containing regimens in HIV-1-infected patients have experienced a global increase. Recently, WHO has emphasized the need to fast-track the transition to dolutegravir (DTG)-based antiretroviral (ARV) treatments. However, continued surveillance of INSTI resistance is recommended. In this study, clinical, epidemiological, and virological features associated with INSTI resistance diagnosed in Spain were analyzed. Samples collected between 2008 and 2021 from HIV-1-infected patients were analyzed in integrase, protease, and reverse transcriptase using Sanger population sequencing. ARV drug resistance was evaluated with the Stanford University HIVdb program. Among 2,696 patients, 174 (6.5%) had INSTI resistance, all of them to first-generation INSTIs, and 71 (2.6%) had also resistance to second-generation INSTIs. Of these, only 5 individuals were exposed to DTG as the only INSTI, in whom resistance development was associated with poor treatment adherence and/or resistance to other ARV classes. Of newly HIV-1-diagnosed individuals, 0.92% harbored INSTI-resistant viruses, with low prevalences maintained along time, and only one had low-level resistance to DTG. Persons who inject drugs, age over 39 years, resistance to other ARV classes, and longer time from diagnosis were associated with INSTI resistance (p < 0.001). Non-subtype B INSTI-resistant viruses lacked the Q148H + G140S resistance pathway and showed lower INSTI resistance levels than subtype B viruses. In conclusion, INSTI resistance is uncommon and associated with long-term infections, older age and additional resistance to other ARV drug classes, and is rare in newly diagnosed HIV-1 infections. Our results also support the preferential use of DTG-containing regimens in first-line treatments, although surveillance of INSTI resistance is encouraged.

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 1,158 HIV-1-infected patients from both regions with new diagnoses (NDs) 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 TC membership and to determine 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% 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. Our results show that TCs are important components of the HIV-1 epidemics 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.

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.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThe study was supported by Acción Estratégica en Salud Intramural (AESI) program of Instituto de Salud Carlos III, projects Estudio sobre Vigilancia Epidemiológica Molecular de la Infección por VIH-1 en España, PI16CIII/00033, and Epidemiología Molecular del VIH-1 en España y su Utilidad para Investigaciones Biológicas y en Vacunas, PI19CIII/00042; Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Plan Nacional I+D+I, project RD16ISCIII/0002/0004; and scientific agreements with the Governments of Galicia (MVI 1004/16) and Basque Country (MVI 1001/16).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Research Ethics Committee of Instituto de Salud Carlos III, Majadahonda, Madrid, Spain, gave ethical approval for this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll newly obtained sequences and associated data have been deposited in GenBank under accessions [OK148895][1]-OK148974 [1]: /lookup/external-ref?link_type=GEN&access_num=OK148895&atom=%2Fmedrxiv%2Fearly%2F2022%2F03%2F10%2F2022.03.08.22272034.atom

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.

The analysis of a pol gene fragment encoding protease and part of reverse transcriptase was carried out for 55 sera collected in 2016 and 2018 from HIV-1-infected patients diagnosed in 2014-2018 living in the south of Western Siberia, Russia: Altai Territory (n = 11), Republic of Altai (n = 15), Kemerovo region (n = 18), and Novosibirsk region (n = 11). CRF63_02A was the dominant genetic form (>70%) in the Altai Territory and Kemerovo and Novosibirsk regions, with subsubtype A6 comprising <30% of samples. In the Altai Republic, subsubtype A6 was predominant (53%), with 33% of viruses belonging to CRF63_02A. Four CRF63_02A/A6 unique recombinant forms were identified in the Altai Territory, Kemerovo Region, and the Altai Republic. A majority (11 of 15) of CRF63_02A viruses from Kemerovo were grouped in a cluster. Antiretroviral (ARV) drug resistance mutations were found in 6 (14%) of 43 drug-naive patients. This study provides new insights in HIV-1 molecular epidemiology and prevalence of transmitted ARV drug resistance mutations in Southwestern Siberia.

Circulating recombinant forms (CRFs) contribute substantially to the HIV-1 pandemic. Among 105 CRFs described in the literature, 16 are BF intersubtype recombinants, most of South American origin, of which CRF12_BF is the most widely spread. A BF recombinant cluster identified in Bolivia was suggested to represent a new CRF_BF. Here we find that it belongs to a larger cluster incorporating 39 viruses collected in 7 countries from 3 continents, 22 of them in Spain, most from Bolivian or Peruvian individuals, and 12 in South America (Bolivia, Argentina, and Peru). This BF cluster comprises three major subclusters, two associated with Bolivian and one with Peruvian individuals. Near full-length genome sequence analyses of nine viruses, collected in Spain, Bolivia, and Peru, revealed coincident BF mosaic structures, with 13 breakpoints, 6 and 7 of which coincided with CRF12_BF and CRF17_BF, respectively. In a phylogenetic tree, they grouped in a clade closely related to these CRFs, and more distantly to CRF38_BF and CRF44_BF, all circulating in South America. These results allowed to identify a new HIV-1 CRF, designated CRF89_BF. Through phylodynamic analyses, CRF89_BF emergence was estimated in Bolivia around 1986. CRF89_BF is the fifth CRF member of the HIV-1 recombinant family related to CRF12_BF.

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.

Multiple SARS-CoV-2 introductions have been detected in Spain, and at least four resulted in the emergence of locally transmitted clusters that originated not later than mid-February, with further dissemination to many other countries around the world, and a few weeks before the explosion of COVID-19 cases detected in Spain during the first week of March. The majority of the earliest variants detected in Spain branched in the clade 19B (D614 viruses), which was the most prevalent clade during the first weeks of March, pointing to a founder effect. However, from mid-March to June 2020, G614-bearing viruses (clades 20A, 20B, and 20C) overcame D614 variants in Spain, probably as a consequence of an evolutionary advantage of this substitution in the spike protein. A higher infectivity of G614-bearing viruses than D614 variants was detected, suggesting that this substitution in SARS-CoV-2 spike protein could be behind the variant shift observed in Spain.

Objectives SARS-CoV-2 whole-genome analysis has identified three large clades spreading worldwide, designated G, V and S. This study aims to analyze the diffusion of SARS-CoV-2 in Spain/Europe.Methods Maximum likelihood phylogenetic and Bayesian phylodynamic analyses have been performed to estimate the most probable temporal and geographic origin of different phylogenetic clusters and the diffusion pathways of SARS-CoV-2.Results Phylogenetic analyses of the first 28 SARS-CoV-2 whole genome sequences obtained from patients in Spain revealed that most of them are distributed in G and S clades (13 sequences in each) with the remaining two sequences branching in the V clade. Eleven of the Spanish viruses of the S clade and six of the G clade grouped in two different monophyletic clusters (S-Spain and G-Spain, respectively), with the S-Spain cluster also comprising 8 sequences from 6 other countries from Europe and the Americas. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated in the city of Wuhan, China, around November 24, 2019, with a 95% highest posterior density (HPD) interval from October 30-December 17, 2019. The origin of S-Spain and G-Spain clusters were estimated in Spain around February 14 and 18, 2020, respectively, with a possible ancestry of S-Spain in Shanghai.Conclusions Multiple SARS-CoV-2 introductions have been detected in Spain and at least two resulted in the emergence of locally transmitted clusters, with further dissemination of one of them to at least 6 other countries. These results highlight the extraordinary potential of SARS-CoV-2 for rapid and widespread geographic dissemination.### Competing Interest StatementThe authors have declared no competing interest.

In Western Europe, the HIV-1 epidemic among men who have sex with men (MSM) is dominated by subtype B. However, recently, other genetic forms have been reported to circulate in this population, as evidenced by their grouping in clusters predominantly comprising European individuals. Here we describe four large HIV-1 non-subtype B clusters spreading among MSM in Spain. Samples were collected in 9 regions. A pol fragment was amplified from plasma RNA or blood-extracted DNA. Phylogenetic analyses were performed via maximum likelihood, including database sequences of the same genetic forms as the identified clusters. Times and locations of the most recent common ancestors (MRCA) of clusters were estimated with a Bayesian method. Five large non-subtype B clusters associated with MSM were identified. The largest one, of F1 subtype, was reported previously. The other four were of CRF02_AG (CRF02_1; n = 115) and subtypes A1 (A1_1; n = 66), F1 (F1_3; n = 36), and C (C_7; n = 17). Most individuals belonging to them had been diagnosed of HIV-1 infection in the last 10 years. Each cluster comprised viruses from 3 to 8 Spanish regions and also comprised or was related to viruses from other countries: CRF02_1 comprised a Japanese subcluster and viruses from 8 other countries from Western Europe, Asia, and South America; A1_1 comprised viruses from Portugal, United Kingom, and United States, and was related to the A1 strain circulating in Greece, Albania and Cyprus; F1_3 was related to viruses from Romania; and C_7 comprised viruses from Portugal and was related to a virus from Mozambique. A subcluster within CRF02_1 was associated with heterosexual transmission. Near full-length genomes of each cluster were of uniform genetic form. Times of MRCAs of CRF02_1, A1_1, F1_3, and C_7 were estimated around 1986, 1989, 2013, and 1983, respectively. MRCA locations for CRF02_1 and A1_1 were uncertain (however initial expansions in Spain in Madrid and Vigo, respectively, were estimated) and were most probable in Bilbao, Spain, for F1_3 and Portugal for C_7. These results show that the HIV-1 epidemic among MSM in Spain is becoming increasingly diverse through the expansion of diverse non-subtype B clusters, comprising or related to viruses circulating in other countries.

This work is aimed at assessing the presence of positive selection and/or shifts of the evolutionary rate in a fast-expanding HIV-1 subtype F1 transmission cluster affecting men who have sex with men in Spain. We applied Bayesian coalescent phylogenetics and selection analyses to 23 full-coding region sequences from patients belonging to that cluster, along with other 19 F1 epidemiologically-unrelated sequences. A shift in the overall evolutionary rate of the virus, explained by positively selected sites in the cluster, was detected. We also found one substitution in Nef (H89F) that was specific to the cluster and experienced positive selection. These results suggest that fast transmission could have been facilitated by some inherent genetic properties of this HIV-1 variant.