Date Published: November 24, 2015
Publisher: Public Library of Science
Author(s): Elena Delgado, María Teresa Cuevas, Francisco Domínguez, Yolanda Vega, Marina Cabello, Aurora Fernández-García, Marcos Pérez-Losada, María Ángeles Castro, Vanessa Montero, Mónica Sánchez, Ana Mariño, Hortensia Álvarez, Patricia Ordóñez, Antonio Ocampo, Celia Miralles, Sonia Pérez-Castro, María José López-Álvarez, Raúl Rodríguez, Matilde Trigo, Julio Diz-Arén, Carmen Hinojosa, Pablo Bachiller, Silvia Hernáez-Crespo, Ramón Cisterna, Eugenio Garduño, Lucía Pérez-Álvarez, Michael M Thomson, Chiyu Zhang.
We recently reported the rapid expansion of an HIV-1 subtype F cluster among men who have sex with men (MSM) in the region of Galicia, Northwest Spain. Here we update this outbreak, analyze near full-length genomes, determine phylogenetic relationships, and estimate its origin. For this study, we used sequences of HIV-1 protease-reverse transcriptase and env V3 region, and for 17 samples, near full-length genome sequences were obtained. Phylogenetic analyses were performed via maximum likelihood. Locations and times of most recent common ancestors were estimated using Bayesian inference. Among samples analyzed by us, 100 HIV-1 F1 subsubtype infections of monophyletic origin were diagnosed in Spain, including 88 in Galicia and 12 in four other regions. Most viruses (n = 90) grouped in a subcluster (Galician subcluster), while 7 from Valladolid (Central Spain) grouped in another subcluster. At least 94 individuals were sexually-infected males and at least 71 were MSM. Seventeen near full-length genomes were uniformly of F1 subsubtype. Through similarity searches and phylogenetic analyses, we identified 18 viruses from four other Western European countries [Switzerland (n = 8), Belgium (n = 5), France (n = 3), and United Kingdom (n = 2)] and one from Brazil, from samples collected in 2005–2011, which branched within the subtype F cluster, outside of both Spanish subclusters, most of them corresponding to recently infected individuals. The most probable geographic origin and age of the Galician subcluster was Ferrol, Northwest Galicia, around 2007, while the Western European cluster probably emerged in Switzerland around 2002. In conclusion, a recently expanded HIV-1 subtype F cluster, the largest non-subtype B cluster reported in Western Europe, continues to spread among MSM in Spain; this cluster is part of a larger cluster with a wide geographic circulation in diverse Western European countries.
The HIV-1 epidemic among men who have sex with men (MSM) has experienced a notable upsurge in recent years in many countries  associated with increased high risk behavior in this population [1,2]. This has been frequently accompanied by the emergence of local HIV-1 transmission clusters [3–16], whose expansion is mostly driven by onward transmission from individuals with recent infection who are unaware of their HIV status [3,4,8,10–13]. In Western Europe and North America, HIV-1 clusters associated with MSM are usually of subtype B, the clade initially introduced and largely predominant in this population . However, several non-subtype B clusters associated with HIV-1 transmission among MSM have been recently reported in Western countries [14,18–21]. The largest one is an F1 subsubtype cluster of Brazilian ancestry which has rapidly spread among MSM in the region of Galicia, Northwest Spain . Since the original report, covering samples collected up to April 2011, the size of this cluster has increased considerably and its geographic range has expanded. Here we update the information on this cluster, analyze near full-length genomes, determine phylogenetic relationships with viruses from other countries, and estimate its geographic and temporal origin.
Among HIV-1 infections diagnosed in Spain in 2009–2013, we identified 87 viruses of subtype F, mainly from Galicia, that formed a clade for the PR-RT sequence (Fig 1a). None of the 1,660 HIV-1 samples collected during 1999–2008 in Galicia and previously sequenced by us branched in this cluster. Three viruses of the subtype F cluster (one from Galicia and two from Valladolid, in central Spain) branched off basally to the rest, which formed a subcluster. We will refer to this subcluster as the Galician subcluster. Similarly, we will refer to the entire subtype F cluster, comprising viruses from Spain and other Western European countries and one from Brazil [identified in a previous  or in this study (see below)] as the Western European subtype F cluster. For one virus from Galicia, X3049, whose PR-RT bulk sequence was of subtype B (Fig 1a), the sequence electropherogram showed numerous mixed positions with minor peaks overlapping major peaks, most of them corresponding to positions where B subtype and F cluster consensuses differ. Suspecting dual B/F1 infection, we obtained 19 clones for the amplified PR-RT fragment, all of which were of subtype B; however, nested PCR with F1 subsubtype-specific primers yielded an amplicon whose sequence branched within the Galician F subcluster (Fig 1a), indicating the presence of a dual B subtype/F cluster infection. Another virus from Galicia, X3461, also showed numerous mixed electropherogram peaks only in protease at positions differing between B subtype and the F cluster. Nineteen PR-RT clones of this virus, analyzed by bootscanning, revealed an infection with diverse variants, with 18 clones being BF1 recombinant and one of subtype B (S1 Fig). Most recombinant clones had 5’-F/B-3’ structures, with breakpoints near the protease-RT junction in 12 and at around position 120 of protease in 7, and one had a B/F/B structure, with breakpoints at around positions 120 and 290 of protease (S1 Fig); the subtype B fragment of the recombinant clones derived from the subtype B strain represented by a single clone (S1 and S2 Figs). A phylogenetic tree of protease of the recombinant clones of X3461 having the largest subtype F fragment showed branching in the subtype F cluster (S3 Fig). Three additional viruses from Galicia (X2890, X2955, and X3253) that could not be RT-PCR-amplified for PR-RT but were amplified for protease, also branched within the subtype F cluster for this segment (S3 Fig). Assignment of protease sequences to the Galician subcluster was uncertain because the subcluster was not well supported in the protease tree (S3 Fig). Analysis of ARV drug resistance mutations only revealed a minor population with protease M46I, associated with low level resistance to Nelfinavir, in a patient who was under treatment with atazanavir/ritonavir.
The most remarkable finding of this study is probably that the Galician HIV-1 subtype F cluster previously reported by us  is part of a larger cluster that has independently spread in at least three other Western European geographic areas, as evidenced by local clustering of sequences from Valladolid, central Spain (7 infections) (Fig 1b), Switzerland (7 infections) (Fig 5b and 5c) (although clustering of 3 Swiss viruses is not strongly supported in PR-RT, they share K103N drug resistance mutation , and in p24gag the 4 Swiss viruses group with a 96% bootstrap value), and Belgium (5 infections, with a sixth virus from Galicia clustering with them) (Fig 6c and 6d). Additionally, 2 newly diagnosed infections from the UK branching within the Western European subtype F cluster are closely related to each other (Fig 6a), and 3 viruses from France, with uncertain mutual relations, also branch within the Western European subtype F cluster (Fig 6c and 6d). The fact that most of the viruses are from recent diagnoses, with 7 infections from Switzerland  and 2 from France  being from primary or recent infections, also indicates that viruses of the Western European subtype F cluster are propagating throughout multiple local networks. The available data indicate that transmission takes place among MSM, since all Spanish cases studied by us are sexually-infected men, at least 71 MSM, as are 3 infections from Switzerland (2 from MSM)  and 2 from Belgium (both from MSM) . Considering that all 100 infections of the cluster studied by us were detected in men, we strongly suspect that those cases from Spain reported to be transmitted via heterosexual or unspecified sexual contact also correspond to MSM.