Characterization of CRF56_cpx, a new circulating B/CRF02/G recombinant form identified in MSM in France
Leoz, Mariea,b; Feyertag, Felixc; Charpentier, Charlotted; Delaugerre, Constancee; Wirden, Marcf; Lemee, Veroniquea; Plantier, Jean-Christophea,b
aLaboratoire associé au CNR du VIH, hôpital Ch. Nicolle, CHU de Rouen
bGRAM EA 2656, Faculté de Médecine-Pharmacie, Université de Rouen, Rouen, France
cComputational and Evolutionary Biology research group, Faculty of Life Science, University of Manchester, Manchester, UK
dAssistance Publique – Hopitaux de Paris, Groupe Hospitalier Bichat Claude Bernard, Service de Virologie
eAssistance Publique – Hopitaux de Paris, Groupe Hospitalier Saint Louis, Service de Virologie,
fAssistance Publique – Hopitaux de Paris, Groupe Hospitalier Pitié Salpêtrière, Service de Virologie, Paris, France.
Correspondence to Jean-Christophe Plantier, Laboratoire de Virologie, Institut de Biologie Clinique, Hôpital Charles Nicolle, CHU de Rouen, 1 rue de Germont, 76031 Rouen, France. Tel: +33 2 32 88 14 62; fax: +33 2 32 88 04 30; e-mail: firstname.lastname@example.org
Received 15 January, 2013
Revised 7 May, 2013
Accepted 10 May, 2013
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Several B/CRF02_AG Unique Recombinant Forms (URFs) have previously been identified in France. Here we show that one of them (URF5_B/02/G) is emerging in MSM, a high-risk population where HIV incidence and number of superinfections are increasing. We describe this new Circulating Recombinant Form, CRF56_cpx, estimate the time to its most recent common ancestor, investigate its origins and show that it probably shares common ancestors with strains from the East Mediterranean.
HIV type 1 group M (HIV-M) shows a high genetic diversity and has been subdivided into nine subtypes (A–D, F–H, J, K). Recombination, which is frequent during HIV replication, produced intersubtype recombinant forms, at least 55 of which have spread and been described as Circulating Recombinant Forms (CRFs) . The reasons why a particular recombinant form can spread into a population are probably diverse, including epidemiological opportunities, fitness and ability to escape diverse host pressures.
The geographical distribution of HIV-M subtypes and CRFs has first reflected the epidemiological history of the pandemic, as for subtype B predominance in North America and Western Europe , but travelling and mixing of populations makes the molecular epidemiology of HIV-M constantly evolving . In France, subtype B now accounts for only 50% of new diagnosed infections, and CRF02_AG has become the most prevalent non-B form . Subtype B and CRF02_AG were first associated with different risk groups (MSM and Sub Saharan migrants, respectively), but they are now cocirculating in the MSM population, a high-risk group in which we recently described B/CRF02 Unique Recombinant Forms (URFs) . Here, we show that one of these, URF5 (CRF02/B/G), was identified in three new patients living in Paris, allowing us to define the new CRF56_cpx.
Patients A (previously reported as infected by URF5), B, C and D were MSM, aged to 21–32 years when sampled in Infectious Diseases units of three hospitals in Paris. They were diagnosed recently (years 2009 to 2011), three of them during Primary HIV Infection (supplementary Table 1, http://links.lww.com/QAD/A365). Plasma samples of the new patients, showing the same recombination pattern as URF5 based on routine resistance sequencing of the Protease and Reverse Transcriptase, were analyzed.
Single Genome Analysis (SGA) was performed on the Protease and partial Reverse Transcriptase regions (963 bp) as previously described  for identifying putative parental strains, and comparing the quasi-species circulating in the patients. The three new strains were characterized by sequencing the near-full-length genomes, using an overlapping nested reverse transcriptase-PCR strategy as previously described .
Intrapatient and mean interpatient genetic distances were estimated between SGA sequences, and interpatient distances between the near-full-length sequences (supplementary methods, http://links.lww.com/QAD/A365). Subtyping, recombination, phylogenetic and evolutionary analyses were performed using Los Alamos National Laboratory HIV Blast and highlighter (http://http://www.hiv.lanl.gov/), NCBI genotyping (http://http://www.ncbi.nlm.nih.gov/), simplot 3.5.1, MEGA5  and BEAST v1.7  (supplementary methods, http://links.lww.com/QAD/A365).
The SGA analysis showed no parental strain in any sample, demonstrating the absence of superinfection and suggesting a direct transmission of the recombinant strain to each patient. Phylogenetic analysis showed that the sequences from patients A, B and C formed individual clusters nested within the patient D sequences (supplementary Figure 1a, http://links.lww.com/QAD/A365), and quasi-species analysis showed patient-specific mutations (supplementary Figure 1b, http://links.lww.com/QAD/A365), demonstrating distinguishable strains and intrapatient evolution. Intrapatient genetic distances ranged from 0.001 to 0.004, consistent with short duration of infections (supplementary Table 1, http://links.lww.com/QAD/A365). Interpatient mean distances ranged from 0.003 to 0.008 for the SGA sequences, and from 0.006 to 0.008 for the near-full-length genomes. Taking into account the recent infections (2009–2011) and the absence of direct link between the four patients, these results suggest a recent diffusion of this new form.
Near-full-length characterizations confirmed a complex recombination pattern, involving subtypes B, G and CRF02_AG with 16 breakpoints (Fig. 1a), common to the four strains. Thus, we proposed to name this new Circulating Recombinant Form CRF56_cpx, according to the international HIV nomenclature guidelines . Time to the Most Recent Common Ancestor of the four strains was estimated in regions deriving from subtype B, G or CRF02 (supplementary Figure 2a, http://links.lww.com/QAD/A365), with a mean result around 2007 consistent with recent diffusion.
Interestingly, using HIV Blast as a screening tool for possible related strains, we found CRF56_cpx to be close to CY200, a CRF02/B/G URF sampled in 2007 in a MSM patient infected in Cyprus . They shared at least five breakpoints and significantly clustered together in regions belonging either to subtype B, G or CRF02 (Fig. 1, and Bayesian Markov Chain Monte Carlo analysis, not shown). Still, CY200 and CRF56_cpx also presented unshared breakpoints, so it seems unlikely that one could have been a direct ancestor for the other. They would rather share a CRF02/B/G common ancestor, which would be dating from the 1990s (supplementary Figure 2b, http://links.lww.com/QAD/A365). Whether this putative parental recombinant strain emerged in France, Cyprus or elsewhere is uncertain.
We also found 12 CRF02/B sequences, encompassing the Protease and partial Reverse Transcriptase, to be close to our sequences. All 12 were sampled in Greece in 2002–2005 from newly infected patients . Phylogenetic analyses confirmed that they clustered together in the Reverse Transcriptase region, deriving from subtype B (Fig. 1b), but in the Protease, the Greek recombinant sequences involved various CRF02_AG parental strains with no link to that of CRF56_cpx, suggesting multiple recombination events. CRF56_cpx could not be related directly to one of the recombinant forms described in Greece yet, but would rather be linked to the Greek parental subtype B strain.
The distribution of HIV subtypes and CRFs across the world is constantly evolving , leading to an increase of strains cocirculation and recombination. Recombinant forms become more frequent and complex, and now dominate the epidemic in some parts of the world . The growing diversity resulting from recombination must be taken into account as numerous factors can be impacted, from viral properties to host–virus interactions, patients monitoring or vaccine design. Here, we describe a new recombinant form, complex (involving more than two subtypes) and of second generation (involving at least a recombinant parental strain), which is closely related to several recombinant forms from the Eastern Mediterranean region.
Now that bridges have been established between different risk groups, HIV diversity and recombination are increasing in French MSM [13,14]. This could favour the emergence of CRFs in this population, as reported before in England . Whether the recent diffusion of CRF56_cpx in our patients was the result of a favourable epidemiologic context, or particular fitness or adaptation properties is to be investigated. The small intrapatient and interpatient distances are compatible with the hypothesis of a transmission cluster in the MSM population of Paris, which is particularly at-risk, but no direct links were identified between the patients. Moreover, patient A, even if linked to Paris, lives in South Eastern France, leading to a risk of a larger geographical spread.
Our findings confirm the growing complexity of HIV-1 molecular epidemiology and underline the need for maintaining surveillance and reinforcing prevention of HIV transmission in MSM. Despite the global HIV incidence decrease in other populations and large use of antiretroviral drugs, HIV prevalence and incidence remains high in this population , as well as the level of sexual risk behaviours , which at least two of our patients reported. Similar trends have been observed in many countries , underlining the risk of high transmission bursts in MSM. Current French molecular surveillance systems [13,19,20] will, thus, be useful to monitor the spread of CRF56_cpx or other emerging forms in the population.
We thank Brian T. Foley from the Los Alamos National Laboratory, Los Alamos, NM, USA, for useful comments and critical reading of the manuscripts.
We also thank Juliette Leleu for her helpful technical assistance and Dr B. Phung (Infectious Diseases Unit, Hospital Bichat-Claude Bernard, Paris, France).
We thank the Institut de Veille Sanitaire (InVS) for financial support.
Role of each of the authors: C.C., C.D., M.W. and V.L. identified the samples and collected epidemiological data. F.F. performed the Bayesian evolutionary analysis. M.L. performed molecular and phylogenetic analyses. M.L. and J-C.P. conceived of and designed the experiments, and wrote the manuscript.
Conflicts of interest
There are no conflicts of interest.
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