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Divergent gemycircularvirus in HIV-positive blood, France.

To the Editor: Gemycircularviruses are a group of recently discovered single-stranded DNA viruses, found initially in fungi in 2010 (1). These "myco-like" viruses have a genome ranging from 2.1 to 2.3 kb, containing 2 opposite open reading frames that probably code for a capsid protein (CP) and a spliced replication-associated protein (Rep). Related viruses have been subsequently identified in animal blood and fecal matter, raw and treated sewage, and insects and plant material, suggesting that gemycircularviruses may represent a large group of viruses exhibiting considerable genetic diversity (2-8). The presence of these viruses was recently extended to humans after gemycircularvirus sequences were identified in human blood and brain tissue (multiple sclerosis patient), cerebrospinal fluid, and fecal matter (8,9).

While investigating the virome content of an HlV-positive blood donation, we identified several gemycircular-virus-related sequences. The initial metagenomic approach involved an HIV-1-positive plasma sample (B genotype, [approximately equal to]530 copies/mL) obtained from the French blood agency national plasma bank in Tours, France. A 4-mL aliquot was prepared for metagenomic analysis after filtration, concentration, and nucleases treatment. Next, particle-protected nucleic acids were recovered and used for the preparation of a next-generation sequencing library and its subsequent analysis (online Technical Appendix, http://wwwnc.cdc. gov/EID/article/21/11/15-0486-Techapp1.pdf). Gemycircularvirus sequences identified among reads (1,680 vs. 82,560 reads total; [approximately equal to]2%) were assembled into a resulting full-length sequence (GemyC1c) by using CodonCode Aligner version 5.1 (CodonCode Corporation, Centerville, MA, USA). This sequence was verified by using back-to-back specific primers, and the amplicon was cloned and sequenced according to the Sanger method.

The analysis of the GemyC1c sequence (2,109 nt, GenBank accession no. KP987887) revealed a genome divergent from those already available in databases, despite a similar genomic organization (Figure, panel A) and assignment to gemycircularviruses after BLAST (http://blast.ncbi.nlm.nih. gov/Blast.cgi) analysis of putative CP and Rep proteins. This divergence was demonstrated by the phylogenetic analysis of the deduced CP (Figure, panel B), which exhibited [approximately equal to]72% and [approximately equal to]44% aa pairwise identity with the 2 closest gemycircularvirus CP sequences available in GenBank (gemycircularvirus c from mongoose feces [Conceicao-Neto N., unpub. data] and HCBI8.215 from cattle blood, respectively). Moreover, GemyC1c CP exhibited [approximately equal to]30% pairwise identity with viral sequences identified previously in humans (BZ1 from feces, SL1 from cerebrospinal fluid, MSSI2.225 from blood). The deduced spliced Rep (major Rep1 and minor Rep2), seen in such viruses, contained putative rolling circle motifs I (LFTYS), II (HLHAFVD), and III (YATKD) retrieved from gemycircularviruses (4).

We subsequently investigated the presence of GemyC1c DNA in 128 HIV-positive plasma samples (French blood agency national plasma bank) along with 256 HIV-negative plasma samples (healthy blood donors, southeastern France; mean donor age 38 years; 136 men; 1 man:1.13 women). Plasma samples were prepared as described previously (10), and extracted nucleic acids were tested for GemyC1c DNA by using a specific PCR that included negative, positive, and extraction controls (online Technical Appendix).

Application of the above GemyC1c DNA detection system did not generate any positive signal in the 384 plasma samples in this study, suggesting that the presence of this virus in the blood of the populations tested was a rare occurrence. However, it is possible that other, divergent, GemyClc-related sequences could be present in human blood but remain undetectable by the molecular assay used; the development of universal gemycircularvirus PCR systems is now expected.

Gemycircularviruses are potentially very stable in the environment. Because an unknown part of this group is able to infect fungi, possible contamination from the laboratory environment or nucleic acid extraction methods must be considered. The fact that the same genome was never identified in other libraries generated in our laboratory supports the absence of local contamination; of note, we were also able to detect GemyC1c DNA with PCR by using another plasma aliquot extracted by an alternative method (NucliSENS magnetic extraction; bioMerieux, Marcy l'Etoile, France) In addition, the systematic elimination of the first 35 mL of each blood donation, associated with filtration procedures and control of the temperature of stored plasma (-25[degrees]C), contributes to the reduction of bacterial/fungal contamination during blood collection. However, it is not possible to state that the GemyClc sequence would belong to a human-tropic virus because an association of the virus with an unknown fungus is plausible. Thus, the presence of fungi in the gut, with fungi/virions having traversed the gut lining, or circulating in blood should be considered. Such aspects should prompt future investigations of the effective replication of gemycircularviruses in human or other mammalian cells.

Our discovery of the GemyClc by a sequence-independent molecular approach was informative for several reasons: 1) this viral sequence would have been undetectable by PCR according to the high genetic divergence existing between GemyClc and other gemycircularviruses identified; 2) this finding adds clues to the identification of potential new co-infections occurring in HIV-infected persons; and 3) this finding underlines the need to investigate the virome content of blood samples in a research context of new microbes as potential threats for transfusion. Further studies aimed at exploring genetic diversity and natural history of gemycircularviruses in human hosts are needed.

Rathviro Uch, Pierre-Edouard Fournier, Catherine Robert, Caroline Blanc-Tailleur, Vital Galicher, Romain Barre, Frangois Jordier, Philippe de Micco, Didier Raoult, Philippe Biagini

Author affiliations: Centre National de la Recherche Scientifique, Marseilles (R. Uch, P.-E. Fournier, C. Robert, C. Blanc-Tailleur, V. Galicher, R. Barre, F. Jordier, P. de Micco, D. Raoult, P. Biagini); Institut Hospitalo-Universitaire Mediterranee-Infection, Marseille, France (R. Uch, P.-E. Fournier, C. Robert, C. Blanc-Tailleur, R. Barre, F. Jordier, P. de Micco, D. Raoult, P. Biagini); Aix-Marseille Universite, Marseille (R. Uch, P.-E. Fournier, C. Robert, C. Blanc-Tailleur, V. Galicher, R. Barre, F. Jordier, P. de Micco, D. Raoult, P. Biagini); Unite Mixte de Recherche 7268 ADES, Marseilles (R. Uch, V. Galicher, R. Barre, F. Jordier, P. de Micco, P. Biagini); Etablissement Franpais du Sang, Marseilles (R. Uch, V. Galicher, R. Barre, F. Jordier, P. de Micco, P. Biagini); Unite de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseilles (P.-E. Fournier, C. Robert, C. Blanc-Tailleur, D. Raoult)


This work was supported by grant APR 2013.10 from the Etablissement Franjais du Sang (Paris, France) and by the Mediterranee-Infection foundation.


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Address for correspondence: Philippe Biagini, Equipe "Emergence et co-evolution virale," UMR 7268 ADES, Aix-Marseille Universite, EFS, CNRS, 27 Blvd Jean Moulin, 13005 Marseille, France; email:
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Title Annotation:LETTERS
Author:Uch, Rathviro; Fournier, Pierre-Edouard; Robert, Catherine; Blanc-Tailleur, Caroline; Galicher, Vita
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Date:Nov 1, 2015
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