# Novel Single-Stranded DNA Circular Viruses in Pericardial Fluid of Patient with Recurrent Pericarditis

Sébastien Halary, Raja Duraisamy, Laura Fancello, Sonia Monteil-Bouchard, Priscilla Jardot, Philippe Biagini, Frédérique Gouriet, Didier Raoult, Christelle Desnues

<figure><img src="image_1.png" /></figure>

## To cite this version:

Sébastien Halary, Raja Duraisamy, Laura Fancello, Sonia Monteil-Bouchard, Priscilla Jardot, et al. Novel Single-Stranded DNA Circular Viruses in Pericardial Fluid of Patient with Recurrent Pericarditis. Emerging Infectious Diseases, 2016, 22 (10), pp.1839 - 1841. 10.3201/eid2210.160052. hal-01447667

HAL Id: hal-01447667

https://hal.science/hal-01447667

Submitted on 13 Oct 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
was not the case in our study. Genetic differences in cir-
culating CHIKV lineages could also explain poor testing
performance. Furthermore, the OnSite Chikungunya IgM
Combo CE POC test uses a recombinant antigen covering
the 226 residues of the E1 gene from CHIKV variant A226;
recent studies on CHIKV protein characterization showed
that more sensitive serologic assays can be obtained using
specific early-phase E2 glycoprotein as antigens (3).

The successful use of rapid immunochromatography-
based assays with monoclonal antibodies to detect viral
diseases (e.g., dengue) has encouraged the development of
rapid immunoassays for CHIKV antigens, and preliminary
results for these assays seem promising (6). External qual-
ity assessment programs for POC tests and quality controls
consisting of standardized positive serum could also be
helpful for improving the performance of diagnostic tests.

In conclusion, returning travelers are sentinels of the
rapidly changing epidemiology of CHIKV; thus, they re-
quire a prompt diagnosis and careful surveillance for their
possible role in subsequent autochthonous disease trans-
mission. Implementation of user-friendly, rapid, and easily
deliverable POC tests for a prompt and accurate laboratory
diagnosis is therefore needed to improve patient manage-
ment and disease control measures.

Acknowledgments

We thank the Carlo De Negri Foundation for their support (N.650/007B/2011) and staff of the Unit of Serology and Molecular Biology, Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, for their excellent technical assistance.

**References**

1. Kosasih H, Widjaja S, Surya E, Hadiwijaya SH, Butarbutar DP, Jaya UA, et al. Evaluation of two IgM rapid immunochromatographic tests during circulation of Asian lineage chikungunya virus. Southeast Asian J Trop Med Public Health. 2012;43:55-61.

2. Rianthavorn P, Wuttirattanakowit N, Prianantathavorn K, Limpaphayom N, Theamboonlers A, Poovorawan Y. Evaluation of a rapid assay for detection of IgM antibodies to chikungunya. Southeast Asian J Trop Med Public Health. 2010;41:92-6.

3. Yap G, Pok KY, Lai YL, Hapuarachchi HC, Chow A, Leo YS, et al. Evaluation of chikungunya diagnostic assays: differences in sensitivity of serology assays in two independent outbreaks. PLoS Negl Trop Dis. 2010;4:e753. http://dx.doi.org/10.1371/journal.pntd.0000753

4. Blacksell SD, Tanganuchitcharnchai A, Jarman RG, Gibbons RV, Paris DH, Bailey MS, et al. Poor diagnostic accuracy of commercial antibody-based assays for the diagnosis of acute chikungunya infection. Clin Vaccine Immunol. 2011;18:1773-5. http://dx.doi.org/10.1128/CVI.05288-11

5. Prat CM, Flusin O, Panella A, Tenebray B, Lanciotti R, Leparc-Goffart I. Evaluation of commercially available serologic diagnostic tests for chikungunya virus. Emerg Infect Dis. 2014;20:2129-32. http://dx.doi.org/10.3201/eid2012.141269

6. Okabayashi T, Sasaki T, Masrinoul P, Chantawat N, Yoksan S, Nitatpattana N, et al. Detection of chikungunya virus antigen by

a novel rapid immunochromatographic test. J Clin Microbiol.
2015;53:382-8. http://dx.doi.org/10.1128/JCM.02033-14

Address for correspondence: Elisa Burdino, Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Corso Svizzera 164, I-10149 Torino, Italy; email: elisa.burdino@libero.it

Novel Single-Stranded DNA
Circular Viruses in Pericardial
Fluid of Patient with
Recurrent Pericarditis

Sébastien Halary, Raja Duraisamy,
Laura Fancello, Sonia Monteil-Bouchard,
Priscilla Jardot, Philippe Biagini,
Frédérique Gouriet, Didier Raoult,
Christelle Desnues

Author affiliations: Institut Hospitalo-Universitaire Méditerranée-Infection, Marseille, France (S. Halary, R. Duraisamy, L. Fancello, S. Monteil-Bouchard, P. Jardot, P. Biagini, F. Gouriet, D. Raoult, C. Desnues); Aix-Marseille Université, Marseille (S. Halary, R. Duraisamy, L. Fancello, S. Monteil-Bouchard, P. Jardot, P. Biagini, F. Gouriet, D. Raoult, C. Desnues); Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille (S. Halary, R. Duraisamy, L. Fancello, S. Monteil-Bouchard, P. Jardot, F. Gouriet, D. Raoult, C. Desnues); KU Leuven, Leuven, Belgium (L. Fancello); Unité Mixte de Recherche, Marseille (P. Biagini); Etablissement Français du Sang, Marseille (P. Biagini); Centre National de la Recherche Scientifique, Marseille (P. Biagini, C. Desnues)

DOI: http://dx.doi.org/10.3201/eid2210.160052

**To the Editor:** Circular replication initiation protein (Rep)-encoding single-stranded DNA (ssDNA) (CRESS-DNA) genomes are found in diverse group II virus families, which all possess a conserved Rep-encoding gene and a nonenveloped icosahedral capsid, except geminiviruses, which have twinned particles (1). Gemycircu-larvirus (GcV) were initially discovered in fungi, but a growing number of new species has been characterized by metagenomics in air, sewage, insects, and feces from a broad range of vertebrates (1–5). GcVs have also been found in the brain and serum of humans with multiple sclerosis; in the cerebrospinal fluid of a patient with encephalitis; and in several blood samples, including those from an HIV-positive blood donor (6–8). We report the presence of 2 divergent GcVs and a novel CRESS-DNA virus (CV) in 2 pericardial fluid samples from a patient with idiopathic recurrent pericarditis.
The patient, a 14-year-old girl who had thoracic scoliosis surgery in 2007, was admitted to the hospital in 2009 for pleuropneumonia and pericarditis, which required pericardial drainage twice within 3 weeks (samples PF₁ and PF₂, respectively). She had thrombocytopenia, a leukocyte count within the normal range, and a high C-reactive protein level. Biochemical and cytologic testing, bacterial cultures, and PCR of pericardial fluid samples for cytomegalovirus, varicella zoster and herpes simplex viruses, parvovirus B19, fungal 18S rRNA, bacterial 16S rRNA, and *Mycobacterium tuberculosis* were negative. Upon approval from the Institut Fédératif de Recherche IFR48 Ethics Committee (Marseille, France) and written informed consent from the patient's parents, we submitted the drainage samples for further investigation.

Virus particles in 0.45-µm filtrates were purified and analyzed by metagenomics as described (9); resulting contigs were aligned against the National Center for Biotechnology Information nonredundant protein database using blastx (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Three contigs were of viral origin (viral first hit, E-value ≤1E-03), all belonging to the ssDNA circular viruses. We obtained complete genomic sequences by PCR with ad hoc primer pairs and Sanger sequencing technology (online Technical Appendix Table, http://wwwnc.cdc.gov/EID/article/22/10/16-0052-Techapp1.pdf).

We annotated genomes using GeneMark (heuristic parameters; http://exon.gatech.edu/GeneMark/) and EMBOSS palindrome (http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome). Analysis of PF1 enabled characterization of HV-GcV1 (GenBank accession no. KU343136) (Figure). This genome displayed a typical GcV architecture, with a 2,264-nt circular DNA molecule carrying a capsid gene on 1 strand and 2 genes on the opposite strand, which coded for Rep1 (involved in replication initiation) and Rep2 (involved in replication termination), respectively. A putative hairpin structure showed a nanonucleotide motif, which was thought to be the Rep target, TAATGTTAT. A fourth gene with no homologs in databases was predicted upstream of the capsid gene. Phylogenetic inference from concatenated Rep placed this virus close to another GcV (found in sewage) in a clade comprising 2 other human-associated GcVs (online Technical Appendix Figure 1).

PF₂ contained 2 other viruses: HV-GcV2 (GenBank accession no. KU343137), another GcV, and HV-CV1 (GenBank accession no. KU343138), a novel CRESS-DNA virus. HV-GcV2 (2,262 nt) shares the same stem-loop motif and genomic structure with HV-GcV1, with the exception of the *rep* gene, which is coded by a single open reading frame. HV-GcV2 proteins share low sequence similarity with HV-GcV1 proteins (33% for capsid and 46% for Rep, as determined by blastp [https://blast.ncbi.nlm.nih.gov/

Blast.cgi]). HV-GcV2 belongs to another clade of the phylogenetic tree that also contains sewage- and bird feces-associated viruses (online Technical Appendix Figure 1). HV-CV1 (2,951 nt) possesses characteristics of CRESS-DNA genomes. Phylogenetic analysis of REP sequences showed

<figure><img src="image_3.png" /></figure>

**Figure.** Genomic features of gemycircularviruses HV-GcV1 and HV-GcV2 and of a novel circular single-stranded DNA (ssDNA) virus, HV-CV1, including hairpin structure and predicted open reading frames. Cap, capsid; Hyp, hypothetical protein with unknown function; Rep, replication initiation protein.
that HV-CV1 and its closest homologue, an ssDNA circular virus of unknown taxon discovered in an Antarctic shelf pond, are distantly related to other CRESS-DNA viruses (online Technical Appendix Figure 2). HV-GcV2 and HV-CV1 displayed no capsid protein similarity between them or with any other virus, as determined by blastp. Annotation of the HV-CV1 capsid gene required use of HHBlits (https://toolkit.tuebingen.mpg.de/hhblits), a more sensitive algorithm (E-value 1.2E-06, probability of 97.2%).

PCR confirmed the absence of HV-GcV1 in PF₂ and HV-GcV2 and HV-CV1 in PF₁, suggesting multiple infections before each pericarditis event or a rapid fluctuation in the load of all 3 persisting viruses. An additional blastx search on 53 other virus metagenomes sequenced from pericardial fluids after pericarditis events failed to retrieve these sequences. To exclude the possibility of sample contamination during procedures, we simultaneously treated a sample with the same reagents and kits used for PF₁ and PF₂ and surveyed it by PCR; results were negative. All metagenomes are publically available in the METAVIR (http://metavir-meb.univ-bpclermont.fr) directory under the pericardial fluids heading.

No relationship between these viruses and pericarditis was established. However, the fact that some CRESS-DNA viruses are animal pathogens (10) and the growing number of GcVs found in human samples in pathologic contexts (6,7) indicate that the viral genomes described here might replicate in human cells, possibly as opportunistic pathogens (8). On the other hand, although diagnostic tests ruled out fungal or bacterial infections, we should still consider the possibility that these viruses infect other uncharacterized organisms. The genomes described here will assist further studies of the prevalence of these viruses in human populations.

This work was funded in part by French National Research Agency grant number ANR-13-JSV6-0004 to C.D. and a Méditerranée Infection fellowship to R.D.

## References

1. Rosario K, Dayaram A, Marinov M, Ware J, Kraberger S, Stainton D, et al. Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta). J Gen Virol. 2012;93:2668–81. http://dx.doi.org/10.1099/vir.0.045948-0

2. Yu X, Li B, Fu Y, Jiang D, Ghabrial SA, Li G, et al. A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus. Proc Natl Acad Sci U S A. 2010;107:8387–92. http://dx.doi.org/10.1073/pnas.0913535107

3. Whon TW, Kim MS, Roh SW, Shin NR, Lee HW, Bae JW. Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere. J Virol. 2012;86:8221–31. http://dx.doi.org/10.1128/JVI.00293-12

4. Kraberger S, Argüello-Astorga GR, Greenfield LG, Galilee C, Law D, Martin DP, et al. Characterisation of a diverse range of circular replication-associated protein encoding DNA viruses recovered from a sewage treatment oxidation pond. Infect Genet Evol. 2015;31:73–86. http://dx.doi.org/10.1016/j. meegid.2015.01.001

5. Sikorski A, Massaro M, Kraberger S, Young LM, Smalley D, Martin DP, et al. Novel myco-like DNA viruses discovered in the faecal matter of various animals. Virus Res. 2013;177:209–16. http://dx.doi.org/10.1016/j.viruses.2013.08.008

6. Lamberto I, Gunst K, Müller H, Zur Hausen H, de Villiers EM. Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients. Genome Announc. 2014;2:pii: e00848-14. http://dx.doi.org/10.1128/ genomeA.00848-14

7. Phan TG, Mori D, Deng X, Rajindrajith S, Ranawaka U, Fan Ng TF, et al. Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage. Virology. 2015;482:98–104. http://dx.doi.org/10.1016/j. virol.2015.03.011

8. Uch R, Fournier PE, Robert C, Blanc-Tailleur C, Galicher V, Barre R, et al. Divergent gemycircularvirus in HIV-positive blood, France. Emerg Infect Dis. 2015;21:2096–8. http://dx.doi.org/10.3201/ eid2111.150486

9. Fancello L, Monteil S, Popgeorgiev N, Rivet R, Gouriet F, Fournier PE, et al. Viral communities associated with human pericardial fluids in idiopathic pericarditis. PLoS ONE. 2014;9:e93367. http://dx.doi.org/10.1371/journal.pone.0093367

10. Segalés J, Allan GM, Domingo M. Porcine circovirus diseases. Anim Health Res Rev. 2005;6:119–42. http://dx.doi.org/10.1079/ AHR2005106

Address for correspondence: Christelle Desnues, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Aix-Marseille Université, Marseille, France; email: christelle.desnues@univ-amu.fr

# Reemergence of Japanese Encephalitis in South Korea, 2010–2015

Jun-Sang Sunwoo,<fnref n="1" /> Keun-Hwa Jung, Soon-Tae Lee, Sang Kun Lee, Kon Chu

Author affiliation: Seoul National University Hospital, Seoul, South Korea

DOI: http://dx.doi.org/10.3201/eid2210.160288

**To the Editor:** Japanese encephalitis (JE) is caused by a virus transmitted by *Culex tritaeniorhynchus* mosquitoes. JE was the major public health concern in South Korea until the late 1960s, with several thousand cases reported annually. The national vaccination program with the inactivated mouse brain-derived Nakayama strain was initiated in 1983 and targeted children <15 years of age. During 1983–2000, annual booster vaccinations were given to children <15 years of age, but in 2000, the booster schedule was changed to 2 doses (1 dose each) for children 6 and 12 years of age. The live attenuated JE vaccine SA

<footnote n="1">¹Current affiliation: Soonchunhyang University Seoul Hospital, Seoul, South Korea.</footnote>