LETTERS

Usutu in Bats, (brain tissue only). Direct strains with the 2011 bird-derived sequencing of the PCR amplicons re- strain BH65/11–02–03 from Germa- Germany, 2013 vealed that the USUV sequences were ny, forming a distinct group within the To the Editor: Usutu virus related to the recently described bird- phylogenetic tree (group Europe 3) (USUV) is an arthropod-borne fla- derived USUV strain BH65/11–02–03 (Figure). A partial envelope and NS5- vivirus that belongs to the Japanese from Germany (2). Attempts to isolate gene–based phylogenetic analysis that serocomplex (1). USUV the bat USUV strains in cell culture used more available sequences from circulates between ornithophilic were not successful, probably because databases yielded the same topology mosquito vectors (mainly spp. of autolysis. However, the complete (data not shown). mosquitoes) and avian amplification genome sequences of both bat USUV Pipistrellus bats are highly preva- hosts (2). Migratory birds play a key strains (BAT1USUTU-BNI, KJ859682; lent in Germany. Their geographic role in the introduction of USUV into BAT2USUTU-BNI, KJ859683) were range overlaps with that of the USUV new areas (3). USUV has recently then determined directly from the brain epizootic. Thus, considerable interac- been introduced from Africa into samples by using primers (online Tech- tions between birds, mosquitoes, and Europe, causing epizootics among nical Appendix, http://wwwnc.cdc.gov/ bats could have occurred. The ami- wild birds and Usutu in humans EID/article/20/10/14-0909-Techapp1. no acid replacements (A1236V and (4–6). The detection and isolation of pdf) designed from multiple align- L1549F) detected in the NS genes of USUV from different bird and mam- ments of USUV genomes obtained Pipistrellus bat–derived USUV strains malophilic mosquitoes during the from databases. and the bird-derived USUV strain epizootic in Germany raise questions The 2 genomes had an identi- from Germany suggest an adaptive regarding the USUV range (2,3). cal size of 11,065 nt with a 96-nt 5′ evolution, which probably occurred Bats have been considered natural nontranslated region and a 664-nt 3′ during the introduction of the virus reservoir hosts of a wide diversity nontranslated region. The single open into Germany. of , including several flavivi- reading frame encodes a polyprotein of Although the role of these mu- ruses (7,8). Their ability to fly and 3,434 aa. Both bat USUV strains had tations is not known, similar muta- their social behavior enable efficient 99.9% nt and 99.8% aa identity. We tions in the related maintenance, spread, and evolution compared the 2 bat USUV strains with modulated the host antiviral response of viruses. those detected in mosquitos, birds, by inhibition of interferon signaling In September and October 2013, and humans from Europe and Africa; (10). Our results suggest that bats in southwest Germany, 2 dead bats the sequence identities of nucleotides probably contribute to the epizootic were found within ≈15 km of each varied from 78.3% to 99.3% and of rather than act as a silent reservoir other (bat 1, Ludwigshafen am Rhein, amino acids from 90.8% to 99.3%. for the virus. In contrast, infections 49°28′34′′N 8°26′46′′E; bat 2, Wald- The sequence identity matrix with the of bats might be merely coincidental see, 49°23′44′′N 8°26′27′′E), corre- USUV strain BH65/11–02–03 from to what may well be broader infec- sponding to the previously described Germany was 99.3% for nucleotides tions of vertebrates in the epizootic USUV-endemic area (2,3). A full and 99.2% for amino acids. Compari- area. However, for confirmation of necropsy was conducted on each bat, son of the Pipistrellus bat USUV com- this hypothesis, further investigations and samples were collected for virus plete polyprotein sequence with mos- are required. Although the detected detection, histologic analysis, and bat quito and bird-derived strains showed bat-derived sequences are somehow species determination. 2 aa substitutions—one (A1236V) in distinct from sequences of other Total DNA and RNA were extract- the nonstructural protein (NS) 2a and USUV strains, a spillover infection ed from tissue samples (brain, liver, the other (L1549F) in the NS3 gene— from birds or another, yet unrecog- lung, and heart) and subjected to reverse which were detected only in the bird- nized, host cannot be ruled out. The transcription PCR for rhabdovirus and derived USUV strain BH65/11–02–03 detection of the virus exclusively (2). Histologic analysis of the from Germany. In addition, 2 addi- in brain tissue suggests that USUV tissue samples was not successful be- tional unique amino acid substitutions might have a higher tropism for the cause of autolysis. Use of a cytochrome (A1841V and K1870M) in the NS3 nervous system in bats, as opposed b–specific PCR and direct sequencing protein gene of the BAT1USUTU-BNI to the pantropism observed in birds of the PCR amplicons genetically iden- strain were also identified. Bayesian (2). The detection of USUV in bats tified each bat as a common pipistrelle and maximum-likelihood phylogenet- raises questions for further research, (Pipistrellus pipistrellus) (9). ic analyses of the full-length sequenc- including the potential role of bats as The bat samples were nega- es revealed the close relationship of reservoirs in Africa and transmission tive for rhabdoviruses but positive for the Pipistrellus bat–derived USUV by mosquito vectors.

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Figure. Maximum-likelihood phylogenetic tree of Pipistrellus bat Usutu viruses (USUV) based on full-length nucleotide sequences and showing the phylogenetic placement of the bat-derived USUV compared with human-, mosquito-, and bird-derived strains. The phylogenetic analyses were performed by using PhyML 3.0 (http://www.atgc-montpellier.fr/phyml/versions.php) with 1,000 pseudo- replicates and parallel Bayesian Markov chain Monte Carlo tree-sampling methods based on 2 runs consisting of 4 chains of 1,000,000 with a burn-in of 25% using MrBayes 3.1.2 (http://mrbayes.sourceforge.net/). The Akaike information criterion was chosen as the model selection framework and the general time-reversible model of sequence evolution with gamma-distributed rate variation among sites as the best model. Numbers at the nodes indicate maximum-likelihood bootstrap replicates (>70%) and parallel the posterior probability values (clade credibilities >90%). Boldface indicates USUV strains from Pipistrellus bats in Germany in 2013 (this study). Strain names, GenBank accession numbers, and countries of origin for sequences used to construct the tree are indicated on the branches. Scale bar indicates mean number of nucleotide substitutions per site.

Acknowledgments References 6. Allering L, Jöst H, Emmerich P, Günther S, Lattwein E, Schmidt M, et al. Detection We thank Claudia Poggensee, Alex- of Usutu virus infection in a healthy blood 1. Kuno G, Chang GJ, Tsuchiya KR, andra Bialonski, Birgit Hüsing, Corinna donor from south-west Germany, 2012. Karabatsos N, Cropp CB. Phylogeny of the Thomè, Sabine Köhler, and Mathis Peters- Euro Surveill. 2012;17:20341. genus Flavivirus. J Virol. 1998;72:73–83. 7. Calisher CH, Childs JE, Field HE, en for excellent technical assistance. 2. Becker N, Jöst H, Ziegler U, Eiden M, Holmes KV, Schountz T. Bats: impor- Höper D, Emmerich P, et al. Epizootic tant reservoir hosts of emerging viruses. We dedicate this letter to the late emergence of Usutu virus in wild and Clin Microbiol Rev. 2006;19:531–45. Ursula Herrmann (1927–2014), who made captive birds in Germany. PLoS ONE. http://dx.doi.org/10.1128/CMR.00017-06 2012;7:e32604. http://dx.doi.org/10.1371/ this study possible. 8. Liu S, Li X, Chen Z, Chen Y, Zhang Q, journal.pone.0032604 Liao Y, et al. Comparison of genomic and 3. Jöst H, Bialonski A, Maus D, Sambri V, amino acid sequences of eight Japanese Eiden M, Groschup MH, et al. Isolation Daniel Cadar, Norbert Becker, encephalitis virus isolates from bats. Arch of Usutu virus in Germany. Am J Trop Renata de Mendonca Campos, Virol. 2013;158:2543–52. http://dx.doi. Med Hyg. 2011;85:551–3. http://dx.doi. org/10.1007/s00705-013-1777-5 Jessica Börstler, Hanna Jöst, org/10.4269/ajtmh.2011.11-0248 9. Burkett-Cadena ND, Graham SP, and Jonas Schmidt-Chanasit 4. Weissenböck H, Bakonyi T, Rossi G, Hassan HK, Guyer C, Eubanks MD, Mani P, Nowotny N. Usutu Author affiliations: Bernhard Nocht Institute Katholi CR, et al. Blood feeding patterns virus, Italy, 1996. Emerg Infect Dis. for Tropical Medicine, Hamburg, Germany of potential vectors of the genus 2013;19:274–7. http://dx.doi.org/10.3201/ (D. Cadar, J. Börstler, H. Jöst, J. Schmidt- Culex targeting ectothermic hosts. Am J eid1902.121191 Trop Med Hyg. 2008;79:809–15. Chanasit); German Mosquito Control 5. Vilibic-Cavlek T, Kaic B, Barbic L, 10. Liu WJ, Wang XJ, Mokhonov VV, Pem-Novosel I, Slavic-Vrzic V, Lesnikar V, Association, Speyer, Germany (N. Becker); Shi PY, Randall R, Khromykh AA. In- et al. First evidence of simultaneous Federal University of Rio de Janeiro, Rio de hibition of interferon signaling by the occurrence of West Nile virus and Usutu Janeiro, Brazil (R. de Mendonca Campos); New York 99 strain and Kunjin subtype virus neuroinvasive disease in humans of West Nile virus involves blockage of and German Centre for Infection Research, in Croatia during the 2013 outbreak. STAT1 and STAT2 activation by nonstruc- Infection. 2014 May 5 [Epub ahead of Hamburg (H. Jöst, J. Schmidt-Chanasit) tural proteins. J Virol. 2005;79:1934–42. print]. http://dx.doi.org/10.1007/s15010- http://dx.doi.org/10.1128/JVI.79.3.1934- DOI: http://dx.doi.org/10.3201/eid2010.140909 014-0625-1 1942.2005

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Address for correspondence: Jonas Schmidt- On May 25, 2014, a healthy After CHIKV infection was Chanasit, Bernhard Nocht Institute for Tropical 60-year-old woman returned to French confirmed, the case was immediately Medicine, WHO Collaborating Centre for Polynesia after a 6-month stay with reported to health authorities in Arbovirus and Haemorrhagic Fever Reference her husband’s family in Guadeloupe, French Polynesia. control and Research, Bernhard-Nocht-Strasse 74, 20359 where she had been in contact with measures were immediately im- Hamburg, Germany; email: [email protected] family members who reportedly had plemented and included individual . On the first night after protection against mosquito bites arriving back home in French Polynesia, (mosquito repellents) for the patient she noted headache, transient high and her close social and family fever, and mild arthralgia of the knees. contacts and collective protection The next day, she sought care from (insecticide spraying and breeding her general practitioner for weakness, site elimination) targeting the house headache, and severe polyarthralgia of the patient and the areas that she (wrists, fingers, knees, toes). Physical had visited. Written informed consent Chikungunya Virus examination revealed only swollen was obtained from the patient before Imported into fingers and toes; the patient was not publication of this case report. febrile. Blood samples were collected, Arbovirus diseases are endemic French Polynesia, and the patient was administered to French Polynesia. 2014 acetaminophen and corticosteroids. serotypes 1 and 3 have been co- Her headache persisted until day 3, and circulating since 2013 (8); and, from To the Editor: Chikungunya arthralgia persisted until day 4. October 2013 through April 2014, a virus (CHIKV) is an emerging Laboratory tests revealed large outbreak of infection arthropodborne of the lymphopenia (589 × 106 cells/L) and occurred (9). Because this case provides family Togaviridae (1). The most slightly elevated C-reactive protein evidence of the possible emergence of common clinical manifestations of (14.2 mg/L) and liver enzyme levels CHIKV in French Polynesia, health infection with CHIKV are abrupt onset (aspartate aminotransferase 44 IU/L, authorities and health care workers in of fever, headache, back pain, myalgia, gamma-glutamyl transferase 58 IU/L). French Polynesia were immediately and arthralgia affecting mainly the CHIKV infection was confirmed by a alerted and prepared to detect local extremities; in ≈50% of patients, specific real-time reverse transcription transmission of CHIKV infection. a rash develops, and relapsing and PCR (rRT-PCR) with previously CHIKV is transmitted by mos- incapacitating arthralgia is common reported primers and probe (7) and quitoes of the species, especially (1). Three CHIKV lineages have been by partial sequencing of the E1 gene Ae. aegypti and Ae. albopictus (1,2). The characterized: West African, Asian, (GenBank accession no. KJ939333). risk for emergence of a chikungunya and East/Central/South African (1,2). Phylogenetic analysis (Figure) showed outbreak in French Polynesia is high The strain currently circulating in that the virus strain isolated from the because of the presence of 2 potential the Caribbean belongs to the Asian patient was most closely related to strains vectors: Ae. aegypti mosquitoes, lineage (2). isolated in the British Virgin Islands vectors of CHIKV in New Caledonia In the Pacific region in 2011, in 2014 (VG14/99659, accession no. (3) and in Guadeloupe (2), and Ae. a CHIKV outbreak was reported KJ451624), Yap State, Federated States polynesiensis mosquitoes, potential in New Caledonia (3). Additional of Micronesia, in 2013 (FM13/3807, CHIKV vectors as suggested by outbreaks have been reported in accession no. KJ451622), and Zhejiang experimental infections (10). Papua New Guinea in June 2012 (4), Province, China, in 2012 (CN12/chik- The role of foreign travel in Yap State in August 2013 (5), and sy, accession no. KF318729), with spreading between French in the Kingdom of Tonga in April 100%, 99.89%, and 99.78% homology, overseas territories is highlighted 2014 (6). In the Caribbean region, respectively, thereby confirming its by the observation that the CHIKV- cases of CHIKV infection were inclusion in the Asian lineage. A blood infected patient reported here returned reported in the French part of Saint sample from the patient was inoculated from Guadeloupe and by a previous Martin Island in December 2013, into Vero and C6/36 report that the 2013 outbreak of dengue after which CHIKV rapidly spread cells. Cells were incubated for 6 days, virus type 3 in French Polynesia to other Caribbean islands, including after which time both supernatants was caused by a virus introduced Guadeloupe (2), where by the end of were removed and tested. RT-PCR, as from French Guiana (8). May 2014 it had caused an estimated described above, gave positive results was reported in French Polynesia 23,100 infections. for CHIKV. in October 2013; within the next 6

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