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    Locations of study sites in Germany and phylogenetic analysis of Usutu virus (USUV) strains. For each trapping site, proportions and number of collected mosquitoes are given as a pie chart. Mosquitoes in the pool that were positive for USUV were trapped at Weinheim (underlined). The Bayesian phylogenetic tree was inferred with MrBayes and is based on partial protein envelope nucleotide sequences (length = 1,215 nucleotides) of USUV. For each sequence, the GenBank accession number, strain designation, and strain origin are shown. Posterior probabilities are shown on each node, if higher than 0.50. Scale bar indicates number of nucleotide substitutions per site.

  • 1.

    Adam F, Diguette JP, Virus d'Afrique (Base de Données). Centre Collaborateur OMS de Référence et de Recherche pour les Arbovirus et les Virus de Fièvres Hémorrhagiques (CRORA). Dakar, Sengal: Institut Pasteur de Dakar. Available at: http://www.pasteur.fr/recherche/banques/CRORA/.

    • Search Google Scholar
    • Export Citation
  • 2.

    Weissenbock H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder B, Nowotny N, 2002. Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis 8: 652656.

    • Search Google Scholar
    • Export Citation
  • 3.

    Chvala S, Bakonyi T, Bukovsky C, Meister T, Brugger K, Rubel F, Nowotny N, Weissenböck H, 2007. Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003–2005. Vet Microbiol 122: 237245.

    • Search Google Scholar
    • Export Citation
  • 4.

    Bakonyi T, Erdelyi K, Ursu K, Ferenczi E, Csorgo T, Lussy H, Chvala S, Bukovsky C, Meister T, Weissenböck H, Nowotny N, 2007. Emergence of Usutu virus in Hungary. J Clin Microbiol 45: 38703874.

    • Search Google Scholar
    • Export Citation
  • 5.

    Busquets N, Alba A, Allepuz A, Aranda C, Ignacio Nuñez J, 2008. Usutu virus sequences in Culex pipiens (Diptera: Culicidae), Spain. Emerg Infect Dis 14: 861863.

    • Search Google Scholar
    • Export Citation
  • 6.

    Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Maioli G, Galletti G, Gelati A, Barbieri I, Tamba M, Lelli D, Carra E, Cordioli P, Angelini P, Dottori M, 2010. Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009. PLoS ONE 5: e14324.

    • Search Google Scholar
    • Export Citation
  • 7.

    Cavrini F, Gaibani P, Longo G, Pierro AM, Rossini G, Bonilauri P, Gerundi GE, Di Benedetto F, Pasetto A, Girardis M, Dottori M, Landini MP, Sambri V, 2009. Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy, August–September 2009. Euro Surveill 14: pii: 19448.

    • Search Google Scholar
    • Export Citation
  • 8.

    Pecorari M, Longo G, Gennari W, Grottola A, Sabbatini AM, Tagliazucchi S, Savini G, Monaco F, Simone M, Lelli R, Rumpianesi F, 2009. First human case of Usutu virus neuroinvasive infection, Italy, August–September 2009. Euro Surveill 14: pii: 19446.

    • Search Google Scholar
    • Export Citation
  • 9.

    Buckley A, Dawson A, Moss SR, Hinsley SA, Bellamy PE, Gould EA, 2003. Serological evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK. J Gen Virol 84: 28072817.

    • Search Google Scholar
    • Export Citation
  • 10.

    Buckley A, Dawson A, Gould EA, 2006. Detection of seroconversion to West Nile virus, Usutu virus and Sindbis virus in UK sentinel chickens. Virol J 3: 71.

    • Search Google Scholar
    • Export Citation
  • 11.

    Linke S, Niedrig M, Kaiser A, Ellerbrok H, Müller K, Müller T, Conraths FJ, Mühle RU, Schmidt D, Köppen U, Bairlein F, Berthold P, Pauli G, 2007. Serologic evidence of West Nile virus infections in wild birds captured in Germany. Am J Trop Med Hyg 77: 358364.

    • Search Google Scholar
    • Export Citation
  • 12.

    Jöst H, Bialonski A, Storch V, Günther S, Becker N, Schmidt-Chanasit J, 2010. Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany. J Clin Microbiol 48: 19001903.

    • Search Google Scholar
    • Export Citation
  • 13.

    Jöst H, Bialonski A, Schmetz C, Günther S, Becker N, Schmidt-Chanasit J, 2011. Isolation and phylogenetic analysis of Batai virus, Germany. Am J Trop Med Hyg 84: 241243.

    • Search Google Scholar
    • Export Citation
  • 14.

    Becker N, Petric D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A, 2010. Mosquitoes and Their Control. Second edition. Heidelberg, Germany: Springer.

  • 15.

    Cook S, Diallo M, Sall AA, Cooper A, Holmes EC, 2005. Mitochondrial markers for molecular identification of Aedes mosquitoes (Diptera: Culicidae) involved in transmission of arboviral disease in West Africa. J Med Entomol 42: 1928.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Isolation of Usutu Virus in Germany

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  • German Mosquito Control Association, Waldsee, Germany; Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; Microbiology Unit, Regional Reference Centre for Microbiological Emergencies, Bologna, Italy; Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Institute for Novel and Emerging Infectious Diseases, Greifswald, Germany

Usutu virus (USUV) is a mosquito-borne flavivirus that emerged 2001 in Austria and caused deaths in wild birds. In Germany, 70,378 female mosquitoes were captured in 2009 and 2010 and assayed for USUV. Virus was isolated in cell culture from one pool of Culex pipiens pipiens mosquitoes trapped exclusively in August 2010 in Weinheim, Germany. Subsequent phylogenetic analysis demonstrated a close relationship between the isolated USUV strain from Germany and a USUV strain from Austria, which was detected in a dead blackbird in 2004.

Usutu virus (USUV) is an arthropod-borne, single-stranded RNA virus and belongs to the Japanese encephalitis virus group within the family Flaviviridae. This virus was first isolated in 1959 from Culex neavei mosquitoes collected in Ndumu, Natal, South Africa.1 Vectors of USUV are ornithophilic mosquitoes of the genera Culex, Coquillettidia, Mansonia, and Culiseta.1

Wild birds are the principal reservoir for USUV and migratory birds play a key role in the introduction of USUV into new areas. Outside Africa, USUV emerged in 2001 in Vienna, Austria and caused deaths in blackbirds (Turdus merula) and great gray owls (Strix nebulosa).2 In 2002, USUV was still circulating in Austria, demonstrating that USUV has managed to overwinter in a local bird-mosquito cycle in central Europe.3 More recently, USUV-specific RNA or antigen was also detected in birds or mosquitoes in Hungary, Switzerland, Italy, and Spain.46 In the summer of 2009, USUV-related illness were reported in two immunocompromised patients in Italy.7,8 Despite these cases, the potential of USUV to cause human disease needs further investigations.

In 2002, an extensive analysis of serum samples from healthy birds captured in the United Kingdom was performed and demonstrated that a significant number of the serum samples contained neutralizing antibodies against USUV.9 This study was subsequently followed by a seroconversion study of chickens from the time of hatching until the age of eight or nine months and seroconversion to USUV was detected.10 Neutralizing antibodies against USUV were also demonstrated in serum samples of wild birds captured during 2002–2005 in Germany.11 In the United Kingdom and in Germany, no obvious reduction in the bird population was observed during this period. However, it was not known if USUV circulate in German mosquito populations. Therefore, the natural vectors of USUV in Germany also remain unknown.

Mosquitoes were collected during July–September 2009 and April–September 2010 at 11 sites in Germany (Figure 1). Trappings were made with CO2-baited encephalitis vector surveillance traps (BioQuip, Compton, CA) and at the Weinheim trapping site (Figure 1) with gravid traps designed according to the Centers for Disease Control and Prevention (Atlanta, GA) gravid trap model 1712 (John W. Hock Company, Gainesville, FL). All collected mosquitoes were frozen at –70°C and transported to the laboratory.

Figure 1.
Figure 1.

Locations of study sites in Germany and phylogenetic analysis of Usutu virus (USUV) strains. For each trapping site, proportions and number of collected mosquitoes are given as a pie chart. Mosquitoes in the pool that were positive for USUV were trapped at Weinheim (underlined). The Bayesian phylogenetic tree was inferred with MrBayes and is based on partial protein envelope nucleotide sequences (length = 1,215 nucleotides) of USUV. For each sequence, the GenBank accession number, strain designation, and strain origin are shown. Posterior probabilities are shown on each node, if higher than 0.50. Scale bar indicates number of nucleotide substitutions per site.

Citation: The American Society of Tropical Medicine and Hygiene 85, 3; 10.4269/ajtmh.2011.11-0248

Mosquito identification, RNA extraction, and virus isolation were performed as described.12,13 Extracted RNA was analyzed by using a newly designed USUV-specific real-time reverese transcription–polymerase chain reaction (RT-PCR) with primers USUTU F (5′-CGTTCTCGACTTTGACTA-3′, nucleotide positions 3294–3311; nucleotide positions are given according to numbering in USUV strain Vienna, GenBank accession no. AY453411), USUTU R (5′-GCTAGTAGTAGTTCTTATGGA-3′, nucleotide positions 3384–3364), probe USUTU P (5′-FAM-ACCGTCACAATCACTGAAGCAT-BHQ1-3′, nucleotide positions 3325–3346, FAM, 6-carboxyfluorescein; BHQ-1, black hole quencher 1). The target was a 91-nucleotide region of the nonstructural protein 1 gene. Real-time RT-PCR was used as described.12,13

A total of 70,378 female mosquitoes (16,057 in 2009 and 54,321 in 2010) were captured, identified to species, and pooled according to species (25 mosquitoes per pool).14 The most abundant mosquito was Aedes vexans (46.6%) followed by Ochlerotatus sticticus (32.0%) and Culex pipiens/Cx. torrentium (15.0%) (Table 1). USUV-specific RNA was detected in one pool (pool no. 1477) of Cx. pipiens/Cx. torrentium captured in the middle of August 2010 at Weinheim (Figure 1). Compared with 2009, the number of collected Cx. pipiens/Cx. torrentium was nearly identical in 2010 (Table 1). The USUV infection rate was low because only 1 of 422 Cx. pipiens/Cx. torrentium pools from Weinheim was positive for USUV.

Table 1

Mosquito species and numbers of females per species caught in Germany, 2009 and 2010

Mosquito speciesNo. (%) mosquitoes collected
20092010Total
Culex pipiens/Cx. torrentium*5,163(32.2)5,383(9.9)10,546(15.0)
Aedes vexans4,366(27.2)28,384(52.3)32,750(46.6)
Ae. cinereus311(2.0)2,329(4.3)2,640(3.8)
Ochlerotatus annulipes594(3.7)32(< 0.1)626(0.8)
Oc. cantans0(0)294(0.5)294(0.4)
Oc. communis0(0)82(0.1)82(0.1)
Oc. punctor0(0)41(< 0.1)41(< 0.1)
Oc. rusticus0(0)654(1.2)654(0.9)
Oc. sticticus1,086(6.8)16,280(30.0)17,366(32.0)
Culiseta annulata438(2.7)363(0.7)801(1.1)
Anopheles claviger3,626(22.6)288(0.5)3,914(5.6)
An. maculipennis473(3.6)164(0.3)637(0.9)
An. plumbeus0(0)27(< 0.1)27(< 0.1)
Total16,05754,32170,378

Sibling species Cx. pipiens and Cx. torrentium were not tested separately because female adults cannot be distinguished morphologically.

Morphologic species determination of the mosquitoes included in the pool that were positive for USUV was refined by sequence analysis of cytochrome oxidase c subunits 1 and 2 (CO1 and CO2) of mitochondrial DNA according to a published protocol.15 Sequence analysis of CO1 and CO2 demonstrated that the mosquitoes included in pool 1477 belong exclusively to Cx. pipiens pipiens. Inoculation of C6/36 cells with pool 1477 caused a cytopathic effect after 144 hours, and USUV-specific RNA was detected by real-time RT-PCR in the supernatant of the infected cell culture after five passages. Moreover, electron microscopy of the infected cell culture showed enveloped viral particles ≈60 nm in diameter.

For extensive phylogenetic analysis, a part of the envelope protein coding sequence (nt positions 1159–2527) was amplified by RT-PCR in two overlapping fragments with reported primers.3 Subsequent phylogenetic analysis by Bayesian inference showed a close relationship between novel USUV strain 1477 and the USU338-04 strain from Austria, which detected in a dead blackbird (Turdus merula) in 2004 (Figure 1).

Our study demonstrates the potentially emerging USUV in southwestern Germany. The virus most likely spread from Austria to Germany rather than being independently introduced from Africa. This finding is the first direct detection of USUV in Germany, although neutralizing antibodies were detected in wild birds in Germany.11 No obvious increase in deaths of birds were detected in Germany in 2009 and 2010, which suggested a low prevalence or virulence of circulating USUV. However, in Italy, USUV-related illness in humans was reported even though the USUV infection rate in mosquitoes was rather low.6

USUV strain 1477 was detected in a pool of Cx. pipiens pipiens mosquitoes in Weinheim, a small city in the upper Rhine valley and the only trapping site that represented an urban ecosystem. The density of blackbirds and Cx. pipiens pipiens mosquitoes in such an urban ecosystems is much higher compared with an agro-ecosystems or forest. This might explain why USUV was not found in other regions of Germany covered by our trapping sites.

In conclusion, after detection of several arboviruses that are new to Germany in 200912,13 another important arbovirus was demonstrated to circulate in Germany in 2010. Thus, mosquito-based surveillance for arboviruses in Germany is important for public health because it provides data about arbovirus activity and distribution. Further studies will be conducted to estimate the medical importance of USUV in southwestern Germany.

  • 1.

    Adam F, Diguette JP, Virus d'Afrique (Base de Données). Centre Collaborateur OMS de Référence et de Recherche pour les Arbovirus et les Virus de Fièvres Hémorrhagiques (CRORA). Dakar, Sengal: Institut Pasteur de Dakar. Available at: http://www.pasteur.fr/recherche/banques/CRORA/.

    • Search Google Scholar
    • Export Citation
  • 2.

    Weissenbock H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder B, Nowotny N, 2002. Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis 8: 652656.

    • Search Google Scholar
    • Export Citation
  • 3.

    Chvala S, Bakonyi T, Bukovsky C, Meister T, Brugger K, Rubel F, Nowotny N, Weissenböck H, 2007. Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003–2005. Vet Microbiol 122: 237245.

    • Search Google Scholar
    • Export Citation
  • 4.

    Bakonyi T, Erdelyi K, Ursu K, Ferenczi E, Csorgo T, Lussy H, Chvala S, Bukovsky C, Meister T, Weissenböck H, Nowotny N, 2007. Emergence of Usutu virus in Hungary. J Clin Microbiol 45: 38703874.

    • Search Google Scholar
    • Export Citation
  • 5.

    Busquets N, Alba A, Allepuz A, Aranda C, Ignacio Nuñez J, 2008. Usutu virus sequences in Culex pipiens (Diptera: Culicidae), Spain. Emerg Infect Dis 14: 861863.

    • Search Google Scholar
    • Export Citation
  • 6.

    Calzolari M, Bonilauri P, Bellini R, Albieri A, Defilippo F, Maioli G, Galletti G, Gelati A, Barbieri I, Tamba M, Lelli D, Carra E, Cordioli P, Angelini P, Dottori M, 2010. Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009. PLoS ONE 5: e14324.

    • Search Google Scholar
    • Export Citation
  • 7.

    Cavrini F, Gaibani P, Longo G, Pierro AM, Rossini G, Bonilauri P, Gerundi GE, Di Benedetto F, Pasetto A, Girardis M, Dottori M, Landini MP, Sambri V, 2009. Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy, August–September 2009. Euro Surveill 14: pii: 19448.

    • Search Google Scholar
    • Export Citation
  • 8.

    Pecorari M, Longo G, Gennari W, Grottola A, Sabbatini AM, Tagliazucchi S, Savini G, Monaco F, Simone M, Lelli R, Rumpianesi F, 2009. First human case of Usutu virus neuroinvasive infection, Italy, August–September 2009. Euro Surveill 14: pii: 19446.

    • Search Google Scholar
    • Export Citation
  • 9.

    Buckley A, Dawson A, Moss SR, Hinsley SA, Bellamy PE, Gould EA, 2003. Serological evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK. J Gen Virol 84: 28072817.

    • Search Google Scholar
    • Export Citation
  • 10.

    Buckley A, Dawson A, Gould EA, 2006. Detection of seroconversion to West Nile virus, Usutu virus and Sindbis virus in UK sentinel chickens. Virol J 3: 71.

    • Search Google Scholar
    • Export Citation
  • 11.

    Linke S, Niedrig M, Kaiser A, Ellerbrok H, Müller K, Müller T, Conraths FJ, Mühle RU, Schmidt D, Köppen U, Bairlein F, Berthold P, Pauli G, 2007. Serologic evidence of West Nile virus infections in wild birds captured in Germany. Am J Trop Med Hyg 77: 358364.

    • Search Google Scholar
    • Export Citation
  • 12.

    Jöst H, Bialonski A, Storch V, Günther S, Becker N, Schmidt-Chanasit J, 2010. Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany. J Clin Microbiol 48: 19001903.

    • Search Google Scholar
    • Export Citation
  • 13.

    Jöst H, Bialonski A, Schmetz C, Günther S, Becker N, Schmidt-Chanasit J, 2011. Isolation and phylogenetic analysis of Batai virus, Germany. Am J Trop Med Hyg 84: 241243.

    • Search Google Scholar
    • Export Citation
  • 14.

    Becker N, Petric D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A, 2010. Mosquitoes and Their Control. Second edition. Heidelberg, Germany: Springer.

  • 15.

    Cook S, Diallo M, Sall AA, Cooper A, Holmes EC, 2005. Mitochondrial markers for molecular identification of Aedes mosquitoes (Diptera: Culicidae) involved in transmission of arboviral disease in West Africa. J Med Entomol 42: 1928.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Jonas Schmidt-Chanasit, Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Strasse 74, D-20359 Hamburg, Germany. E-mail: jonassi@gmx.de

Authors' addresses: Hanna Jöst, German Mosquito Control Association, Ludwigstrasse 99, D-67165 Waldsee Germany, E-mail: hanna.joest@gmx.de. Alexandra Bialonski, Deborah Maus, Stephan Günther, and Jonas Schmidt-Chanasit, Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Strasse 74, D-20359 Hamburg, Germany, E-mails: bialonski@bni-hamburg.de, maus@bnitm.de, guenther@bni-hamburg.de, and jonassi@gmx.de. Vittorio Sambri, Microbiology Unit, Regional Reference Centre for Microbiological Emergencies, Azienda Ospedaliero–Universitaria di Bologna, Policlinico S.Orsola-Malpighi, Bologna Italy, E-mail: vittorio.sambri@unibo.it. Martin Eiden and Martin H. Groschup, Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Institute for Novel and Emerging Infectious Diseases, Südufer 10, D-17493 Greifswald, Insel Riems, Germany, E-mails: martin.eiden@fli.bund.de and martin.groschup@fli.bund.de. Norbert Becker, German Mosquito Control Association, Ludwigstrasse 99, D-67165 Waldsee, Germany and University of Heidelberg, Im Neuenheimer Feld 230 D-69120 Heidelberg, Germany, E-mail: norbertfbecker@web.de.

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