Author's personal copy Journal of Clinical Virology 46 (2009) 270–274 Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007 Maria Logotheti a , Vasiliki Pogka a , Elina Horefti a , Konstantinos Papadakos a , Maria Giannaki b , Anastasia Pangalis b , Dionyssios Sgouras a,∗ , Andreas Mentis a a National Enterovirus/Poliovirus Reference Laboratory, Hellenic Pasteur Institute, Vas. Sofias 127, 11521, Athens, Greece b Department of Microbiology, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece article info Article history: Received 2 June 2009 Received in revised form 22 July 2009 Accepted 22 July 2009 Keywords: Enterovirus Aseptic meningitis Echovirus 4 VP1 NASBA abstract Background: Aseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV). Objective: Identification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007. Study design: In total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains. Results: EV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006. Conclusions: It is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods. © 2009 Elsevier B.V. All rights reserved. 1. Background Aseptic meningitis is the most commonly observed CNS infec- tion characterized by acute onset of meningeal inflammation signs and symptoms, in which common bacterial agents cannot be iden- tified in the CSF. 1 Aseptic meningitis is usually affecting children and is mainly attributed to Non-Polio Enteroviruses, 2 transmit- ted through the faecal-oral and potentially through the respiratory route. 3,4 Meningitis outbreaks typically occur in restricted geo- graphical areas or communities during summer and autumn Abbreviations: ECV, echovirus; EV, enterovirus; CBV, Coxsackie B virus; CSF, cerebrospinal fluid; NASBA, nucleic acid sequence-based amplification assay; CPE, cytopathic effect; PCR, polymerase chain reaction; RT, reverse transcriptase. ∗ Corresponding author. Tel.: +30 2106478824; fax: +30 2106423498. E-mail address: sgouras@pasteur.gr (D. Sgouras). leading to increased hospital admissions for short periods 5 while only few winter outbreaks have been reported. 6 In Europe, increased incidence of ECV13 and ECV30 was recorded in viral meningitis outbreaks during 2000–2001. 7 Most recent enteroviral meningitis outbreaks occurred in France in 2005 8 and Spain, 9 Kosovo 10 and Latvia 11 in 2006. Strains identi- fied were mainly ECV30 and Coxsackie B5 and to a less extent the ECV2, 4, 6, 9, 11, 13, 14, 18, 20, 29, Coxsackie B4 and Coxsackie A9 serotypes. 8,9,11 In Greece, the ECV6, 11, 15, 30 and Coxsackie B5 serotypes were the etiological agents of recently recorded aseptic meningitis outbreaks. 6,12,13 2. Objectives The present study aimed to the detection and characterization of the EV involved in an aseptic meningitis outbreak in Greece during the summer of 2007. 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.07.019