Journal of Medical Virology 81:309–316 (2009) Prevalence of Tick-Borne Encephalitis Virus in Ixodes Ricinus From a Novel Endemic Area of North Eastern Italy P. D’Agaro, 1 E. Martinelli, 1 P. Burgnich, 1 F. Nazzi, 2 S. Del Fabbro, 2 A. Iob, 3 M. Ruscio, 4 P. Pischiutti, 3 and C. Campello 1 * 1 Department of Public Medicine Sciences, U.C.O. Hygiene and Preventive Medicine, University of Trieste, and Institute of Child Health IRCCS Burlo Garofolo, Trieste, Italy 2 Department of Biology and Plant Protection, University of Udine, Udine, Italy 3 Department of Prevention, ‘A.S.S.3 Alto Friuli’, Gemona del Friuli, Italy 4 San Daniele Hospital, San Daniele del Friuli, Italy In Alpine area of extreme North Eastern Italy the first autochthonous case of TBE was reported in 1998 and was followed by 45 cases during the period 2001–2007, thus defining this area as definitely endemic. An ecological survey eval- uated the tick density and the Tick-borne ence- phalitis virus (TBEV) infection prevalence in tick collected in selected sites. In addition, TBE strains were characterized by sequencing and phyloge- netic analysis. Overall, 2,361 ticks (2,198 nymphs and 163 adults) of the Ixodes ricinus L. species collected during 2005 and 2006 were examined. Five samples were positive for TBEV, correspond- ing to an overall prevalence rate of 0.21%. When analyzed by place, TBEV was discovered in three sites where the highest tick density was found. The difference of prevalence between high and low density areas tested to be statistically significant (P ¼ 0.028). Phylogenetic analysis showed that four sequences clustered with the Neudoerfl prototype, while the other clustered with the Isosaari 17 strain and with a number of Slovenian isolates. In addition, a sequence detected in archival samples from one human case segregated with another variant, namely the Swedish Toro ¨ strain. J. Med. Virol. 81:309– 316, 2009. ß 2008 Wiley-Liss, Inc. KEY WORDS: TBEV; prevalence; Ixodes rici- nus; sequencing; Italy INTRODUCTION Tick-borne encephalitis virus (TBEV) is the agent of a systemic acute infection in which the neurological disorder, that is, meningoencephalitis, is part of a composite disease pattern with a typical biphasic course and a wide degree of severity. TBEV has been detected frequently in a broad area ranging from Japan to Central Europe, crossing through Siberia. Phylogenetic analysis has revealed that TBE viruses underwent a progressive spreading from East to West, starting from a common ancestor [Zanotto et al., 1995]. Currently, three main lineages of TBEV are described, namely the European, the Far Eastern and the Siberian subtypes, genetically divergent and show- ing different biological characteristics, distinct patterns of geographic distribution and vector involvement [Ecker et al., 1999]. The European subtype, isolated in Central Europe and European Russia, is transmitted by Ixodes ricinus L. and is associated with a milder disease (case–fatality ratio of 1–2%). The Far Eastern subtype reflects the distribution of its vector, Ixodes persulcatus Schultze, being detected in Russia, China, and Japan as well as in Baltic countries and in Finland [Dumpis et al., 1999; Jaaskelainen et al., 2006; Gubler et al., 2007]. The Siberian subtype is closer phylogenetically to the Far Eastern subtype than to the European subtype [Hayasaka et al., 2001], sharing both I. persulcatus as vector and a high virulence (5 – 20% case – fatality ratio), though this figure may be biased by the selective registration of severe cases [Poponnikova, 2006; Lind- quist and Vapalahti, 2008]. A mixed circulation of TBEV subtypes has been described in Estonia, Latvia, and recently also in Finland where the vectors’ distribution was overlapping [Lundkvist et al., 2001; Suss et al., 2002; Golovljova et al., 2004; Jaaskelainen et al., 2006]. Grant sponsor: Institute of Child Health IRCCS Burlo Garofolo; Grant number: R.C. 73/05; Grant sponsor: Azienda Servizi Sanitari n. 3 Alto Friuli. *Correspondence to: C. Campello, Department of Public Medicine Sciences, University of Trieste, Via Dell’Istria 65/1, 34147 Trieste, Italy. E-mail: campello@burlo.trieste.it Accepted 23 September 2008 DOI 10.1002/jmv.21389 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2008 WILEY-LISS, INC.