Short communication Evidence of recombination in Tula virus strains from Serbia Valentina Nikolic a , Novica Stajkovic b , Gorana Stamenkovic c , Radovan Cekanac b , Predrag Marusic d , Marina Siljic a , Ana Gligic e , Maja Stanojevic a,⇑ a University of Belgrade Faculty of Medicine, Belgrade, Serbia b Military Medical Academy, Belgrade, Serbia c University of Belgrade, Institute for Biological Research ‘‘Sinisa Stankovic’’, Belgrade, Serbia d Institute of Public Health, Zajecar, Serbia e Institute of Virology, Vaccines and Sera Torlak, Belgrade, Serbia article info Article history: Available online xxxx Keywords: Recombination Tula virus Phylogenetic analysis Molecular evolution abstract Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nat- ure of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phy- logenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, sup- ported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Hantaviruses, members of the Bunyaviridae family, are envel- oped viruses consisting of tripartite single-stranded RNA genome. Each of the three segments, large (L), medium (M) and small (S) en- codes an RNA-dependent RNA polymerase, envelope glycoproteins (Gn, Gc) and a nucleocapsid protein, respectively (Elliott et al., 1991). Hantaviruses are unique in the Bunyaviridae family in not being transmitted by an arthropod vector. Their natural hosts are rodents belonging to four different subfamilies, including Sig- modontinae, Arvicolinae, Murinae and Neotominae (Kang et al., 2011). Worldwide circulation of over 20 serotypes of hantaviruses, causative agents of human zoonosis, is closely associated with the geographical distribution of their natural hosts (Jonsson et al., 2010). Old world hantaviruses: Dobrava-Belgrade (DOBV), Seoul (SEOV), Puumala (PUUV) and Tula (TULV) are found to be present in Europe, where DOBV, SEOV, PUUV are causing hemorrhagic fe- ver with renal syndrome (HFRS) (Plyusnin et al., 1995; Vapalahti et al., 2003). Human infection caused by TULV has been reported, but no clear evidence of clinical disease associated with this virus exists so far (Clement et al., 2003; Klempa et al., 2003a; Schultze et al., 2002). TULV is widespread across Eurasia, including France, Germany, the Netherlands, Austria, Slovenia, Croatia, Hungary, Poland and Russia, where numerous mammal species have been shown to be reservoirs for TULV, including Microtusarvalis, Microtussubterraneus, Microtusrossiaemeridionalis, Microtusagrestis, Microtusgregalis, Arvicola amphibius and Lagurus lagurus (Schlegel et al., 2012; Schmidt-Chanasit et al., 2010). In the Balkans, Serbia is the first country where TULV was detected, in European pine vole, M. subterraneus (Pitymys subterraneus) trapped in 1987 (Song et al., 2002). Genetic drift and reassortment have been main proposed mechanisms for genetic diversity of hantaviruses as segmented negative-sense RNA viruses (Plyusnin et al., 2002). Recent findings suggested occurrence of homologous recombination in these viruses. The aim of the current study is phylogenetic characteriza- tion of TULV strains from Serbia, one newly detected in M. arvalis and the other previously described in M. subterraneus. 2. Materials and methods 2.1. Reverse transcription PCR (RT-PCR) and sequencing Eight European common voles, M. arvalis, were trapped in central Serbia in 2007, approximately 150 km south of Belgrade (Fig 1.). The 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.08.020 ⇑ Corresponding author. Address: University of Belgrade Faculty of Medicine, Institute of Microbiology and Immunology, Dr Subotica 1, 11000 Belgrade, Serbia. Tel.: +381 11 3643379; fax: +381 11 3643360. E-mail address: mstanojevic@med.bg.ac.rs (M. Stanojevic). Infection, Genetics and Evolution xxx (2013) xxx–xxx Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Please cite this article in press as: Nikolic, V., et al. Evidence of recombination in Tula virus strains from Serbia. Infect. Genet. Evol. (2013), http://dx.doi.org/ 10.1016/j.meegid.2013.08.020