Phylogeographic Characterization of Tick-Borne Encephalitis Virus from Patients, Rodents and Ticks in Slovenia

Rodents and Ticks in Slovenia. PLoS ONE 7(11): e48420. doi:10.1371/journal.pone.0048420 Phylogeographic Characterization of Tick-Borne Encephalitis Virus from Patients, Rodents and Ticks in Slovenia Luka Fajs 0 Emina Durmis i 0 Natas a Knap 0 Franc Strle 0 Tatjana Avs ic -Z upanc 0 Ulrike Gertrud Munderloh, University of Minnesota, United States of America 0 1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 2 Department of Infectious Diseases, University Medical Center Ljubljana , Ljubljana , Slovenia Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing infections of the central nervous system in central Europe. Previous studies have shown that TBEV exhibits pronounced genetic variability, which is often correlated to the geographical origin of TBEV. Genetic variability of TBEV has previously been studied predominantly in rodents and ticks, while information about the variability in patients is scarce. In order to understand the molecular relationships of TBEV between natural hosts, vectors and humans, as well as correlation between phylogenetic and geographical clustering, sequences of TBEV E and NS5 protein genes, were obtained by direct sequencing of RT-PCR products from TBE-confirmed patients as well as from rodents and ticks collected from TBE-endemic regions in Slovenia. A total of 27 partial E protein gene sequences representing 15 human, 4 rodent and 8 tick samples and 30 partial NS5 protein gene sequences representing 17 human, 5 rodent and 8 tick samples were obtained. The complete genome sequence of TBEV strain Ljubljana I was simultaneously obtained. Phylogenetic analysis of the E and NS5 protein gene sequences revealed a high degree of TBEV variability in patients, ticks and rodents. Furthermore, an evident correlation between geographical and phylogenetic clustering was shown that was independent of the TBEV host. Moreover, we show the presence of a possible recombination event in the TBEV genome obtained from a patient sample, which was supported with multiple recombination event detection methods. This is the first study that simultaneously analyzed the genetic relationships of directly sequenced TBEV samples from patients, ticks and rodents and provides the largest set of patient-derived TBEV sequences up to date. In addition, we have confirmed the geographical clustering of TBEV sequences in Slovenia and have provided evidence of a possible recombination event in the TBEV genome, obtained from a patient. - Funding: This study was partially funded by European Commission (European Virus Archive, FP7 CAPACITIES project, GA no. 228292 and EU grant GOCE-2003010284 EDEN). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tick-borne encephalitis virus (TBEV) is the most important arboviral agent in central Europe, responsible for thousands of cases of central nervous system infections every year. TBEV is a member of the family Flaviviridae, genus Flavivirus [1]. TBEV genome comprises a single-stranded positive-sense RNA of approximately 11 kb, which encodes a polyprotein that is cleaved into three structural and seven non-structural proteins [2]. Phylogenetic analyses of TBEV isolates identified three TBEV subtypes: European (TBEV-Eu), Far Eastern (TBEV-Fe) and Siberian (TBEV-Sib) [3]. TBEV is transmitted by Ixodes spp. ticks. Primary vector of the TBEV-Eu subtype strains is Ixodes ricinus. TBE is maintained in the ecosystem between tick and vertebrate hosts. The most important vertebrate reservoirs in Europe are rodents. Rodents serve as both maintaining and amplifying hosts [1]. Although TBEV viral load is often low or undetectable, they play an important role in the circulation of the virus in nature as they enable co-feeding of infected and uninfected ticks [4]. In TBE-endemic areas the virus is distributed patchily in geographically limited areas, known as TBE-foci [1,5]. Within such foci, several genetic variants of TBEV have been identified [612]. Furthermore, several studies have identified geographical clustering of specific TBEV variants within small geographic areas [68]. Gaumann et al. for example performed phylogenetic analysis of 71 TBEV found in I. ricinus in Switzerland and identified 10 phylogenetic clades of closely related TBEV, some of which showed regional clustering [6]. Furthermore, Weidmann et al. have also identified the presence of several viral variants in ticks in Czech Republic and Germany. Their analysis also showed regional clustering of TBEV variants and a correlation between the genetic and geographic distances of TBEV isolates. They were thereby able to determine the direction of spread of TBEV in Central Europe in a westerly direction [7]. These studies were focused on determination of genetic variability of TBEV in rodents and ticks, but little is known about genetic variability of TBEV in patients in endemic areas. While the incidence of disease has been shown to be correlated with the regional prevalence of TBEV in ticks and rodents [8,13,14] limited information is available on the relatedness of TBEV strains among different hosts in endemic areas. Durmisi et al. [8] have shown for example that TBEV is genetically related in ticks and patients in Slovenia. The aim of our study was to expand the knowledge about the genetic relatedness of TBEV in rodents, ticks and patients in Slovenia. Furthermore, we aim to determine the overall genetic variability of TBEV in Slovenia and assess if there is a correlation between phylogenetic and geographic clustering of TBEV variants in Slovenia in all three hosts. While previous studies were focused on molecular determination of virus isolates, direct sequencing of RT-PCR products was employed in our study in order to avoid the occurrence of genetic variation due to virus culturing or cloning. Thereby we were able to obtain a more realistic view of the genetic variability of TBEV in Slovenia. We report the largest number of patient-derived TBEV sequences to date, and show that geographical clustering of TBEV variants is correlated to the phylogenetic clustering and is independent of the host. We also provide evidence of a possible recombination event in the TBEV genome obtained from a patient. Complete genome sequence of TBEV strain Ljubljana I was determined and deposited in the GenBank database under accession number JQ654701. Genome organization analysis of the 11,092 bp long sequence revealed that it was identical to that of other members of the TBEV group. Phylogenetic analysis revealed that strain TBEV Ljubljana I belongs to the TBE-Eu subtype (Figure 1) and is most closely related to the Central European TBEV strains Salem and 263 both on the nucleotide level and th (...truncated)