1. Molecular analysis of Rabies virus circulating in terrestrial mammals in the Slovak Republic R. Franka 1,2, M.Mojzis 2, I. Kuzmin 1, A. Velasco Villa 1, P. Yager 1, S. Jerg 2 and C.E.Rupprecht 1 1 Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA, USA 2 State Veterinary Institut Zvolen, National Reference Laboratory for Rabies, Slovak Republic 4. Results 3. Material and Methods 2. Introduction 1. Abstract Rabies of terrestrial mammals remains a problem in eastern Europe despite active oral vaccination campaigns. To date, the red fox (Vulpes vulpes) and the raccoon dog (Nyctereutes procyonoides) are the main hosts of rabies virus (RABV) in Europe. In this preliminary study, we sequenced part of the nucleoprotein (N) gene of RABV, obtained from 22 red foxes, 6 cats, 1 wild cat, 4 dogs, 2 lynxes, 1 goat, 4 stone martens, 1 fitch and 1 badger collected during 2000 to 2003 in the Slovak Republic. A phylogenetic analysis was undertaken, using for comparison RABV sequences originating from terrestrial animal species from other European countries, which were available from GenBank. Sequences were edited using BioEdit software, and multiple alignments were carried out using the Clustal X package. Neighbor joining analysis for 1000 bootstrap replicates was performed using MEGA version 2.1. The analysis revealed that 96% of RABV samples were related with the lineage previously described as the ‘Eastern Europe’ group (EE). The remaining 4% were joined to the lineage described as the ‘Central Europe’ group (CE). No significant associations with the year of occurrence, host species, or a particular geographic location were found within the set of Slovak samples analyzed. In our set of data we did not identify isolates similar to the vaccine strain SAD-Bern which is currently used for oral rabies vaccination in the country. Key words: rabies, phylogenetic, Slovakia, Slovak Republic, epizootiology, epidemiology, 5. Conclusions LITERATURE CITED The main hosts of the rabies in Europe are the red fox (Vulpes vulpes) and the raccoon dog (Nyctereutes procyonoides), with only sporadic human cases registered in European countries over the last few decades, mainly because of successfully implemented oral vaccination programs and advanced human post-exposure prophylaxis (Toma and Andral, 1977; Bourhy et al., 1999). In the western part of Europe, with the exception of Germany, rabies of terrestrial mammals was successfully eliminated. However, the disease remains enzootic in the eastern part of Europe despite active oral vaccination campaigns. Previous phylogenetic studies have demonstrated an intrinsic molecular diversity of rabies virus variants (Kissi et al., 1995; Bourhy et al., 1999, Kuzmin et al., 2004). Phylogenetic analyses of the rabies virus circulating in European terrestrial mammals showed four geographically defined groups: Western Europe (WE), Central Europe (CE) and Eastern Europe (EE) with the red fox as predominant host; and North- Eastern Europe (NEE) with the red fox and raccoon dog as predominant hosts (Bourhy et al., 1999). Oral vaccination campaigns in a few districts of Slovakia were first implemented during 1992-1993 and extended nationwide since 1994. The first strain used for oral vaccination was Vnukovo-32/107, later replaced by an oral vaccine based on the SAD Bern strain. Despite the oral vaccination campaign, rabies remains enzootic in Slovakia. Red foxes are the only rabies reservoir with frequent spill-over mostly to wildlife and to a lesser extent to domestic animals. The goal of our study was to describe the molecular diversity of rabies virus circulating in different species of terrestrial mammals in the Slovak Republic and to compare them with other lineages circulating in different regions of Europe. BLANCOU, J., M.F.A. AUBERT AND M. ARTOIS. 1991. Fox rabies. In: The Natural History of Rabies. G.M. BAER (ed.) Boca Raton. CRC Press, pp. 257–290. BOURHY, H., B. KISSI, L. AUDRY, M. SMRECZAK, M. SADKOWSKA-TODYS, K. KULONEN, N. TORDO, J.F. ZMUDZINSKI, AND E.C. HOLMES. 1999. Ecology and evolution of rabies virus in Europe. Journal of General Virology 80:2545-2557. DEAN, D.J., M.K. ABELSETH, AND P. ATANASIU. 1996. The fluorescent antibody test. In: Laboratory techniques in rabies, 4th Edition, F.-X. MESLIN, M.M. KAPLAN, AND H. KOPROWSKI (eds.). World Health Organization, Geneva, Switzerland, pp.88-93. HOLMES, C.E., C.H. WOELK, R. KASSIS AND H. BOURHY. 2002. Genetic constraints and the adaptive evolution of rabies virus in nature. 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Samples from different species of terrestrial mammals from different regions of the Slovak Republic, collected during 2000 – 2003, were initially screened by the direct fluorescent antibody test (Dean et al., 1996) (Fig.1). Total RNA was extracted from 42 positive brains using TRIzol® Reagent (Invitrogen). The reverse transcriptase polymerase chain reaction (RT-PCR) was performed with a primer set to nucleoprotein (N) gene – 21g and 304 (Trimarchi and Smith, 2002). A fragment of 940nts within the coding region of the N gene (position 70 – 1010 according to PV strain), obtained from 42 different terrestrial animals (22 red foxes, 6 cats, 1 wild cat, 4 dogs, 2 lynxes, 1 goat, 4 stone martens, 1 fitch and 1 badger), was sequenced. The phylogenetic analysis was undertaken by the Neighbor Joining method using the MEGA computer program, version 2.1. For comparison, RABV sequences originating from different mammals from Europe, Asia and Africa, available from GenBank, were included. European bat lyssavirus sequences were used as an outgroup (Bourhy et al., 1999; Kuzmin at al., 2004). Bootstrap values of more than 70% were regarded as providing support for phylogenetic grouping. Identical or highly similar samples from Slovakia were excluded from the tree and only representatives from each subgroup were left.  96% of RABV samples belonged to one clade previously described as the ‘Eastern Europe’ (EE) group, and the remaining 4% were joined to the lineage described as the ‘Central Europe’ (CE). The statistical support observed for these two groups was 95% and 99%, respectively (Fig. 2). The phylogeny showed clear separation between the EE and north-eastern Europe (NEE) clade (Bourhy et al., 1999), which might be explained by the existence of a physical barrier – Carpathian mountain (High Tatras) on the north border of the Slovak Republic and Poland. Thus, the Slovakian isolate 94250SLK collected from a cat in 1994, described by Bourhy et al. (1999) as a member of NEE clade, could be explained as the result of translocation of the animal through the mountain barrier.  The sequence identity of the Slovakian samples with respect to other EE clade members collected over 17 years ranged between 98% and 100%, for the nucleoprotein gene fragment studied.  We have not found any significant differences according to the year of occurrence, host species, or a particular geographic location within eastern Europe.  In our dataset we did not identify isolates similar to the vaccine strain SAD-Bern used for oral rabies vaccination in the country.  No spill-over of European Bat Lyssaviruses from bats to terrestrial mammals was detected in our dataset.  Most of the rabies viruses circulating in different terrestrial mammals in Slovakia belong to the eastern Europe (EE) group.  Absence of distinctions among EE isolates as regards host species or geographic origin suggests that the isolates belong to one circulation circle. Additionally, this virus lineage has been considerably constrained over at least 17 years.  Presence of members of Central Europe (CE) group in the eastern part of the Slovak Republic is surprising and could suggest that this clade is dispersed over a wider geographical area.  Neither vaccine virus, nor European bat lyssavirus spill-overs were detected in our limited dataset. Fig.2 Neighbor joining phylogenetic tree of the 54 rabies virus isolates according to a 940 nucleotide fragment of the nucleoprotein gene (position 70 – 1010 according to PV strain, accession number: M13215). Bootstrap values are presented for the key nodes, and branch lengths are drawn to scale. Isolates from Slovak Republic are bolded. Abbreviations for the isolates from Slovakia are as follows: abbreviation for city isolation followed by id number and two numbers indicating year of collection, followed by specification of species from which samples were collected. Abbreviations for earlier described European and Asian groups (NEE, CE, EE, WE, and A, B, C, D) are given according to Bourhy et al. (1999) and Kuzmin et al. (2004) respectively. SAD Bern is the vaccine strain used for oral vaccination in Slovakia. Fig.1 Locations where samples for our study were collected during period 2000 - 2003 9223FRA74fox 86111YOU86fox 9244FRA92fox 8618POL85raccoondog 9212ALL91fox 9202ALL91fox PO17003fitch PO1903fox 8653YOU86wolf KE14800fox DK24800fox ZV35501cat BA16300fox KE29000cat DK7200fox KE16100cat ZV6601cat ZV21403stonemarten NR19902goat ZV40301lynx NR700fox ZV254003badger RV1596RUSfox 9142EST85raccoondog 9342EST91raccondog 9339EST91raccoondog 3683cRUSsteppefox RV299RUSfox 8681IRAdog 8702IRAwolf 8706ARSfox 9135OMAfox 86107YOU76fox 8658YOU81cattle 8721AFS81human 8807ETH87hyena 9137ALG82dog PV SAD Bern SAD B19 CVS ERA 857rRUSraccoondog KomatsugawaJAPdog 304cRUSsteppefox 9105CAN90fox 4055CANdog RVHKRushuman 9141RUS88arcticfox RV250Rusrodent 8738THAhuman 9007FIN 8615POL EBL1aSK98eserotinus 100 94 77 78 100 95 100 85 69 67 100 99 100 98 72 100 83 97 77 99 96 98 89 69 99 60 80 81 61 92 89 95 0.05 Western Europe Central Europe Eastern Europe North-Eastern Europe Middle East Laboratory vaccine strains Africa 1 B –Arctic like A –Arctic C D European part of Russia EBL 1 EBL 2 South Euro-Asia