**1. Introduction**

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545-550.

230 Encephalitis

Lyssaviruses represent the Lissavirus genus belonging to the family Rhabdoviridae [War‐ rell, Warrell, 2004]. This genus includes 7 genotypes. Genotype 1 is Rabies virus, which is widespread all over the world [Hughes, 2008; Iseni et al., 1998]. Genotypes 2 (Lagos bat vi‐ rus) [Boulger, Porterfield, 1958; Sureau et al., 1980], 3 (Mokola virus) [Shope et al., 1970; Fa‐ milusi et al., 1972; Kemp et al., 1972] and 4 (Duvenhage virus) [Meredith et al., 1971; Van der Merwe, 1982] are widespread in Central and South Africa. In European countries as well as in European part of Russia there are European bat lyssaviruses of subtypes 1 and 2 (EBLV-1 и EBLV-2) belonging to genotypes 5 and 6, which were isolated from bats and humans bit‐ ten by them (for EBLV-1: [Boulger, Porterfield, 1958; Schneider, Cox, 1994; Selimov et al., 1989; Selimov et al., 1991], for EBLV-2: [Lumio et al., 1986; King et al., 1994]). Australian bat lyssavirus (ABLV) belongs to genotype 7, which is also known to be isolated from humans [McCall et al., 2000; Fraser et al., 1996]. Four genotypes have been recently discovered which are Aravan Virus (Kyrgyzstan) [Botvinkin et al., 1996; Kuzmin et al., 1992], Khujand virus (Tajikistan) [Kuzmin et al., 2001], Irkut virus (Eastern Siberia) and West Caucasian Bat virus (Caucasus) [Botvinkin et al., 2003].

The risk of bat virus infection in humans is low. Every three months the single cases of chi‐ ropteran and human virus infection are reported in Europe (France, Spain, Slovenia, Germa‐ ny, Romania, Ukraine, Russia, etc.) [Rab. Bull. Europe, 2008]. The confirmed cases of rabies

© 2013 Leonova et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

following the bat bite were reported in Ukraine and Russia [Botvinkin et al., 2005]. These cases were associated with European bat virus type 1 (EBLV-1). Human cases of Lyssavirus infection in Siberia and Russian Far East were undiscovered so far.

**2.4. Electron Microscopy (EM)**

**2.5. Molecular genetic methods**

man Coulter, USA).

*2.5.1. Extraction of RNA, PCR and sequencing*

supernatant fluid for 5 minutes. The PEK cells (2 X106

FE (Carl Zeiss, Germany) transmission electron microscope.

The passaged PEK cell culture was infected with Ozernoe strain. After one day the monolay‐ er was separated from the glass using 0.2 ml of 0.25% tripsin solution after each removal of

with Ozernoe virus strain, were placed into the combined fixator for 1 h at room tempera‐ ture [Ito, Karnovsky, 1968]. The fixator was prepared on the basis of 0.2 M cacodylate buffer (рН 7.4) with 3% paraformaldehyde and 0.02% picric acid. After centrifugation the cells were postfixed in 1% buffered OsO4 (Serva, USA) at room temperature for 2 hours. Later on, the dehydration of samples was performed in ethanol solution of increasing density and em‐ bedded in epon-araldite resin (Serva, USA). The ultra thin microscopic sections were pre‐ pared with ultramicrotome LKB-V (LKB, Sweden) in a plane parallel to the cells monolayer. Samples were contrasted with lead citrate by standard method and examined with Libra 200

Total RNA was extracted from the brain of infected suckling mice using a RIBO-zol-A kit (AmpliSens, Russia) according to the manufacturer's protocol. Reverse transcription reaction was carried out using random hexanucleotide primers and Reverta-L-10 kit (InterLabSer‐ vice, Russia) according to the manufacturer's recommendations. For initial detection of virus we used primers previously described by Heaton et al. (1997) for nucleoprotein-encoding genes with minor modifications (JW12 – ATGTAACACCCCTACAATGG, JW6(DPL) – CAATTTGCACACATTTTGTG, JW6(M) – CAGTTAGCGCACATCTTATG, JW6(E) – CAGTTGGCACACATCTTGTG). Amplification was performed with common forward pri‐ mer JW12 and one of the backward primers (JW6(DPL), JW6(M), JW6(E)) alternately. The length of amplification product of the lyssavirus nucleoprotein gene fragment was equally 605 bp. The set of primers for complete genome sequencing has been constructed on the ba‐ sis of full genome sequence of strain Irkut (GeneBank Accession EF614260), so the amplified fragments were 600-700 nucleotides in length, and overlapping areas of the adjacent frag‐ ments were 70-100 nucleotides in length. PCR was carried out in the final volume of 20 μl. PCR buffer contained 4.0 – 6.0 mM magnesium chloride, 65 mМ Tris-HСl (pH 8.8), 20 mМ (NH4)2SO4, 0.01% Tween-20, 200 mM of each dNTP, 0.5 units of Taq-polymerase (AmpliS‐ ens, Russia), 10 pmol of each primer and 0.5 – 2 μl of cDNA mixture (template). The amplifi‐ cation was performed with DNA Engine Dyad (MJ Research, USA) using initial denaturation at 96°C for 30 sec followed by 35 cycles of amplification (5 sec – 96°C, 5 sec – 53°C, 1 min – 72°C). All PCR products were analyzed in 0.8 % agarose gel in TAE buffer contained ethidium bromide and DNA amplicons were extracted from gel slices with QIA‐ quick gel extraction kit (Qiagen). In purified PCR products the both strands were directly sequenced using the same set of primers. Sequencing was performed with Genome Lab DTCS-Quick Start Kit (Beckman Coulter, USA) and automated sequencer CEQ-8800 (Beck‐

), after infection for 1, 2 and 3 days

http://dx.doi.org/10.5772/52869

233

The Fatal Case of Lyssavirus Encephalitis in the Russian Far East

This paper reviews the epidemiological and clinico-morphological characteristics of the fatal human case of not previously described lyssavirus infection identified in Asian Russia as well as results of virological and molecular genetic analysis of its infection agent.
