**2. Methods of diagnosis**

#### **2.1. Virological tests**

The brain samples taken postmortem from the patient were used to prepare 10% suspen‐ sion. To isolate the virus two-day-old noninbred white mice were used. These animals were challenged with 0.01 ml of 10% brain suspension both intracerebrally and subcutaneously. The isolated strain was called Ozernoe after the place where the dead patient was infected.

The titer of virus with 10-fold dilution was determined using intracerebral inoculation of the two-day-old and three-week-old white mice as well as pig embryo kidney (PEK) cell cul‐ tures.

#### **2.2. Immunological methods**

The IgG antibodies were determined by ELISA using a Vector-TBE-IgG kit ("Vector-Best", Novosibirsk, Russia). We used the conventional methods for determine hemagglutinating properties of the isolated strain and haemagglutination-inhibition (HI) antibodies in the pa‐ tient's blood.

For the indirect immunofluorescent (IF) antibody method Ozernoe strain was used to infect continuous PEK cell culture; the slides were prepared on the second day after infection. The luminescent sera against human globulins (manufactured by Gamaleya Institute of Epi‐ demiology and Microbiology RAMS, Moscow, Russia) were used to detect the specific lumi‐ nescence of the antigen–antibody complexes. The titer of specific antibodies was determined by the bright green intraplasma granular fluorescence of the specific complex using the endpoint dilutions of the blood serum.

#### **2.3. Morphological methods**

Conventional methods were used for pathohistological examination of the cadaveric materi‐ al fixed in 8% neutral buffered formalin. The samples were fixed in paraffin wax according to the standard technique [Pearse, 1968], and sections with five micron thickness were cut using a hand-driven microtome and transferred to egg albumin coated slides. Then the sec‐ tions were dewaxed in xylene, stained in hematoxylin and eosin, mounted in DPX, and viewed under light microscope and photographed (Axioscope A1, Zeiss, Germany).

#### **2.4. Electron Microscopy (EM)**

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

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

The brain samples taken postmortem from the patient were used to prepare 10% suspen‐ sion. To isolate the virus two-day-old noninbred white mice were used. These animals were challenged with 0.01 ml of 10% brain suspension both intracerebrally and subcutaneously. The isolated strain was called Ozernoe after the place where the dead patient was infected.

The titer of virus with 10-fold dilution was determined using intracerebral inoculation of the two-day-old and three-week-old white mice as well as pig embryo kidney (PEK) cell cul‐

The IgG antibodies were determined by ELISA using a Vector-TBE-IgG kit ("Vector-Best", Novosibirsk, Russia). We used the conventional methods for determine hemagglutinating properties of the isolated strain and haemagglutination-inhibition (HI) antibodies in the pa‐

For the indirect immunofluorescent (IF) antibody method Ozernoe strain was used to infect continuous PEK cell culture; the slides were prepared on the second day after infection. The luminescent sera against human globulins (manufactured by Gamaleya Institute of Epi‐ demiology and Microbiology RAMS, Moscow, Russia) were used to detect the specific lumi‐ nescence of the antigen–antibody complexes. The titer of specific antibodies was determined by the bright green intraplasma granular fluorescence of the specific complex using the end-

Conventional methods were used for pathohistological examination of the cadaveric materi‐ al fixed in 8% neutral buffered formalin. The samples were fixed in paraffin wax according to the standard technique [Pearse, 1968], and sections with five micron thickness were cut using a hand-driven microtome and transferred to egg albumin coated slides. Then the sec‐ tions were dewaxed in xylene, stained in hematoxylin and eosin, mounted in DPX, and

viewed under light microscope and photographed (Axioscope A1, Zeiss, Germany).

well as results of virological and molecular genetic analysis of its infection agent.

infection in Siberia and Russian Far East were undiscovered so far.

**2. Methods of diagnosis**

**2.2. Immunological methods**

point dilutions of the blood serum.

**2.3. Morphological methods**

**2.1. Virological tests**

tures.

232 Encephalitis

tient's blood.

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 supernatant fluid for 5 minutes. The PEK cells (2 X106 ), after infection for 1, 2 and 3 days 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 FE (Carl Zeiss, Germany) transmission electron microscope.

#### **2.5. Molecular genetic methods**

#### *2.5.1. Extraction of RNA, PCR and sequencing*

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‐ man Coulter, USA).

#### *2.5.2. Phylogenetic analysis*

Phylogenetic trees were constructed by: (i) maximum-parsimony (MP) using algorithms from the DNAPARS and PROTPARS programs of the PHYLIP package; (ii) neighbour-join‐ ing (NJ) using the evolutionary distance correction statistics of Kimura (1980) and Tajima & Nei (1984); and (iii) maximum-likelihood (ML) using the PAUP\* phylogenetic program [Swofford, 2001].

en into the Primorye Clinical Hospital No.1 of Vladivostok. On her arrival at hospital department of resuscitation and intensive care the patient was in deep sopor. She was found to have three-finger stiff neck. Kernig's sign was not observed. Pupils with the diameter of 3 mm were sluggish in respond to the light, D<S. The diagnosis set was infectious (virus) me‐ ningoencephalitis of unknown origin, an acute stage with a severe course attended by deep flaccid paresis and bulbar syndrome. On September 14, 2007, the patient was examined by a neurologist who indicated poor general state and depressed consciousness up to the level of superficial coma. The skin of arms, legs and upper shoulder girdle was covered with punc‐ tulated hemorrhagic rash (D=1–1,5 mm). There was Kernig's sign of 160° – 170° on both sides. Pupils were mydriatic, D=S; pupillary reactions were lively. Amyotonia of limbs was observed. Deep reflexes were very weak, D=S; pathological reflexes were not recorded. An assumption of rhabdovirus infection was made upon epidemiological anamnesis, presence of hemorrhagic rash on the skin, ecchymoses at sites of injection, bloody vagina discharges, hypersalivation, and neurologic symptomatology, as well as lack of vaccination against ra‐

bies. Development of infection was probably caused by abrupt hypothermia.

**Parameters 13.09.2007 21.09.2007** Hemoglobin 149,7 g/l 116,7 g/l Erythrocytes 4,9.109cells/l 3,5.109cells/l

Hematocrit 48 32

(DNI) delta neutrophil index (normal - 0.06) 0,2 0,4

**Table 1.** Dynamics of the hematological parameters.

ESR 20 mm/h 65 mm/h

Leukocytes 10-13.109 cells/l 10-13.109 cells/l

(LI) Leukocyte index of intoxication (normal - 0,3-1,5 u.) 3,8 u. 8,3 u. (high toxicity)

mm/hr to 65 mm/hr). At all times a stable leukocytosis (10 – 13 ×109

116.7 g/L), erythrocyte content (from 4.9 ×109

intoxication.

As seen in Tabl. 1, blood test revealed the decrease in hemoglobin level (from 149.7 g/L to

hematocrit (from 48 to 32) and increase in erythrocyte sedimentation rate (ESR) (from 20

served. To estimate a degree of neutrophil shift in blood which reflects the severity of patho‐ logic process, an index of neutrophil shift, which normally is 0.06, was counted. On arrival of the patient at hospital department the index of neutrophil shift was 0.2 that is 3.3 times higher than normal. At the day when patient died the index of neutrophil shift was 0.4, that is 6.7 times higher than normal. The value of leukocyte intoxication index (LII) by Calf-Ca‐ liph is representative for the estimation of an intoxication degree. Normally, the LII is 0.3 – 1.5 units and the values ≥1.5 indicate the intoxication. After the hospitalization of the patient the LII was 3.8 units, and by the time of death it was 8.3 units indicating the high stage of

cells per liter to 3.5 ×109 cells per liter), the

The Fatal Case of Lyssavirus Encephalitis in the Russian Far East

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

235

cells per liter) was ob‐

Bootstrap resampling analysis [Felsenstein, 1985] was carried out using 1000 data replica‐ tions to evaluate the robustness of the phylogenetic groupings observed. Bootstrap values gave a strong evidence for a particular phylogenetic grouping [Hillis, Bull, 1993]. All ABL nucleotide sequences obtained in this study have been submitted to GeneBank and their ac‐ cession numbers are listed in Table 1. All other lyssavirus nucleotide sequences used for phylogenetic analysis and sequence comparison were obtained from GenBank; their acces‐ sion numbers and appropriate references are listed in Table 1.
