**3. The results of a comprehensive study of virus infection case**

#### **3.1. Clinical and epidemiological diagnosis**

The patient was a twenty-year-old girl named Zh. who was the resident of Ozernoye village (Yaroslavsky Region, Primorye Territory, Far East of Russia). She's got an acute disease on the 10th of September, 2007 (medical record No11063). The epidemiological anamnesis con‐ tained two facts to be paid attention to. Firstly, on July 12 – 13, 2007 the patient was bitten by a tick into lumbar region while being in forest zone of Yaroslavsky Region. Prior to this, she had been vaccinated against tick-borne encephalitis (TBE) and had routine revaccinations. Secondly, on August 10, 2007 in Ozernoe the girl ran into the bat which entered into the house. Being frightened by a loud music the bat has bumped into the girl and wounded her underlip left two thin parallel slightly bleeding stripes. The bat species was unknown. The wounds healed in a short period of time. During one month the girl's mother who was a health-care worker, did not notice any inflammatory infiltration in the wound or increase of regional lymph nodes. The girl felt well and did not have any complaints. She was not vacci‐ nated against rabies.

One month later (on September 10, 2007) the girl got an acute disease caused by hypother‐ mia (she sopped in the rain). Her body temperature rose up to 38°C and intensive headache, repeated vomiting, diplopia, head and hand tremor occurred. On September 11, 2007 she was hospitalized to infectious disease ward of Ussuriysk hospital. The patient has got worse: the general brain symptomatology (constricting diffuse headache and multiple vom‐ iting without any relief) and toxic syndrome increased (the body temperature reached 38.6°); the photophobia occurred. The bulbar disturbances (chocking when swallowing, bar‐ yphonia) added and increased; profuse discharge of phlegm from the upper respiratory tract was noticed; meningeal symptoms and depression of consciousness (soporific state) oc‐ curred. On September 13, 2007, the patient with a diagnosis of meningoencephalitis was tak‐ 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.

As seen in Tabl. 1, blood test revealed the decrease in hemoglobin level (from 149.7 g/L to 116.7 g/L), erythrocyte content (from 4.9 ×109 cells per liter to 3.5 ×109 cells per liter), the hematocrit (from 48 to 32) and increase in erythrocyte sedimentation rate (ESR) (from 20 mm/hr to 65 mm/hr). At all times a stable leukocytosis (10 – 13 ×109 cells per liter) was ob‐ 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 intoxication.


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

*2.5.2. Phylogenetic analysis*

[Swofford, 2001].

234 Encephalitis

nated against rabies.

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

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‐

The patient was a twenty-year-old girl named Zh. who was the resident of Ozernoye village (Yaroslavsky Region, Primorye Territory, Far East of Russia). She's got an acute disease on the 10th of September, 2007 (medical record No11063). The epidemiological anamnesis con‐ tained two facts to be paid attention to. Firstly, on July 12 – 13, 2007 the patient was bitten by a tick into lumbar region while being in forest zone of Yaroslavsky Region. Prior to this, she had been vaccinated against tick-borne encephalitis (TBE) and had routine revaccinations. Secondly, on August 10, 2007 in Ozernoe the girl ran into the bat which entered into the house. Being frightened by a loud music the bat has bumped into the girl and wounded her underlip left two thin parallel slightly bleeding stripes. The bat species was unknown. The wounds healed in a short period of time. During one month the girl's mother who was a health-care worker, did not notice any inflammatory infiltration in the wound or increase of regional lymph nodes. The girl felt well and did not have any complaints. She was not vacci‐

One month later (on September 10, 2007) the girl got an acute disease caused by hypother‐ mia (she sopped in the rain). Her body temperature rose up to 38°C and intensive headache, repeated vomiting, diplopia, head and hand tremor occurred. On September 11, 2007 she was hospitalized to infectious disease ward of Ussuriysk hospital. The patient has got worse: the general brain symptomatology (constricting diffuse headache and multiple vom‐ iting without any relief) and toxic syndrome increased (the body temperature reached 38.6°); the photophobia occurred. The bulbar disturbances (chocking when swallowing, bar‐ yphonia) added and increased; profuse discharge of phlegm from the upper respiratory tract was noticed; meningeal symptoms and depression of consciousness (soporific state) oc‐ curred. On September 13, 2007, the patient with a diagnosis of meningoencephalitis was tak‐

sion numbers and appropriate references are listed in Table 1.

**3.1. Clinical and epidemiological diagnosis**

**3. The results of a comprehensive study of virus infection case**

Moreover, the determination of total serum immunoglobulin levels (IgM, IgG, IgA) in the patient's blood (on the arrival at hospital) showed the dis-immunoglobulinemia with IgM level 2.4 times higher than normal (3.15 g/L and 1.30 g/L, respectively), and IgG and IgA lev‐ els 1.5 and 1.8 times, respectively, lower than normal. The number of circulating immune complexes (CIC) of small sizes was 1.7 times higher than normal (123 and 72 arbitrary units, respectively). The results of hematomancy indicated increasing intensity of intoxication, overall inflammatory reaction, and multiple organ failure.

vascular walls, as well as thickening and hyalinosis of septa were found. In the liver the ex‐ tended distrophic and necrobiotic changes of parenchyma coupled with diffuse sparse prolif‐ eration of Kupffer cells and friable polymorphic cellular infiltration along the portal tracts followed by destruction of blood vessel walls were presented. In the kidney a severe necrotic glomerulonephrosis was found. In the cortical substance a necrosis and destruction of the ves‐ sels in malpighian tufts, as well as hemorrhages were observed. In the medullary substance a vascular plethora and hemorrhages were found. Also a total necrobiosis and tubular epitheli‐ um necrosis were observed along the nephrons. The results obtained by histological study in‐ dicated the severe multipleorgan pathology coupled with systemic destructive-dystrophic changes of blood vessels with predominance of edematous and destructive changes in CNS and parenchymatous organs. The pathological process was accompanied by the development of a severe immunodeficiency and a suppression of cell-mediated inflammatory response.

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237

**Figure 1. The pathomorphological changes in brain of patient Zh. deceased because of Lyssavirus infection.** A – a sparse perivascular found round some lumen; B, C – lymphohistiocytic infiltrate significant vascular disruption of the microvasculature with the fibrinoid necrosis of vascular wall and the presence of fibrin in the vascular lumen; D – er‐ ythrostasis; E – hyaline thrombi and a small amount of mononuclear cells in many vascular lumen; F – neuronophagic

nodules like "rabies nodules" in the subcortical brain. Gemotoxiline-eozine; X 200.

Despite the intensive therapy (antibacterial, antiviral, neurometabolic, symptomatic, and ar‐ tificial lung ventilation (ALV) via bronchostomy) the patient was getting worse every day due to the increase of general brain symptoms coupled with multiple organ failure, bilateral hypostatic pneumonia, arterial blood pressure (ABP) fall up to 60/40 mm Hg, and apparent tachycardia of 140 – 160 beats per minute. On the eleventh day, September 21, 2007, the dis‐ ease resulted in fatal outcome.

#### **3.2. Pathologic diagnosis**

The pathologic diagnosis was an acute stage of meningoencephalitis as an underlying disease with complications of edema, swelling and dislocation of brain as well as bilateral hypostatic pneumonia and parenchymatous degeneration of myocardium, liver and kidney. The post‐ mortem analysis indicated an acute spongy endema in brain and hemocirculatory disorders in all parts of the central nervous system (CNS) (Fig. 1). A significant vascular disruption of the microvasculature was due to plethora, fibrinoid necrosis of vascular wall and the presence of fibrin in the vascular lumen (1 a, b, c), as well as erythrostasis (1 d), hyaline thrombi (1 f) and a small amount of mononuclear cells in many vascular lumen. Around some lumen a sparse perivascular lymphohistiocytic infiltrate was observed (1 a). The spongy edema was found in all parts of the brain. It was the mostly evident in the cerebellum with fiber dissociation of the medullary substance; also the small diapedetic hemorrhages were found. In the cerebellar cor‐ tex a total loss of Purkinje cells was observed without any evident proliferation of Bergmann glia. Besides, a dramatic depletion and atrophy of granular layer was found. The mild prolifer‐ ating and infiltrating components of inflammation stood out particularly due to severe de‐ structive changes of the inflammatory process structure in the CNS. Along with the vascular disruption a total chromatolysis and necrobiosis of nerve cells were found, as well as the for‐ mation of many neuronophagic nodules like "rabies nodules" mainly in the subcortical brain (Fig. 1g). In the lung a vascular plethora, erythrostasis, red thrombi in vascular lumen, de‐ structive changes of many vascular walls and an evident serohemorrhagic edema of pulmona‐ ry tissue were presented. A polymorphic cellular exudate was observed in the lumen of some large bronchi. In the lung parenchyma a damage of alveolar septa followed by formation of emphysematous areas filled with serofibrinous contents were found. A focal inflammatory polymorphic cellular infiltration was observed. In the spleen the pathohistological changes characterizing a severe immunodeficiency state as delymphatization of white pulp follicles which were not almost visualized were found. At the same time, lymphocyte aggregations were identified only in periarteriolar follicular area (T-dependent zone). A cellular depletion with nonuniform tissue atrophy and denudation of stroma were observed in red pulp. Also the vascular plethora, erythrostasis, fibrinoid swelling, fibrinoid necrosis and hyalinosis of vascular walls, as well as thickening and hyalinosis of septa were found. In the liver the ex‐ tended distrophic and necrobiotic changes of parenchyma coupled with diffuse sparse prolif‐ eration of Kupffer cells and friable polymorphic cellular infiltration along the portal tracts followed by destruction of blood vessel walls were presented. In the kidney a severe necrotic glomerulonephrosis was found. In the cortical substance a necrosis and destruction of the ves‐ sels in malpighian tufts, as well as hemorrhages were observed. In the medullary substance a vascular plethora and hemorrhages were found. Also a total necrobiosis and tubular epitheli‐ um necrosis were observed along the nephrons. The results obtained by histological study in‐ dicated the severe multipleorgan pathology coupled with systemic destructive-dystrophic changes of blood vessels with predominance of edematous and destructive changes in CNS and parenchymatous organs. The pathological process was accompanied by the development of a severe immunodeficiency and a suppression of cell-mediated inflammatory response.

Moreover, the determination of total serum immunoglobulin levels (IgM, IgG, IgA) in the patient's blood (on the arrival at hospital) showed the dis-immunoglobulinemia with IgM level 2.4 times higher than normal (3.15 g/L and 1.30 g/L, respectively), and IgG and IgA lev‐ els 1.5 and 1.8 times, respectively, lower than normal. The number of circulating immune complexes (CIC) of small sizes was 1.7 times higher than normal (123 and 72 arbitrary units, respectively). The results of hematomancy indicated increasing intensity of intoxication,

Despite the intensive therapy (antibacterial, antiviral, neurometabolic, symptomatic, and ar‐ tificial lung ventilation (ALV) via bronchostomy) the patient was getting worse every day due to the increase of general brain symptoms coupled with multiple organ failure, bilateral hypostatic pneumonia, arterial blood pressure (ABP) fall up to 60/40 mm Hg, and apparent tachycardia of 140 – 160 beats per minute. On the eleventh day, September 21, 2007, the dis‐

The pathologic diagnosis was an acute stage of meningoencephalitis as an underlying disease with complications of edema, swelling and dislocation of brain as well as bilateral hypostatic pneumonia and parenchymatous degeneration of myocardium, liver and kidney. The post‐ mortem analysis indicated an acute spongy endema in brain and hemocirculatory disorders in all parts of the central nervous system (CNS) (Fig. 1). A significant vascular disruption of the microvasculature was due to plethora, fibrinoid necrosis of vascular wall and the presence of fibrin in the vascular lumen (1 a, b, c), as well as erythrostasis (1 d), hyaline thrombi (1 f) and a small amount of mononuclear cells in many vascular lumen. Around some lumen a sparse perivascular lymphohistiocytic infiltrate was observed (1 a). The spongy edema was found in all parts of the brain. It was the mostly evident in the cerebellum with fiber dissociation of the medullary substance; also the small diapedetic hemorrhages were found. In the cerebellar cor‐ tex a total loss of Purkinje cells was observed without any evident proliferation of Bergmann glia. Besides, a dramatic depletion and atrophy of granular layer was found. The mild prolifer‐ ating and infiltrating components of inflammation stood out particularly due to severe de‐ structive changes of the inflammatory process structure in the CNS. Along with the vascular disruption a total chromatolysis and necrobiosis of nerve cells were found, as well as the for‐ mation of many neuronophagic nodules like "rabies nodules" mainly in the subcortical brain (Fig. 1g). In the lung a vascular plethora, erythrostasis, red thrombi in vascular lumen, de‐ structive changes of many vascular walls and an evident serohemorrhagic edema of pulmona‐ ry tissue were presented. A polymorphic cellular exudate was observed in the lumen of some large bronchi. In the lung parenchyma a damage of alveolar septa followed by formation of emphysematous areas filled with serofibrinous contents were found. A focal inflammatory polymorphic cellular infiltration was observed. In the spleen the pathohistological changes characterizing a severe immunodeficiency state as delymphatization of white pulp follicles which were not almost visualized were found. At the same time, lymphocyte aggregations were identified only in periarteriolar follicular area (T-dependent zone). A cellular depletion with nonuniform tissue atrophy and denudation of stroma were observed in red pulp. Also the vascular plethora, erythrostasis, fibrinoid swelling, fibrinoid necrosis and hyalinosis of

overall inflammatory reaction, and multiple organ failure.

ease resulted in fatal outcome.

**3.2. Pathologic diagnosis**

236 Encephalitis

**Figure 1. The pathomorphological changes in brain of patient Zh. deceased because of Lyssavirus infection.** A – a sparse perivascular found round some lumen; B, C – lymphohistiocytic infiltrate significant vascular disruption of the microvasculature with the fibrinoid necrosis of vascular wall and the presence of fibrin in the vascular lumen; D – er‐ ythrostasis; E – hyaline thrombi and a small amount of mononuclear cells in many vascular lumen; F – neuronophagic nodules like "rabies nodules" in the subcortical brain. Gemotoxiline-eozine; X 200.

#### **3.3. The laboratory diagnostics**

The brain samples taken postmortem were used to prepare 10% suspension for the infection of two-day-old noninbred white mice. All mice have fall ill on the seventh day; and during the reisolation the symptoms appeared on the sixth day (physical inactivity and respiratory impairment followed by death) (Fig. 2). With the first passage the incubation period short‐ ened to 5 – 6 days.

virus was used for antigen detection in IIFT. Using the slides with Ozernoe strain antigen the antibodies with titer 1:160 were found in this blood serum that could indicate the close antigen relationship between studied strain and vaccine strain of rabies virus. Based on the obtained data we considered that this fatal case could be prevented with timely course of

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239

The electron microscopy study revealed that the cytoplasm of infected PEK cells contained multivesicular and lamellar bodies. The granular electron-dense structures with fibrillar in‐ clusions were observed around of the cellular nucleus or very close to the plasma membrane during all periods of infection. The number of these structures increased with time and a larger number of them were found at 72 h post-infection (Figs 3b). The granular electrondense structures were localized very close to the plasma membrane or fused to it (Figs 3c). Each infected cell contained from 2 to 8 cytoplasmic inclusions which seemed to be Negri body-like (viral ribonucleoprotein (RNP)) structures as a strongly electron-dense matrix. The assembled viral particles with 100 nm in diameter and variable lengths (approximately 670 nm) were observed around such structures (Figs 3d). The viral particles were also asso‐

**Figure 3. Electron micrographs of cultured PEG cells.** A: infected cell 48 h post-infection (pi). The viral inclusions (arrow) were detected inside the cytoplasm. Nucleus (Nu). Bar = 440 nm; B: infected cell 48 h pi; viral inclusions (ar‐ row) and strands forming (Sf) in cellular cytoplasm. Bar = 500 nm; C: infected cell 72 h pi; cytoplasmic vesicles and viral particles (arrows) near the plasma membrane. Bar = 250 nm. D: the viral particles near Negri body-like (NB) structures

ciated with vesicles close to the endoplasmic reticulum or Golgi apparatus.

vaccine prophylaxis against rabies virus.

**3.4. Electron Microscopy (EM)**

Bar = 440 nm.

**Figure 2.** The clinical picture of infection for 6-7 days in mice infected with a 10% suspension of the brain of dead patient Zh.

The two-day-old white mice challenged intracelebrally showed high susceptibility to the iso‐ lated virus. Its titer content in the brain of the dead patient was 3.7 lg LD50, and on the first passage it has reached 6.0 lg LD50. At the same time, the susceptibility of the three-fourweek-old white mice was much lower; in the first passage the virus titre has hardly reached 2.5 lgLD50. The virus antigen showed an evident hemagglutinating activity; in the brain of the dead patient it was 32 a.u., and in the brain on the suckling mice it was 64-128 a.u. The isolated virus strain was named Ozernoe, and the conclusion about the virus etiology of dis‐ ease was made.

On the fourth day after the onset of disease the patient blood was tested by ELISA for anti‐ bodies against tick-borne encephalitis virus (TBEV) and borrelia because the anamnesis had the fact of tick bite in the endemic part of Primorskiy region. The IgM class antibodies against both pathogens were not found, but IgG antibodies against TBEV were revealed with titer 1:800 that was estimated as antibodies after earlier anti-TBEV vaccination. The he‐ magglutinating antigen was obtained from the brains of infected mice and used in hemag‐ glutination-inhibition reaction test (HIRT); in this test the homologous antibodies were revealed in patient blood sera with titer 1:20. But belonging of this antigen to the particular virus wasn't known, so PEK cells cultures were infected with Ozernoe strain and then the slides with antigen were prepared for virus identification by indirect immunnofluorescence test (IIFT). To get the evidence of homologous antigen specifity in IIFT the blood sera of pa‐ tient Zh. was used. Both in HIRT and in IIFT the titer were low (the titer 1:40 by specific flu‐ orescence in IIFT). Moreover, the blood serum of person triple-immunized against rabies virus was used for antigen detection in IIFT. Using the slides with Ozernoe strain antigen the antibodies with titer 1:160 were found in this blood serum that could indicate the close antigen relationship between studied strain and vaccine strain of rabies virus. Based on the obtained data we considered that this fatal case could be prevented with timely course of vaccine prophylaxis against rabies virus.

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

**3.3. The laboratory diagnostics**

ened to 5 – 6 days.

238 Encephalitis

patient Zh.

ease was made.

The brain samples taken postmortem were used to prepare 10% suspension for the infection of two-day-old noninbred white mice. All mice have fall ill on the seventh day; and during the reisolation the symptoms appeared on the sixth day (physical inactivity and respiratory impairment followed by death) (Fig. 2). With the first passage the incubation period short‐

**Figure 2.** The clinical picture of infection for 6-7 days in mice infected with a 10% suspension of the brain of dead

The two-day-old white mice challenged intracelebrally showed high susceptibility to the iso‐ lated virus. Its titer content in the brain of the dead patient was 3.7 lg LD50, and on the first passage it has reached 6.0 lg LD50. At the same time, the susceptibility of the three-fourweek-old white mice was much lower; in the first passage the virus titre has hardly reached 2.5 lgLD50. The virus antigen showed an evident hemagglutinating activity; in the brain of the dead patient it was 32 a.u., and in the brain on the suckling mice it was 64-128 a.u. The isolated virus strain was named Ozernoe, and the conclusion about the virus etiology of dis‐

On the fourth day after the onset of disease the patient blood was tested by ELISA for anti‐ bodies against tick-borne encephalitis virus (TBEV) and borrelia because the anamnesis had the fact of tick bite in the endemic part of Primorskiy region. The IgM class antibodies against both pathogens were not found, but IgG antibodies against TBEV were revealed with titer 1:800 that was estimated as antibodies after earlier anti-TBEV vaccination. The he‐ magglutinating antigen was obtained from the brains of infected mice and used in hemag‐ glutination-inhibition reaction test (HIRT); in this test the homologous antibodies were revealed in patient blood sera with titer 1:20. But belonging of this antigen to the particular virus wasn't known, so PEK cells cultures were infected with Ozernoe strain and then the slides with antigen were prepared for virus identification by indirect immunnofluorescence test (IIFT). To get the evidence of homologous antigen specifity in IIFT the blood sera of pa‐ tient Zh. was used. Both in HIRT and in IIFT the titer were low (the titer 1:40 by specific flu‐ orescence in IIFT). Moreover, the blood serum of person triple-immunized against rabies

The electron microscopy study revealed that the cytoplasm of infected PEK cells contained multivesicular and lamellar bodies. The granular electron-dense structures with fibrillar in‐ clusions were observed around of the cellular nucleus or very close to the plasma membrane during all periods of infection. The number of these structures increased with time and a larger number of them were found at 72 h post-infection (Figs 3b). The granular electrondense structures were localized very close to the plasma membrane or fused to it (Figs 3c). Each infected cell contained from 2 to 8 cytoplasmic inclusions which seemed to be Negri body-like (viral ribonucleoprotein (RNP)) structures as a strongly electron-dense matrix. The assembled viral particles with 100 nm in diameter and variable lengths (approximately 670 nm) were observed around such structures (Figs 3d). The viral particles were also asso‐ ciated with vesicles close to the endoplasmic reticulum or Golgi apparatus.

**Figure 3. Electron micrographs of cultured PEG cells.** A: infected cell 48 h post-infection (pi). The viral inclusions (arrow) were detected inside the cytoplasm. Nucleus (Nu). Bar = 440 nm; B: infected cell 48 h pi; viral inclusions (ar‐ row) and strands forming (Sf) in cellular cytoplasm. Bar = 500 nm; C: infected cell 72 h pi; cytoplasmic vesicles and viral particles (arrows) near the plasma membrane. Bar = 250 nm. D: the viral particles near Negri body-like (NB) structures Bar = 440 nm.

#### **3.5. Genetic identification**

Firstly, to identify exactly the virus genotype the fragment of N gene was amplificated and sequenced. The bat virus primers including the primers for six genotypes of rabies and rabies-related viruses [Heaton, 1997] were synthesized to identify a virus genome. The amplicon of the expected length was obtained by PCR with the primers 5'-ATG‐ TAACACCCCTACAATGG-3' and 5'-CAATTTGCACACATTTTGTG-3', and then the nu‐ cleotide sequence of amplicon was determined by sequencing. The obtained nucleotide sequence was found to have 95% homology with Irkut strain of bat lyssavirus isolated before from a bat in Eastern Siberia. Eearlier Irkut virus strain had been isolated from a dead Greater Tubenosed Bat *(Murina leucogaster)* in Irkutsk [Botvinkin et al., 2003]. The homology level of this nucleotide sequence with European bat lyssavirus 1 (EBLV-1) is substantially less (from 77 to 76% of identity) and even less with EBLV-2, Duvenhage vi‐ rus and Rabies virus. The complete sequence of N gene of Ozernoe strain determined in our study (GeneBank No FJ905105) has the 93% homology level with Irkut strain, 79% with EBLV-1 strain, 75% with Duvenhage virus, 77% with EBLV-2 and Khujand viruses, 76% with Rabies, Avaran and ABLV viruses, 75% with Lagos, 73% with West Caucasian and 72% with Mokola viruses (Tabl. 2). Since the Ozernoe strain is the first strain of the genotype 8 isolated from a dead human, we have identified its complete genome se‐ quence. For this purpose, the primers for amplification of complete genome fragments were designed by comparison of complete genomes of the Irkut strain and strains of EBLV-1 of the lyssavirus subtype. Then the complete genome sequence of Ozernoe strain was obtained after sequencing and alignment of overlapping fragments (GenBank accession FJ905105). The lengths of each complete viral genome sequences were 11980 bases for Irkut and Ozernoe strains and corresponded to the standard rhabdovirus ge‐ nome organization. Lyssavirus genome consists of negative-sense, single-stranded RNA that encodes five viral proteins: nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G and polymerase L. Comparison with other lyssavirus sequences demon‐ strates variation in levels of homology: the nucleoprotein was the most conserved, and the phosphoprotein - the most variable genes (Table 2).

Accession, bat lyssavirus

EF157976.1, EBLV 1 isolate RV9

EF157977.1, EBLV 2 isolate RV1333

AF418014.1, Australian

JQ685919.1, Rabies isolate NJ2262

EU293108.1, Lagos isolate 0406SEN

EU293117, Mokola isolate 86100CAM

cording to GenBank records.

bat lyssavirus

EU293120.1, Duvenhage isolate 94286SA

Maximum identity,%

77% 79 73 80 74 78

75% 77 70 79 72 76

75% 77 69 80 72 75

75% 76 68 76 71 74

75% 75 68 74 71 75

75 75 72 72 74 71

73 74 74 73 69 71

Complete genome

EF614260.1, Irkut 92% 93% 92% 92 91 92

EF614259.1, Avaran 75% 76 69 78 72 75

EF614261.1, Khujand 75% 76 69 78 72 75

GU170201, Shimoni 77 76 83 74 65 72 EF614258.1, West Caucasian bat virus 70 73 74 73 75 72

**Figure 4.** The phylogenetic tree of Lyssaviruses based on complete genome sequences. Virus names are provided ac‐

**Table 2.** Comparison of strain Ozernoe homology with other Lyssaviruses.

Coding regions of genes

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241

N gene P gene M gene G gene L gene

The comparison of complete genomes of Ozernoe and Irkut strains confirmed that they are closely related. The complete genome sequence of Ozernoe strain was 92% identical to the complete genome of Irkut strain, 77-78% to the EBLV-1 genome, and 75% to the EBLV-2 genome. Moreover, the virus proteins sequences are more homologous than the corresponding genes. For example, the N and L genes of Ozernoe strain are 92% identi‐ cal to Irkut strain sequences, as well as 79% and 77%, respectively, to EBLV-1. At the same time, the corresponding nucleoprotein and polymerase sequences have 98% and 92% homology. Phosphoproteins were found to show the most striking difference with 95% and 70% homology, respectively. Phylogenetic analysis of complete-genome nucleo‐ tide sequences of all lyssavirus genotypes showed that Irkut and Ozernoe strains are lo‐ cated on the same branch of the phylogenetic tree, have a common ancestor and form one cluster.


**Table 2.** Comparison of strain Ozernoe homology with other Lyssaviruses.

**3.5. Genetic identification**

240 Encephalitis

the phosphoprotein - the most variable genes (Table 2).

one cluster.

Firstly, to identify exactly the virus genotype the fragment of N gene was amplificated and sequenced. The bat virus primers including the primers for six genotypes of rabies and rabies-related viruses [Heaton, 1997] were synthesized to identify a virus genome. The amplicon of the expected length was obtained by PCR with the primers 5'-ATG‐ TAACACCCCTACAATGG-3' and 5'-CAATTTGCACACATTTTGTG-3', and then the nu‐ cleotide sequence of amplicon was determined by sequencing. The obtained nucleotide sequence was found to have 95% homology with Irkut strain of bat lyssavirus isolated before from a bat in Eastern Siberia. Eearlier Irkut virus strain had been isolated from a dead Greater Tubenosed Bat *(Murina leucogaster)* in Irkutsk [Botvinkin et al., 2003]. The homology level of this nucleotide sequence with European bat lyssavirus 1 (EBLV-1) is substantially less (from 77 to 76% of identity) and even less with EBLV-2, Duvenhage vi‐ rus and Rabies virus. The complete sequence of N gene of Ozernoe strain determined in our study (GeneBank No FJ905105) has the 93% homology level with Irkut strain, 79% with EBLV-1 strain, 75% with Duvenhage virus, 77% with EBLV-2 and Khujand viruses, 76% with Rabies, Avaran and ABLV viruses, 75% with Lagos, 73% with West Caucasian and 72% with Mokola viruses (Tabl. 2). Since the Ozernoe strain is the first strain of the genotype 8 isolated from a dead human, we have identified its complete genome se‐ quence. For this purpose, the primers for amplification of complete genome fragments were designed by comparison of complete genomes of the Irkut strain and strains of EBLV-1 of the lyssavirus subtype. Then the complete genome sequence of Ozernoe strain was obtained after sequencing and alignment of overlapping fragments (GenBank accession FJ905105). The lengths of each complete viral genome sequences were 11980 bases for Irkut and Ozernoe strains and corresponded to the standard rhabdovirus ge‐ nome organization. Lyssavirus genome consists of negative-sense, single-stranded RNA that encodes five viral proteins: nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G and polymerase L. Comparison with other lyssavirus sequences demon‐ strates variation in levels of homology: the nucleoprotein was the most conserved, and

The comparison of complete genomes of Ozernoe and Irkut strains confirmed that they are closely related. The complete genome sequence of Ozernoe strain was 92% identical to the complete genome of Irkut strain, 77-78% to the EBLV-1 genome, and 75% to the EBLV-2 genome. Moreover, the virus proteins sequences are more homologous than the corresponding genes. For example, the N and L genes of Ozernoe strain are 92% identi‐ cal to Irkut strain sequences, as well as 79% and 77%, respectively, to EBLV-1. At the same time, the corresponding nucleoprotein and polymerase sequences have 98% and 92% homology. Phosphoproteins were found to show the most striking difference with 95% and 70% homology, respectively. Phylogenetic analysis of complete-genome nucleo‐ tide sequences of all lyssavirus genotypes showed that Irkut and Ozernoe strains are lo‐ cated on the same branch of the phylogenetic tree, have a common ancestor and form

**Figure 4.** The phylogenetic tree of Lyssaviruses based on complete genome sequences. Virus names are provided ac‐ cording to GenBank records.
