**4. Discussion**

Though human lyssavirus infection cases are rare at Eurasian continent, the minor cases have appeared again and again during the last decades. As a rule, such cases appear unex‐ pectedly, diagnose with delay and end in fatal outcome. For example, the lyssavirus infec‐ tion case with fatal outcome was described in Scotland in 2003 for the first time in 100 years [Nathwani et al., 2003]. Also, 2 cases of rabies have been confirmed in the United Kingdom in Daubenton's bats (*Myotis daubentonii*): the first in 1996 in Newhaven and the second in September, 2002 in Lancashire; both cases were caused by European bat Lyssavirus (EBLV) type 2a [Whitby et al., 2000; Johnson et al., 2002]. In Europe 3 human deaths from rabies caused by EBLV have been reported [Lumio et al., 1986; Roine et al., 1988; Rabies Bulletin Europe, 1987]. One can say that at present epizootic foci of Lyssaviruses are active and un‐ der certain conditions on epidemeological situations the lyssavirus infection cases could take place.

wound as well as a specific vaccination regardless the time of antirabic treatment seeking according to the recent criteria of World Health Organization (WHO) Expert Consulation on Rabies. Despite the wound on the underlip made by bat the patient was not vaccinated against rabies as a preventive measure. Representatives of the health care service in the Pri‐ morye Territory did not expect that an accidental contact of a human with bat would cause a

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The evidence of crossimmunity between classical rabies virus of genotype 1 and Lyssavirus‐ es of genotypes 5 and 6 (EBLV types 1 and 2) was obtained; however, definitive proof of cross-protection is lacking [Badrane et al., 2001]. In spite of this fact, we are sure that in cases of possible infection the emergency vaccinal prevention against the rabies should be carried out. About this suggestion the IIFT results of blood sera of patient triply vaccinated against rabies could indicate because they demonstrated the high levels of sera specificity to Ozer‐ noe strain antigen. Moreover, it is known that human diploid cells vaccine protects against classic rabies virus strains and Duvenhage virus, but not Mokola virus and Lagos bat virus; Duvenhage virus is neutralized by RIG, but Mokola and Lagos bat viruses are not [Fekadu et al., 1988; Hanlon et al., 2001]. So, based on the cases described above the vaccine against classic rabies could be used for disease prevention caused not only by rabies virus (genotype 1) but by EBLV (types 1 and 2) and Duvenhage virus. Apparently, it could be attributed to fact that Lyssavirus species segregate into two phylogroups. Phylogroup 1 includes Rabies virus (RABV), Duvenhage virus (DUVV), European bat lyssaviruses, type 1 and 2 (EBLV-1 and 2, respectively), and Australian bat lyssavirus (ABLV). Also, Aravan virus (ARAV), Khujand virus (KHUV) and Irkut virus (IRKV) cross-react serologically with the members of phylogroup 1. Phylogroup 2 includes Lagos bat virus (LBV), Mokola virus (MOKV) and Shi‐ moni bat virus (SHIBV). West Caucasian bat virus (WCBV) does not cross-react serologically with any of the two phylogroups (http://www.who-rabies-bulletin.org/about\_rabies/classifi‐ cation.aspx). Certainly, the exact experimental proofs of cross-reacting protection between different Lyssavirus genetic variants should be known to general practitioners to make rap‐ id and correct decision about vaccinal prevention of rabies infection of different etiology. Es‐ pecially, it is very important in cases with peripheral localization of injury place when a lot of time is available for virus elimination with specific antibodies after patient's vaccination.

Unfortunately, the bat species was not identified, because the killed bat was tortured by a dog. The residents of Ozernoe said that dog had died one month later. The dog, as the bat, was not examined. Other cases indicating a spread of the virus were not registered. Howev‐ er, the doctor working in the neighbouring village found a bat at the medical station. The cases when bats enter into houses are not rare and usually not hazardous for human health. At the same time, during the investigation of houses and nonresidential premises in Ozer‐ noe we did not found any permanent inhabitations of bats. We have suggested that it is pos‐

There are 15 known species of chiropteran in the Far East. The most of them are referred to nonmigratory species; they do not fly far wintering in caves near summer habitats forming large assemblages. Some bat species from southern regions of Primorye Territory spend

sible for bats from nearby forests to enter periodically into the houses.

winters in the balks [Tiunov, 1997].

typical rabies clinical picture.

In our study we have shown the first fatal case of lyssavirus infection in Siberia and Russian Far East. The data of epidemiological anamnesis (the patient's underlip was wounded by bat), clinical picture of infection (hypersalivation, chocking, baryphonia, acute meningoence‐ phalitis) as well as the virlogical, morphological and molecular genetic data have given the evidence that this case belonged to lyssavirus infection. The brief description of this case was presented earlier in the Journal "Rabies Bulletin Europe" [Leonova et al., 2009].

We have paid attention to the short incubation period (4 weeks), rapid infection develop‐ ment and fast (in 10 days) fatal outcome. First of all, such infection development could be assosiated with injury localization (head). Thus, the infection fatal case described in Scotland and caused by EBLV-2 was attributed with the bite of patient's finger. The incubation period lasted 19 weeks and fatal outcome came in 14 days after the beginning of disease [Nathwani et al., 2003]. Another fatal case of human rabies caused by Duvenhage bat lyssavirus in Ken‐ ya has also indicated about the significance of injury localization [van Thiel et al., 2009]. Af‐ ter the contact with bat the patient has pointed to the small double parallel bleeding wounds at right part of the nose. Though the wounds were disinfected (rinsed with water and soap and treated with alcohol), the patient has got ill in short incubation period (23 days) and died at 20th day of the disease in spite of intensive therapy.

The mother of patient Zh. said that the underlip of her daughter had two typical parallel scratches that were made by bat with the anterior teeth. It means that the saliva of infected animal was in the wound. It should be noted that the appearance of the drops of blood indi‐ cated blood vessel damage in dermal layer. But the patients could be mistaken during the contact with bat and told about scratching with leg claws which couldn't form double strip (the bat leg has only one claw) in contrast to skin damage by teeth. The character of skin damage by anterior teeth in a form of double strip should always be the fact of possible vi‐ rus injection with bat saliva into human blood and requires the drastic measures for the ra‐ bies infection prevention.

Therefore, such head injury followed by the vessel damage is considered to have the third severity level which requires the introduction of antirabic immunoglobulin around the wound as well as a specific vaccination regardless the time of antirabic treatment seeking according to the recent criteria of World Health Organization (WHO) Expert Consulation on Rabies. Despite the wound on the underlip made by bat the patient was not vaccinated against rabies as a preventive measure. Representatives of the health care service in the Pri‐ morye Territory did not expect that an accidental contact of a human with bat would cause a typical rabies clinical picture.

**4. Discussion**

242 Encephalitis

take place.

Though human lyssavirus infection cases are rare at Eurasian continent, the minor cases have appeared again and again during the last decades. As a rule, such cases appear unex‐ pectedly, diagnose with delay and end in fatal outcome. For example, the lyssavirus infec‐ tion case with fatal outcome was described in Scotland in 2003 for the first time in 100 years [Nathwani et al., 2003]. Also, 2 cases of rabies have been confirmed in the United Kingdom in Daubenton's bats (*Myotis daubentonii*): the first in 1996 in Newhaven and the second in September, 2002 in Lancashire; both cases were caused by European bat Lyssavirus (EBLV) type 2a [Whitby et al., 2000; Johnson et al., 2002]. In Europe 3 human deaths from rabies caused by EBLV have been reported [Lumio et al., 1986; Roine et al., 1988; Rabies Bulletin Europe, 1987]. One can say that at present epizootic foci of Lyssaviruses are active and un‐ der certain conditions on epidemeological situations the lyssavirus infection cases could

In our study we have shown the first fatal case of lyssavirus infection in Siberia and Russian Far East. The data of epidemiological anamnesis (the patient's underlip was wounded by bat), clinical picture of infection (hypersalivation, chocking, baryphonia, acute meningoence‐ phalitis) as well as the virlogical, morphological and molecular genetic data have given the evidence that this case belonged to lyssavirus infection. The brief description of this case

We have paid attention to the short incubation period (4 weeks), rapid infection develop‐ ment and fast (in 10 days) fatal outcome. First of all, such infection development could be assosiated with injury localization (head). Thus, the infection fatal case described in Scotland and caused by EBLV-2 was attributed with the bite of patient's finger. The incubation period lasted 19 weeks and fatal outcome came in 14 days after the beginning of disease [Nathwani et al., 2003]. Another fatal case of human rabies caused by Duvenhage bat lyssavirus in Ken‐ ya has also indicated about the significance of injury localization [van Thiel et al., 2009]. Af‐ ter the contact with bat the patient has pointed to the small double parallel bleeding wounds at right part of the nose. Though the wounds were disinfected (rinsed with water and soap and treated with alcohol), the patient has got ill in short incubation period (23 days) and

The mother of patient Zh. said that the underlip of her daughter had two typical parallel scratches that were made by bat with the anterior teeth. It means that the saliva of infected animal was in the wound. It should be noted that the appearance of the drops of blood indi‐ cated blood vessel damage in dermal layer. But the patients could be mistaken during the contact with bat and told about scratching with leg claws which couldn't form double strip (the bat leg has only one claw) in contrast to skin damage by teeth. The character of skin damage by anterior teeth in a form of double strip should always be the fact of possible vi‐ rus injection with bat saliva into human blood and requires the drastic measures for the ra‐

Therefore, such head injury followed by the vessel damage is considered to have the third severity level which requires the introduction of antirabic immunoglobulin around the

was presented earlier in the Journal "Rabies Bulletin Europe" [Leonova et al., 2009].

died at 20th day of the disease in spite of intensive therapy.

bies infection prevention.

The evidence of crossimmunity between classical rabies virus of genotype 1 and Lyssavirus‐ es of genotypes 5 and 6 (EBLV types 1 and 2) was obtained; however, definitive proof of cross-protection is lacking [Badrane et al., 2001]. In spite of this fact, we are sure that in cases of possible infection the emergency vaccinal prevention against the rabies should be carried out. About this suggestion the IIFT results of blood sera of patient triply vaccinated against rabies could indicate because they demonstrated the high levels of sera specificity to Ozer‐ noe strain antigen. Moreover, it is known that human diploid cells vaccine protects against classic rabies virus strains and Duvenhage virus, but not Mokola virus and Lagos bat virus; Duvenhage virus is neutralized by RIG, but Mokola and Lagos bat viruses are not [Fekadu et al., 1988; Hanlon et al., 2001]. So, based on the cases described above the vaccine against classic rabies could be used for disease prevention caused not only by rabies virus (genotype 1) but by EBLV (types 1 and 2) and Duvenhage virus. Apparently, it could be attributed to fact that Lyssavirus species segregate into two phylogroups. Phylogroup 1 includes Rabies virus (RABV), Duvenhage virus (DUVV), European bat lyssaviruses, type 1 and 2 (EBLV-1 and 2, respectively), and Australian bat lyssavirus (ABLV). Also, Aravan virus (ARAV), Khujand virus (KHUV) and Irkut virus (IRKV) cross-react serologically with the members of phylogroup 1. Phylogroup 2 includes Lagos bat virus (LBV), Mokola virus (MOKV) and Shi‐ moni bat virus (SHIBV). West Caucasian bat virus (WCBV) does not cross-react serologically with any of the two phylogroups (http://www.who-rabies-bulletin.org/about\_rabies/classifi‐ cation.aspx). Certainly, the exact experimental proofs of cross-reacting protection between different Lyssavirus genetic variants should be known to general practitioners to make rap‐ id and correct decision about vaccinal prevention of rabies infection of different etiology. Es‐ pecially, it is very important in cases with peripheral localization of injury place when a lot of time is available for virus elimination with specific antibodies after patient's vaccination.

Unfortunately, the bat species was not identified, because the killed bat was tortured by a dog. The residents of Ozernoe said that dog had died one month later. The dog, as the bat, was not examined. Other cases indicating a spread of the virus were not registered. Howev‐ er, the doctor working in the neighbouring village found a bat at the medical station. The cases when bats enter into houses are not rare and usually not hazardous for human health. At the same time, during the investigation of houses and nonresidential premises in Ozer‐ noe we did not found any permanent inhabitations of bats. We have suggested that it is pos‐ sible for bats from nearby forests to enter periodically into the houses.

There are 15 known species of chiropteran in the Far East. The most of them are referred to nonmigratory species; they do not fly far wintering in caves near summer habitats forming large assemblages. Some bat species from southern regions of Primorye Territory spend winters in the balks [Tiunov, 1997].

In Eastern Siberia lyssavirus isolated for the first time from a bat (*Murina leucogaster*) was reported in September, 2002. The bat, which looked healthy, entered into the house in Ir‐ kutsk. It was caught and observed. On the tenth day of its observation the symptoms of dis‐ ease appeared which has resulted in fatal outcome. The virus called Irkut [Botvinkin et al., 2003] was isolated from the brain of the dead animal. During the strain isolation the infected suckling mice had 18 days of the incubation period, and among these mice only one fell ill. At the second passage the incubation period varied from 9 to 18 days. Moreover, it should be noted that the bat infected with Irkut strain wounded the girl, but the illness did not de‐ velop due to the timely vaccination against rabies.

nome sequence. The primers for amplification of genome fragments were designed by com‐ parison of Irkut strain complete genome and EBLV-1 strains of the lyssavirus subtype. The comparison of complete genomes sequences of Ozernoe and Irkut strains confirmed that they are closely related. The virus proteins are more homologous than the corresponding genes. For example, N and L genes of Ozernoe strain are 92% identical to Irkut strains, as well as 79% and 77% to EBLV-1, respectively. At the same time, the corresponding nucleo‐ protein and polymerase have 98% and 92% homology, and the phosphoproteins show the

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Irkut and Ozernoe strains are located on the same branch of the phylogenetic tree and form one cluster. The divergence time was estimated to be about 650 years that was calculated upon the rate of evolution within the range from 1.5 x10-4 to 4.3 х 10-4 dS/site/year [Badrane, Tordo, 2001; Hughes, 2008]. It is interesting to note that Irkut and Ozernoe strains are so closely related despite of their divergence time and distance between their isolation places

So we have demonstrated a real possibility of hazardous lyssavirus infection occurrence in cases when humans accidentally meet chiropteran. This is the first confirmed fatal case of

The clinical and epidemiological characteristics of fatal human case of lyssavirus infection identified fort he first time in the Asian part of Russia were provided. The evidence that this case belongs to Lyssavirus infection in the terms of etiology was obtained based on the data of epidemiological anamnesis, clinical picture of infection as well as the virological, morpho‐ logical, and molecular genetic studies. The pathologic diagnosis was the acute stage of me‐ ningoencephalitis as underlying disease with complications of edema, swelling and dislocation of brain, the formation of neuronophagic nodules like "rabies nodules" mainly in the subcortical brain as well as bilateral hypostatic pneumonia and parenchymatous degen‐ eration of myocardium, liver and kidney. The study by electron microscopy revealed that the viral particles of 100 nm in diameter could also be associated with the vesicles close to the endoplasmic reticulum or Golgi apparatus. The complete genome sequence of Ozernoe strain is 92% identical to the complete genome of Irkut strain and 77-78% to EBLV-1 ge‐ nome. The phylogenetic analysis based on the complete genome sequences revealed that Ozernoe strain, isolated from the brain of the dead patient, and Irkut strain, isolated in Ir‐ kutsk from the brain of a bat, are located on the same branch of the phylogenetic tree and have the common ancestor. So the real possibility of serious Lyssavirus infection in cases

when people have accidentally encountered with chiropteran was demonstrated.

This case has ended in fatal outcome because of the fact that doctors have never registered patients with a lyssavirus infection in the Far East before. Therefore, it was difficult to make the right decision for emergency vaccination against rabies after the accidence as it is usually

most striking difference with 95% and 70% homology, respectively.

lyssavirus infection on the territory of the Asian continent.

(about 3,000 km).

**5. Conclusion**

Both cases in Irkutsk and Primorye Territory confirm that usually sick animals enter into hu‐ man dwellings. First of all such animals are of a special danger and require to take measures which protect people from the possible accidental contacts with them. Other cases of lyssa‐ virus infection in Europe reported by Botvinkin et al. (2003) look very similar to these ones.

Unfortunately, the final diagnosis in studied case of infection was stated postmortem. The isolation of Ozernoe strain revealed the following facts to pay attention to: a short incuba‐ tion period up to 6-7 days; the examined suckling mice of a mouse family fell ill at the same time. If the suckling mice were challenged intracerebrally, the virus titre in the brain of the dead patient was evaluated as 3.7 lg LD50. In the first passage the titre of Ozernoe strain in the mice challenged intracerebrally reached 6 lg LD50. Hаemagglutinins were found not only in brain of the infected mice, but also in the brain of the dead patient. These data indicate a high virus concentration which caused profound brain damages and the overall visceral in‐ jury followed by impairment of vital functions and resulted in the imminent fatal outcome. The postmortem analysis indicated the changes characteristic for rabies: extensive destruc‐ tive changes of neurons, formation of many neuronophagic nodules like "rabies nodules", an evident spongy endema of the medullary substance. At the same time, a clearly defined in‐ flammatory reaction around blood vessels was not found. This fact together with the found changes in lymphoid organs indicated the immunodeficiency state, which was undoubtedly associated with the virus infection.

The presence of extracellular viral particles with morphology similar to the structures of Rabies virus on cell surface membranes and spreads forming the electron-dense sub‐ stance in cellular cytoplasm were revealed during the ultrastructural study. Extracellular viral particles in the environment were distinctly observed and had the morphological signs typical for the group of enveloped viruses with spikes on the surfaces [Iseni, 1998]. The oval and dense bodies (with diameter about 2550 nm) were determinated in cyto‐ plasm and mainly in extracellular environment of PEG cells culture infected by strain Ozernoe. These structures were described earlier as Negri bodies in neurons infected by Rabies virus [Velandia et al., 2007].

To identify exactly the virus genotype the fragment of N gene was amplificated and se‐ quenced. Moreover, the search for homologous sequences in GeneBank using BLAST pro‐ gram has given the absolute evidence that the closest relative of Ozernoe strain is Irkut strain isolated earlier from the bat in Eastern Siberia. Since the Ozernoe strain is the first strain of the genotype 8 isolated from a dead human, we have determined its complete ge‐ nome sequence. The primers for amplification of genome fragments were designed by com‐ parison of Irkut strain complete genome and EBLV-1 strains of the lyssavirus subtype. The comparison of complete genomes sequences of Ozernoe and Irkut strains confirmed that they are closely related. The virus proteins are more homologous than the corresponding genes. For example, N and L genes of Ozernoe strain are 92% identical to Irkut strains, as well as 79% and 77% to EBLV-1, respectively. At the same time, the corresponding nucleo‐ protein and polymerase have 98% and 92% homology, and the phosphoproteins show the most striking difference with 95% and 70% homology, respectively.

Irkut and Ozernoe strains are located on the same branch of the phylogenetic tree and form one cluster. The divergence time was estimated to be about 650 years that was calculated upon the rate of evolution within the range from 1.5 x10-4 to 4.3 х 10-4 dS/site/year [Badrane, Tordo, 2001; Hughes, 2008]. It is interesting to note that Irkut and Ozernoe strains are so closely related despite of their divergence time and distance between their isolation places (about 3,000 km).

So we have demonstrated a real possibility of hazardous lyssavirus infection occurrence in cases when humans accidentally meet chiropteran. This is the first confirmed fatal case of lyssavirus infection on the territory of the Asian continent.
