**3. Results and discussion**

For the first time the uniqueness of 886-84 strain was found during the investigation of its serological properties. Trukhina suggested that this strain takes the intermediate place be‐ tween two TBEV serotypes – East-Siberian and Far-Eastern and shows the properties of both serotypes [21].

Then, 886-84 strain was described as a representative of the independent genotype accord‐ ing to criteria developed by our team after the comparing of difference level of 29 strains isolated on different territories of TBEV area [8]. In this study the fragment of E protein gene (positions 567-727 br) was used as a model. It was found that corresponding amino acid se‐ quence of this fragment in 886-84 strain has Leu in position 206 as genotype 3 and Asp in position 234 as genotypes 1 and 2 [10]. At that time we did not find any homologous strains and isolates so the additional data were necessary to separate this TBEV strain into inde‐ pendent genotype.

(mNsc) mice inoculation expressed as lg LD50/ml [18]. Nonlinear mice (6-8 g in weight) were infected into brain with 0.03 ml or subcutaneously with 0.25 ml of inoculate. Animals infect‐ ed intracerebrally were observed during 14 days while animals infected subcutaneously were observed during 21 days. Virus titers were detected by Reed and Muench method. The values of II 1-2.5 meant the high invasive activity of the virus, i.e. the ability of virus to over‐ come the blood-brain barrier to reach central nervous system (CNS) and propagate in it. The values of invasiveness index of equal or more than 3 indicated the lesser invasive activity of

Thermoresistance (Т50) of TBEV strains was tested by Ovchinnikova *et al.* method [17] us‐ ing 24-hour cell culture grown in 96-hole plates at the presence of СО2. The thermoresist‐ ance was determined by inactivation index – difference in lg of titers of virus samples heated at 50°С during 15 minutes or unheated (4°С). In case of titers difference equal or less than 2.0 lg the strain was characterized as Т50+, from 2.1 to 3.0 lg – as medium, equal

Rct42-feature describes the ability of the virus to propagate at supraoptimal temperature. To determine rct42 the 24-hour cell culture grown in 96-hole plates was infected by different vi‐ rus-containing suspensions (10-1 to 10-10). One part of the cell cultures was infected with se‐ lected virus strain and incubated at 37°С, and other cells were infected with the same strain and incubated at 42°С at the presence of СО2. Rct42 was determined on the sixth day after infection as a difference between lg of virus titers after the cultivation in cells at 37˚С and 42˚С. In case of titers difference equal or less than 2,0 lg the strain was characterized as rct<sup>42</sup> -

The cell culture was infected with TBEV strains undergone not more than 4 passages through the white mice brains and 3 cycles of cloning. The plaques appeared on the third or fourth day. The plaque size measuring was performed on the fifth day when they increased and become more sharp and transparent. S-feature was determined as S+ if plaque had the

For the first time the uniqueness of 886-84 strain was found during the investigation of its serological properties. Trukhina suggested that this strain takes the intermediate place be‐ tween two TBEV serotypes – East-Siberian and Far-Eastern and shows the properties of both

.

from 2.1 to 3.0 lg – as medium, equal or more than 3.1 lg – as rct42 <sup>+</sup>

diameter (d)≥2.5 mm; S± at 2.5>d ≥2,0 mm; S– at 2.0>d ≥1,0 mm.

,

the virus strain.

98 Encephalitis

**2.6. Rct42–feature**

**2.7. S-feature**

serotypes [21].

**2.5. Thermoresistance**

or more than 3.1 lg – as Т50-.

**3. Results and discussion**

Comparison of the strain 886-84 complete genome sequence (EF469662) with TBEV sequen‐ ces available in GenBank has shown that it forms an independent branch and does not clus‐ ter with any strains of three main genotypes (Fig.1). It should be noted that nucleotide substitution level was close to the species separation border [11] (Table 2).

**Figure 1.** Phylogenetic tree demonstrating the genetic similarity level of 54 TBEV strains on the base of polyprotein coding region sequences (10242 nr). Genotype 1 cluster - Sofjin [25], AB022703, AB001026, DQ989336, AY182009, AY217093, JF316707, JF316708, FJ997899, EU816450-EU816455, AY169390, FJ906622, GQ228395, FJ402885, FJ402886, DQ862460, GU121642, HQ201303, HQ901367, HQ901366, HM859894, HM859895, JN003205; Genotype 2 cluster - TEU27495, TEU27491, TEU39292, AF091010, EU106868, DQ401140, GV266392, HM535610, HM535611, HM120875, GU183379-GU183381, GU183383; Genotype 3 cluster - L40361, AF527415, DQ486861, FJ968751, JN003206-JN003209, GU183382, GU183384. OHF – Omsk haemorrhagic fever virus.


Thirty unique substitutions were detected in strain 886-84 polyprotein which could proba‐ bly be the "genotype-specific" for "group 886" members. However, since studied polypro‐ tein fragment for "group 886" strains was 1066 ar in length the "genotype-specific"

**Protein С M E NS1**

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Position in polyprotein 98 108 270 688 735 898

Position in protein 158 408 455 122

genotype 1 A V/L/I V K L S

genotype 2 A I V K L S

genotype 3 T T V K L/M S

178-79 A V V K L S

"group 886" V A A R I A

**Comments:** genotype 1 is presented by translated nucleotide sequences X07755, AB022703, AB001026, DQ989336, AY182009, AY217093, JF316707, JF316708, FJ997899, EU816450-EU816455, AY169390, FJ906622, GQ228395, FJ402885, FJ402886, DQ862460, GU121642, HQ201303, HQ901367, HQ901366, HM859894, HM859895, JN003205; genotype 2 - TEU27495, TEU27491, TEU39292, AF091010, EU106868, DQ401140, GV266392, HM535610, HM535611, HM120875, GU183379- GU183381, GU183383; genotype 3 - L40361, AF527415, DQ486861, FJ968751, JN003206-JN003209, GU183382, GU183384. "Group 886" is presented by prototype strain sequence EF469662 (strain 886-84) and GenBank deposited (EU878281-EU878283) and non-deposited 617-90, 711-84 и 740-84 TBEV strains ge‐

**Table 3.** Unique substitutions in the polyprotein fragments (1066 ar) (proteins С, М, Е and part of NS1) of TBEV "group

At present, using MHNA and sequencing methods we have found the group of 13 TBEV isolates with highly homologous genetic structure to the strain 886-84. For eight strains the genome fragments (1650 nr in length) coding proteins C, M, and E protein fragment were determined (GenBank accession numbers EF469662, EU878281-EU878283, JN936341, JN936347, JN936349-JN936350, JN936353-JN936355) (Fig.4). The homology level with the strain 886-84 genome sequence was 98.2-99.8% while the difference level with three main

genotypes ranged from 13.1% (Sofjin) to 16.6% (Neudoerfl).

nome fragments.

886" strains.

uniqueness was confirmed only for 6 substitutions of 30 (Table. 3)

**Comments:** The level of nucleotide and amino acid substitutions within the genotype is marked with grey.

**Table 2.** Nucleotide and amino acid substitution level between different TBEV genotypes and strains 178-79 and 886-84.

The analysis of complete amino acid sequence of strain 886-84 polyprotein confirmed that it's the unique "mixture" of sequences common for genotypes 1, 2 and 3. For example, in the set of 22 positions which clearly differentiate all known TBEV strains into three genotypes the unique amino acids (alanine (A) in position C-108, serine (S) – NS2A-127 and glycine (G) – NS3-258) or interchange with amino acids typical for main TBEV genotypes were found in strain 886-84 polyprotein sequence [7] (Fig. 2).


**Figure 2.** The differences in 22 positions obtained by comparing of 54 TBEV strains polyprotein sequences; **Com‐ ments.** The cells marked with grey identify the amino acid residues corresponding to one of four TBEV genotypes. The unique amino acid for strain 886-84 is marked with black.

Thirty unique substitutions were detected in strain 886-84 polyprotein which could proba‐ bly be the "genotype-specific" for "group 886" members. However, since studied polypro‐ tein fragment for "group 886" strains was 1066 ar in length the "genotype-specific" uniqueness was confirmed only for 6 substitutions of 30 (Table. 3)

Nucleotide substitution level (%) (coding region of polyprotein, 10242 nr)

genotype 3 14,4 15,2 5,4 178-79 11,0 16,0 14,1 886-84 12,5 15,6 13,7 Amino acid substitution level (%) (complete amino acid sequence of polyprotein, 3414 ar.)

genotype 3 5,3 6,2 1,9 178-79 3,1 6,1 5,2 886-84 3,9 6,0 4,2

**Comments:** The level of nucleotide and amino acid substitutions within the genotype is marked with grey.

**Table 2.** Nucleotide and amino acid substitution level between different TBEV genotypes and strains 178-79 and

The analysis of complete amino acid sequence of strain 886-84 polyprotein confirmed that it's the unique "mixture" of sequences common for genotypes 1, 2 and 3. For example, in the set of 22 positions which clearly differentiate all known TBEV strains into three genotypes the unique amino acids (alanine (A) in position C-108, serine (S) – NS2A-127 and glycine (G) – NS3-258) or interchange with amino acids typical for main TBEV genotypes were found in

**Figure 2.** The differences in 22 positions obtained by comparing of 54 TBEV strains polyprotein sequences; **Com‐ ments.** The cells marked with grey identify the amino acid residues corresponding to one of four TBEV genotypes. The

genotype 1 4,3

genotype 1 1,3

strain 886-84 polyprotein sequence [7] (Fig. 2).

unique amino acid for strain 886-84 is marked with black.

886-84.

100 Encephalitis

genotype 2 16,4 2,3

genotype 2 6,9 0,9

genotype 1 genotype 2 genotype 3


**Comments:** genotype 1 is presented by translated nucleotide sequences X07755, AB022703, AB001026, DQ989336, AY182009, AY217093, JF316707, JF316708, FJ997899, EU816450-EU816455, AY169390, FJ906622, GQ228395, FJ402885, FJ402886, DQ862460, GU121642, HQ201303, HQ901367, HQ901366, HM859894, HM859895, JN003205; genotype 2 - TEU27495, TEU27491, TEU39292, AF091010, EU106868, DQ401140, GV266392, HM535610, HM535611, HM120875, GU183379- GU183381, GU183383; genotype 3 - L40361, AF527415, DQ486861, FJ968751, JN003206-JN003209, GU183382, GU183384. "Group 886" is presented by prototype strain sequence EF469662 (strain 886-84) and GenBank deposited (EU878281-EU878283) and non-deposited 617-90, 711-84 и 740-84 TBEV strains ge‐ nome fragments.

**Table 3.** Unique substitutions in the polyprotein fragments (1066 ar) (proteins С, М, Е and part of NS1) of TBEV "group 886" strains.

At present, using MHNA and sequencing methods we have found the group of 13 TBEV isolates with highly homologous genetic structure to the strain 886-84. For eight strains the genome fragments (1650 nr in length) coding proteins C, M, and E protein fragment were determined (GenBank accession numbers EF469662, EU878281-EU878283, JN936341, JN936347, JN936349-JN936350, JN936353-JN936355) (Fig.4). The homology level with the strain 886-84 genome sequence was 98.2-99.8% while the difference level with three main genotypes ranged from 13.1% (Sofjin) to 16.6% (Neudoerfl).

The strains forming this TBE virus variant were isolated from samples collected in Irkutsk region, Buryat Republic and Transbaikalia in 1984-1990. Also it was recently reported about two "group 886" strains isolated on the territory of National Park "Alkhanai" in Duldurgin‐ skiy district, Transbaikalia from *I. persulcatus* tick (in 1999) and one strain from *Myodes ruti‐ lus* (in 2010) [1]. Moreover, the case of meningoencephalitis with lethal outcome was described in Bulganskiy aimak in Mongolia caused by TBEV isolate with genome fragment

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The common feature of above-listed territories is the presence of several landscape forms that could provide rich biodiversity of flora and fauna. The combination of forest land‐ scapes with steppe areas is typical for Ekhirit-Bulagatskiy district of Irkutsk region. Bi‐ churskiy district of Buryat Republic is presented by mountain forest ecosystems as well as submountain and mountain-valley areas including submountain landscapes with local pine woods and steppe-meadows. Barguzinskiy district is located from Barguzin river mouth along Barguzin river valley in mountain-forest zone. Its middle part corresponds an "island" of steppe and forest-steppe landscapes in isolated mountain valley surround‐ ed by mountain-forest area. Krasnochikoiskiy district of Transbaikalia is the eastern fron‐ tier of South-Siberian mountain landscape territory. The basic components of foci territories are similar to ones from Irkutsk region and Buryat Republic south, where the combination of mountain-forest, forest-steppe and steppe landscapes could be observed. The landscape of National Park "Alkhanai" is also very diverse and includes steppes, meadows, forests and rocky mountains. The National Park location on the border of Eur‐ asia boreal forests and Dauria steppes has the special biospheric importance and results in the significant biodiversity because of flora and fauna interpenetration. Bulganskiy ai‐ mak of Mongolia located in Selenga river basin is characterized by forest-steppe, steppe,

Also for "group 886" strains we obtained the data concerning their ecological connections with all elements of transmissible chain. Thus, strains 712-89, 418-90, 606-90, 608-90, 617-90, 636-90, 691-90, 733-90 and 742-90 were isolated from *I. persulcatus* ticks, and 711-84 and

The case of meningoencephalitis with lethal outcome described in Bulganskiy aimak in Mongolia was caused by TBEV isolate highly homologous to strain 886-84. So it demon‐ strates that this TBEV variant may play the role in human infectious pathology. Also the strains isolation during the long period of time (since 1984 to 2010) confirms the stability of

Therefore, "group 886" strains seem to possess all necessary characteristics to separate them into the independent TBEV genotype. Earlier we suggested that two TBEV strains 178-79 and 886-84 are not the members of three known genotypes forming their own branch on phylogenetic tree and may be the representatives of genotypes 4 and 5 [8, 9]. The presented data confirm and develop this hypothesis and also allow to separate "group 886" strains into the new genotype 5 on the base of their properties described in

sequence similar to strain 886-84 [24].

dry steppe zones and river valleys landscapes.

740-84 from gray-sided vole brain.

our work.

its circulation on Eastern Siberia territory.

**Figure 3.** Phylogenetic tree (NJ, Kimura 2), based on TBEV genome fragments sequences (1650 nr in length) (proteins C, M, and E protein fragment).

Our study determined specific areas of habitat for TBEV "group 886" (Fig. 4).

**Figure 4.** TBEV "group 886" area of habitat.

The strains forming this TBE virus variant were isolated from samples collected in Irkutsk region, Buryat Republic and Transbaikalia in 1984-1990. Also it was recently reported about two "group 886" strains isolated on the territory of National Park "Alkhanai" in Duldurgin‐ skiy district, Transbaikalia from *I. persulcatus* tick (in 1999) and one strain from *Myodes ruti‐ lus* (in 2010) [1]. Moreover, the case of meningoencephalitis with lethal outcome was described in Bulganskiy aimak in Mongolia caused by TBEV isolate with genome fragment sequence similar to strain 886-84 [24].

The common feature of above-listed territories is the presence of several landscape forms that could provide rich biodiversity of flora and fauna. The combination of forest land‐ scapes with steppe areas is typical for Ekhirit-Bulagatskiy district of Irkutsk region. Bi‐ churskiy district of Buryat Republic is presented by mountain forest ecosystems as well as submountain and mountain-valley areas including submountain landscapes with local pine woods and steppe-meadows. Barguzinskiy district is located from Barguzin river mouth along Barguzin river valley in mountain-forest zone. Its middle part corresponds an "island" of steppe and forest-steppe landscapes in isolated mountain valley surround‐ ed by mountain-forest area. Krasnochikoiskiy district of Transbaikalia is the eastern fron‐ tier of South-Siberian mountain landscape territory. The basic components of foci territories are similar to ones from Irkutsk region and Buryat Republic south, where the combination of mountain-forest, forest-steppe and steppe landscapes could be observed. The landscape of National Park "Alkhanai" is also very diverse and includes steppes, meadows, forests and rocky mountains. The National Park location on the border of Eur‐ asia boreal forests and Dauria steppes has the special biospheric importance and results in the significant biodiversity because of flora and fauna interpenetration. Bulganskiy ai‐ mak of Mongolia located in Selenga river basin is characterized by forest-steppe, steppe, dry steppe zones and river valleys landscapes.

**Figure 3.** Phylogenetic tree (NJ, Kimura 2), based on TBEV genome fragments sequences (1650 nr in length) (proteins

Our study determined specific areas of habitat for TBEV "group 886" (Fig. 4).

C, M, and E protein fragment).

102 Encephalitis

**Figure 4.** TBEV "group 886" area of habitat.

Also for "group 886" strains we obtained the data concerning their ecological connections with all elements of transmissible chain. Thus, strains 712-89, 418-90, 606-90, 608-90, 617-90, 636-90, 691-90, 733-90 and 742-90 were isolated from *I. persulcatus* ticks, and 711-84 and 740-84 from gray-sided vole brain.

The case of meningoencephalitis with lethal outcome described in Bulganskiy aimak in Mongolia was caused by TBEV isolate highly homologous to strain 886-84. So it demon‐ strates that this TBEV variant may play the role in human infectious pathology. Also the strains isolation during the long period of time (since 1984 to 2010) confirms the stability of its circulation on Eastern Siberia territory.

Therefore, "group 886" strains seem to possess all necessary characteristics to separate them into the independent TBEV genotype. Earlier we suggested that two TBEV strains 178-79 and 886-84 are not the members of three known genotypes forming their own branch on phylogenetic tree and may be the representatives of genotypes 4 and 5 [8, 9]. The presented data confirm and develop this hypothesis and also allow to separate "group 886" strains into the new genotype 5 on the base of their properties described in our work.

Along with the original genome sequences of "group 886" strains we investigated their phe‐ notypic characteristics which are the essential part of the virus nature and properties study and useful for practical virology.

**Strain** № **Isolaion source Life time**

lethal outcome in laboratory animals in 100%.

711-84 *Myodes rufocanus* 5,1±0,49 100 740-84 *Myodes rufocanus* 5,2±0,24 100 712-89 *I. persulcatus* 5,3±0,79 100 780-89 *I. persulcatus* 6,6±0,45 100 691-90 *I. persulcatus* 6,0±0,89 100 418-90 *I. persulcatus* 6,3±0,06 100 733-90 *I. persulcatus* 5,0±0,45 100 742-90 *I. persulcatus* 6,8±0,76 100 886-84 *Myodes rutilus* 6,1±0,35 93,8±1,91 606-90 *I. persulcatus* 5,9±0,27 93,3±1,98 636-90 *I. persulcatus* 5,3±0,44 88,9±2,5 608-90 *I. persulcatus* 6,4±0,89 77,8±3,3 617-90 *I. persulcatus* 5,3±1,03 70±3,65

**Table 5.** The average life time and lethality percent of mice infected with TBEV "group 886" strains.

district of Transbaikalia and Bichurskiy district of Buryat Repblic [5, 15, 22].

the most severe forms of acute TBE resulting in lethal outcome or disability [19].

M.S. Vorob'eva for the strains – candidates for vaccine prototypes [3].

The value of mice average life time after infection with different strains ranged from 5.0±0.45 to 6.8±0.76 days and lethality percent varied from 70±3.65 to 100%. It should be noted that the strains isolated from *I. persulcatus* ticks collected in Krasnochikoiskiy district caused the

Earlier, we have noted that registered and described severe clinical cases of TBE were found in the foci where "group 886" strains were isolated. The foci are located in Krasnochikoiskiy

Recently, the case of meningoencephalitis with lethal outcome was described in Mongolia caused by TBEV isolate possessed 98.5% homology level with strain 886-84 genome sequence. The infection of patient occurred after tick bite in Bulganskiy aimak bordering from south with four natural foci where TBEV "group 886" strains were isolated from collected samples. The patient was hospitalized on 11th day after the tick bite with diagnosis "meningoencephalitis" and died on 11th day of the disease. The presence of TBEV RNA in macromyelon samples, in core andmeninx vasculosa indicates the multilevel localization of lesions which are typical to

Taking into account the genetic and antigenic properties of 886-84 strain, the strain itself and the strains of the group could be considered as a candidates for development of universal vaccines and test-systems effective for different TBEV serotypes (genotypes). So we investi‐ gated "group 886" strains according the complex of criteria suggested by L.S. Vereta and

**(days ± m)**

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**% of lethal cases**

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**(% ± m)**

The pathogenic properties of viruses are the most important biological characteristics. Tak‐ ing into account that TBEV "group 886" strains play the role in human infectious pathology [24] it was extremely important to study their pathogenic potential.

We have estimated the virulence level of TBEV "group 886" strains according two parame‐ ters: the average infectious virus titers after intracerebral or subcutaneous inoculation of mice. Peripheral virus activity was characterized by index of invasiveness (II) (Table 4).


**Table 4.** The index of invasiveness (II) for TBEV "group 886" strains.

The virus titers after intracerebral inoculation of mice ranged from 6.64 to 10.9 lg LD50/ml while after subcutaneous inoculation (peripheral activity) were found to be from 3.8 to 9.8 lg LD50/ml. The determined virus indexes of invasiveness (II) had medium and high values (from 0.8 to 3.04 lg LD50/ml). According to obtained results, six strains from "group 886" had high invasive properties that mean their ability to overcome the blood-brain barrier, pene‐ trate into CNS and propagate in it. The highest invasive properties were observed in three strains isolated from rodents and in one strain isolated from tick collected in Krasnochikois‐ kiy district of Transbaikalia. Two strains (606-90 and 608-90) from Bichurskiy district, Buryat Republic had the lower neuroinvasive activity.

Additionally, to characterize the virulence of TBEV "group 886" strains we determined the average life time and lethality percent of infected mice (Table 5).


**Table 5.** The average life time and lethality percent of mice infected with TBEV "group 886" strains.

Along with the original genome sequences of "group 886" strains we investigated their phe‐ notypic characteristics which are the essential part of the virus nature and properties study

The pathogenic properties of viruses are the most important biological characteristics. Tak‐ ing into account that TBEV "group 886" strains play the role in human infectious pathology

We have estimated the virulence level of TBEV "group 886" strains according two parame‐ ters: the average infectious virus titers after intracerebral or subcutaneous inoculation of mice. Peripheral virus activity was characterized by index of invasiveness (II) (Table 4).

**Strain Isolaion source mNic (lgLD50/мл) mNsc (lgLD50/мл) mNic-mNsc** *II 691-90 I. persulcatus* 7,02 5,1 1,92 +

*418-90 I. persulcatus* 9¸72 7,8 1,52 +

*886-84 Myodes rutilus* 8,58 7,16 1,2 +

*711-84 Myodes rufocanus* 6,75 4,6 1,0 +

*740-84 Myodes rufocanus* 10,2 9,4 0,8 +

*712-89 I. persulcatus* 10,9 9,8 1,1 +

*617-90 I. persulcatus* 6,64 3,8 2,84 ±

*636-90 I. persulcatus* 7,06 4,35 2,71 ±

*608-90 I. persulcatus* 7,9 4,86 3,04 -

*606-90 I. persulcatus* 7,02 4,02 3,0 -

The virus titers after intracerebral inoculation of mice ranged from 6.64 to 10.9 lg LD50/ml while after subcutaneous inoculation (peripheral activity) were found to be from 3.8 to 9.8 lg LD50/ml. The determined virus indexes of invasiveness (II) had medium and high values (from 0.8 to 3.04 lg LD50/ml). According to obtained results, six strains from "group 886" had high invasive properties that mean their ability to overcome the blood-brain barrier, pene‐ trate into CNS and propagate in it. The highest invasive properties were observed in three strains isolated from rodents and in one strain isolated from tick collected in Krasnochikois‐ kiy district of Transbaikalia. Two strains (606-90 and 608-90) from Bichurskiy district, Buryat

Additionally, to characterize the virulence of TBEV "group 886" strains we determined the

**Table 4.** The index of invasiveness (II) for TBEV "group 886" strains.

Republic had the lower neuroinvasive activity.

average life time and lethality percent of infected mice (Table 5).

[24] it was extremely important to study their pathogenic potential.

and useful for practical virology.

104 Encephalitis

The value of mice average life time after infection with different strains ranged from 5.0±0.45 to 6.8±0.76 days and lethality percent varied from 70±3.65 to 100%. It should be noted that the strains isolated from *I. persulcatus* ticks collected in Krasnochikoiskiy district caused the lethal outcome in laboratory animals in 100%.

Earlier, we have noted that registered and described severe clinical cases of TBE were found in the foci where "group 886" strains were isolated. The foci are located in Krasnochikoiskiy district of Transbaikalia and Bichurskiy district of Buryat Repblic [5, 15, 22].

Recently, the case of meningoencephalitis with lethal outcome was described in Mongolia caused by TBEV isolate possessed 98.5% homology level with strain 886-84 genome sequence. The infection of patient occurred after tick bite in Bulganskiy aimak bordering from south with four natural foci where TBEV "group 886" strains were isolated from collected samples. The patient was hospitalized on 11th day after the tick bite with diagnosis "meningoencephalitis" and died on 11th day of the disease. The presence of TBEV RNA in macromyelon samples, in core andmeninx vasculosa indicates the multilevel localization of lesions which are typical to the most severe forms of acute TBE resulting in lethal outcome or disability [19].

Taking into account the genetic and antigenic properties of 886-84 strain, the strain itself and the strains of the group could be considered as a candidates for development of universal vaccines and test-systems effective for different TBEV serotypes (genotypes). So we investi‐ gated "group 886" strains according the complex of criteria suggested by L.S. Vereta and M.S. Vorob'eva for the strains – candidates for vaccine prototypes [3].

The proteins of virus envelope are responsible for hemagglutination and antigenic activity, thermostability and some other properties. All TBEV "group 886" strains have shown the hemagglutination activity (titers in hemagglutination reaction 1:1280-1:10240) in the reaction with goose erythrocytes. In RDPA with cross-adsorbed strain-specific sera 886-84 strain demonstrated the same level of similarity with all TBEV subtypes. The high level of antigen‐ ic cross-reaction with East-Siberian and Far-Eastern subtypes was observed in RDPA and neutralization reaction (NR) tests for 886-84, 711-84 and 740-84 strains [4]. The results of an‐ tigenic typing are presented in Tables 6 and 7.

Thermostability is one of the most important genetic features to which the attention should be paid during the selection of TBEV strains for inactivated vaccines production. The results of inactivation index determination for 13 TBEV "group 886" strains at temperature 50°С

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**Strain Isolaion source lgTCD50/ml at 37˚С lgTCD50/ml at 50˚С Difference**

**Table 8.** The results of thermal resistance determination for TBEV "group 886" strains.

kiy district of Transbaikalia were thermostable.

different temperatures (rct- or ts- feature).

with diameter 1-1.5 mm (S-).

The values of lgTCD50/ml at 37ºС varied from 3.5 to 8.26. According to the thermoresistanse feature all tested strains were divided into three group: thermostable (Т50+) - nine strains; thermolabile (Т50-) – one strain; strains with medium thermoresistance – three strains. It should be noted that all strains isolated from *I. persulcatus* ticks collected in Krasnochikois‐

The genetic features linked with intracellular TBEV proliferation are the cytopathic activity, plaque size and type in cells culture under agar layer (S-feature) and ability to proliferate at

All "group 886" strains caused the destruction of infected cells monolayer on 4th-6th day. The plaque size and type in cells culture under the agar layer differed depending on certain strain. Thus, strain 740-84 formed large plaques 3.0 mm in diameter (S+), strain 886-84 formed plaques of medium size (d=2.0 mm) (S±) and strain 711-84 formed small plaques

*636-90 I. persulcatus* 4,78 3,0 1,78 + *606-90 I. persulcatus* 4,0 2,23 1,77 + *691-90 I. persulcatus* 4,84 3,43 1,41 + *418-90 I. persulcatus* 4,0 2,22 1,78 + *886-84 Myodes rutilus* 7,08 6,9 0,18 + *711-84 Myodes rufocanus* 8,26 7,07 1,2 + *712-89 I. persulcatus* 4,0 2,23 1,77 + *733-90 I. persulcatus* 4,25 2,5 1,75 + *742-90 I. persulcatus* 3,5 2,23 1,27 + *608-90 I. persulcatus* 4,5 2,0 2,5 ± *287-83 I. persulcatus* 5,33 2,5 2,83 ± *740-84 Myodes rufocanus* 7,18 4,78 2,4 ± *617-90 I. persulcatus* 8,0 3,67 4,33 -

(Т50) are shown in Table 8.


**Comments:** the reverse titers of precipitating antibodies are shown; 0-negative result at sera dilution 1:32; \* marks: W – Western antigenic variant; E-S – East-Siberian; F-E – Far Eastern; a/g – antigen.

**Table 6.** Immunotyping of TBEV "group 886" strains by RDPA test.


**Comments:** reversed antibody titers are presented in the table; the significant values of neutralization marked with bold (the significant titers were corresponded to neutralization values of prototype strain with the same sera in ho‐ mologous system). KFD – prototype strain of Kyasanur Forest disease virus. W – TBEV Western antigenic variant; E-S – East-Siberian; F-E – Far Eastern.

**Table 7.** TBEV "group 886" strains neutralization reaction test with antisera against prototype TBEV strains and viruses of tick-borne encephalitis virus complex.

Thermostability is one of the most important genetic features to which the attention should be paid during the selection of TBEV strains for inactivated vaccines production. The results of inactivation index determination for 13 TBEV "group 886" strains at temperature 50°С (Т50) are shown in Table 8.

The proteins of virus envelope are responsible for hemagglutination and antigenic activity, thermostability and some other properties. All TBEV "group 886" strains have shown the hemagglutination activity (titers in hemagglutination reaction 1:1280-1:10240) in the reaction with goose erythrocytes. In RDPA with cross-adsorbed strain-specific sera 886-84 strain demonstrated the same level of similarity with all TBEV subtypes. The high level of antigen‐ ic cross-reaction with East-Siberian and Far-Eastern subtypes was observed in RDPA and neutralization reaction (NR) tests for 886-84, 711-84 and 740-84 strains [4]. The results of an‐

**Strain antigen Serum against strain \*antigenic**

**Sofjin, depleted by а/g 256**

Aina/1448 0 4 4 2 4 0 0 E-S Sofjin 0 0 0 4 0 4-8 0 F-E 256 2-4 0 2 0 0 0 0 W

*740-84* 0 4 4 0 0 0 0 E-S *711-84* 0 4-8 4 2-4 0 4 0 E-S F-E *886-84* 4 4 4 2 2 4-8 0 W, E-S, F-E

**Comments:** the reverse titers of precipitating antibodies are shown; 0-negative result at sera dilution 1:32; \* marks: W

Sofjin **10240** 1280 1280 1280 F-E Lesopark-11 5120 **10240** 10240 1280 E-S 256 10240 2560 **10240** 5120 W KFD 640 640 640 **5120** KFD

740-84 **10240 10240** 2560 1280 F-E, E-S 711-84 **5120 5120** 2560 1280 F-E, E-S 886-84 **5120 10240** 2560 1280 E-S, F-E

**Comments:** reversed antibody titers are presented in the table; the significant values of neutralization marked with bold (the significant titers were corresponded to neutralization values of prototype strain with the same sera in ho‐ mologous system). KFD – prototype strain of Kyasanur Forest disease virus. W – TBEV Western antigenic variant; E-S –

**Table 7.** TBEV "group 886" strains neutralization reaction test with antisera against prototype TBEV strains and viruses

2 4 2-4 0 2 0 0 E-S

**Aina, depleted by а/g 256**

**Strain Immune sera Antigenic typing**

**Sofjin Lesopark 256 KFD results**

**Sofjin, depleted by а/g Lesopark 11**

**Aina, depleted by а/g Lesopark 11**

**Lesopark11, depleted by а/g Sofjin**

**typing results**

tigenic typing are presented in Tables 6 and 7.

**Aina, depleted by а/g Sofjin**

– Western antigenic variant; E-S – East-Siberian; F-E – Far Eastern; a/g – antigen.

**Table 6.** Immunotyping of TBEV "group 886" strains by RDPA test.

**256, depleted by а/g Aina**

**Prototype Strains**

106 Encephalitis

**"group 886" strains**

Lesopark -11

Prototype TBEV strains and viruses of TBEV complex

TBEV "group 886"

East-Siberian; F-E – Far Eastern.

of tick-borne encephalitis virus complex.

strains


**Table 8.** The results of thermal resistance determination for TBEV "group 886" strains.

The values of lgTCD50/ml at 37ºС varied from 3.5 to 8.26. According to the thermoresistanse feature all tested strains were divided into three group: thermostable (Т50+) - nine strains; thermolabile (Т50-) – one strain; strains with medium thermoresistance – three strains. It should be noted that all strains isolated from *I. persulcatus* ticks collected in Krasnochikois‐ kiy district of Transbaikalia were thermostable.

The genetic features linked with intracellular TBEV proliferation are the cytopathic activity, plaque size and type in cells culture under agar layer (S-feature) and ability to proliferate at different temperatures (rct- or ts- feature).

All "group 886" strains caused the destruction of infected cells monolayer on 4th-6th day. The plaque size and type in cells culture under the agar layer differed depending on certain strain. Thus, strain 740-84 formed large plaques 3.0 mm in diameter (S+), strain 886-84 formed plaques of medium size (d=2.0 mm) (S±) and strain 711-84 formed small plaques with diameter 1-1.5 mm (S-).

The most of the viruses proliferate and form mature virus particles in sensitive cells in defi‐ nite temperature limits. A number of authors investigating the proliferation of TBEV strains in cell cultures at different temperatures (rct-feature) came to the conclusion that this marker could be the important phenotypical characteristics of the virus and closely connected with its virulence [13, 14].

"group 886" strains that is the "mixture" of amino acid sequences typical for genotypes

Genetic and Biological Properties of Original TBEV Strains Group Circulating in Eastern Siberia

http://dx.doi.org/10.5772/54087

109

**2.** This TBEV variant can be considered as an independent TBEV genotype 5 (high level of genetic difference compared to other genotypes – more than 12%, the existence of its own natural foci, the ecological connection with all elements of transmissive chain, the

**3.** The ability to cause focal forms of tick-borne encephalitis with lethal outcome and labo‐ ratory results of virulence level evaluation testify the high pathogenic potential of TBEV "group 886" strains. During the study of the genetic markers connected with virus in‐ tracellular reproduction we have found that "group 886" strains have high adaptive ability and can easily accommodate to circulation in different biocenosises and in varie‐

**4.** Some studied "group 886" strains possess the wide spectrum of antigenic properties, hemagglutination and neutralizing activity, high virulence and thermotolerance. They match the basic criteria of strains-candidates chosen for diagnostic and vaccine develop‐

The authors are greatly appreciated to all colleagues collected the field materials and to the staff of Natural-Foci Infection Department: E.V. Arbatskaya, I.V. Voronko, O.Z. Gorin, N.A. Gusarova, G.A. Danchinova, V.M. Kogan, S.I. Lipin, O.V. Melnikova, A.G. Trukhina. Also we acknowledge the partial financial support by Integration interdisciplinary project grant

, Yu.P. Dzhioev1

, A.I. Paramonov1

, S.E. Tkachev4

, O.O. Fedulina1

, L.S. Karan5

,

,

No. 135 from the Siberian Branch of the Russian Academy of Sciences.

, T.V. Demina3

, O.V. Suntsova1

2 Centre for Epidemiology and Hygiene in Irkutsk region, Irkutsk, Russia

3 Irkutsk State Medical University of Russian Ministry of Heath, Irkutsk, Russia

4 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia

5 Central Research Institute of Epidemiology of Rospotrebnadzor RF, Moscow, Russia

1 FSSFE Scientific Centre of Family Health and Human Reproduction Problems SB RAMS,

role in infectious pathology, stability and durational circulation in nature).

1, 2 and 3.

ment.

**Acknowledgements**

**Author details**

E.K. Doroshchenko1

I.V. Kozlova1

A.O. Revizor3

Irkutsk, Russia

ty of landscape-geographical zones.

, M.M. Verkhozina2

and V.I. Zlobin3

, O.V. Lisak1

We investigated rct42 genetic marker for 12 TBEV "group 886" strains. The results are pre‐ sented in Table 9.


**Table 9.** Rct42 genetic marker for TBEV "group 886" strains.

"Group 886" strains demonstrated the high heterogeneity in proliferation ability at 42°С. Five strains propagated more effectively at supraoptimal temperature (42º). Moreover, eight of nine strains isolated from ticks had rct42± feature. The strains isolated from rodents were more heterogenic: strain 740-84 had rct42+ feature, strain 886-84 had rct42- feature and strain 711-84 had rct42± feature. All TBEV "group 886" strains isolated from ticks collected in Kras‐ nochikoiskiy district of Transbaikalia actively propagate at 42ºС.
