**2. Materials and methods**

#### **2.1. TBE virus**

Thirteen TBEV strains from the collection of FSSFE "Scientific Centre of Family Health and Human Reproduction Problems, Institute of Epidemiology and Microbiology SB RAMS", Ir‐ kutsk were investigated in the study. By MHNA genotyping and full genome or fragments sequencing they were classified as "group 886" strains. Detailed information about strains is presented in Table 1.


**Table 1.** Information concerning "group 886" strains of TBE virus, isolated on the Eastern Siberia territory.

### **2.2. Strains genotyping**

conventionally defined as "group 886" [6]. The obtained results confirm the validity of

The unique genetic structure of "group 886" strains also manifests in original phenotype pattern that is quite significant from the scientific point of view. "Group 886" strains can be tested as prototype candidate strains for the design of universal vaccines effective against

The aim of the study was to investigate genetic and biological properties of TBEV "886 group" strains circulating in Eastern Siberia (territories of Irkutsk region, Buryat Repub‐ lic, Transbaikalia) for estimation of their potential as candidates for test-systems and vac‐

Thirteen TBEV strains from the collection of FSSFE "Scientific Centre of Family Health and Human Reproduction Problems, Institute of Epidemiology and Microbiology SB RAMS", Ir‐ kutsk were investigated in the study. By MHNA genotyping and full genome or fragments sequencing they were classified as "group 886" strains. Detailed information about strains is

*886-84* 1984 *Myodes (Clethrionomys*) *rutilus* Irkutsk region, Ekhirit-Bulagatskiy district *711-84* 1984 *Myodes rufocanus* Buryat Republic, Barguzinskiy district *740-84* 1984 *Myodes rufocanus* Buryat Republic, Bichurskiy district *712-89* 1989 *I. persulcatus* Transbaikalia, Krasnochikoyskiy district *780-89* 1989 *I. persulcatus* Buryat Republic, Bichurskiy district *617-90* 1990 *I. persulcatus* Buryat Republic, Bichurskiy district *636-90* 1990 *I. persulcatus* Buryat Republic, Bichurskiy district *608-90* 1990 *I. persulcatus* Buryat Republic, Bichurskiy district *606-90* 1990 *I. persulcatus* Buryat Republic, Bichurskiy district *691-90* 1990 *I. persulcatus* Buryat Republic, Bichurskiy district *418-90* 1990 *I. persulcatus* Transbaikalia, Krasnochikoyskiy district *733-90* 1990 *I. persulcatus* Transbaikalia, Krasnochikoyskiy district *742-90* 1990 *I. persulcatus* Transbaikalia, Krasnochikoyskiy district

**Table 1.** Information concerning "group 886" strains of TBE virus, isolated on the Eastern Siberia territory.

**Isolation source The location of sample collection**

"group 886" certification as possible separate TBEV genotype.

strains of different serotypes (genotypes) and TBEV test-systems.

cine development.

96 Encephalitis

**2.1. TBE virus**

presented in Table 1.

**Strain The year of**

**isolation**

**2. Materials and methods**

We used MHNA with three panels of 40 deoxyoligonucleic probes complemented to frag‐ ments of 10 genes of different TBEV genotypes. The probe description and their localization in TBEV genome was presented earlier by Demina *et al*. [6].

The total RNA extraction from infected mice brains or porcine embryo kidney cells, apply‐ ing RNA onto kapron or cellulose nitrate filters and hybridization with probes were per‐ formed by the common methods [16].

The amplification was carried out with primers complemented to 5'- UTR fragment, to CprM-E-NS1 genes, E gene or E-NS1 genes fragments, synthesized in the Institute of Chemi‐ cal Biology and Fundamental Medicine SB RAS (Novosibirsk, Russia). RT-PCR was performed according to the "BioSan" company (Novosibirsk, Russia) protocol.

The sequence analysis of PCR products was carried out with BigDye Terminators Cycle Se‐ quencing Kit v.3.1 (Applied Biosystems, USA) in DNA Sequencing Center SB RAS, Novosi‐ birsk, Russia. The obtained data was analyzed by Mega 5.0 program [28]. The gene fragments sequences of TBEV strains belonging to the different genetic types from GenBank database were used as a material for comparison. BLAST program (http:// www.ncbi.nlm.nih.gov/blast/) was used for homology search of obtained nucleotide sequen‐ ces with already known fragments of TBEV genomes.

The genome fragments sequences of "group 866" strains obtained during the study have been deposited into GenBank database with access numbers EF469662, EU878281-EU878283, JN936341, JN936347, JN936349-JN936350, JN936353-JN936355.

The sequencing of full genome of 886-84 strain has been performed by Karan *et al*. in Central Research Institute of Epidemiology of Rospotrebnadzor RF, Moscow, Russia.

#### **2.3. Strains immunotyping**

The reaction of diffuse precipitation in agar (RDPA) was carried out by the method developed by Clark [12] with modifications by Rubin [27] and Bochkova [2]. We used immune sera against TBEV prototype strains of three serotypes (Sofjin – Far-Eastern serotype, 256 – Western sero‐ type, Lesopark-11 and Aina/1448 – East-Siberian serotype) exposed to dosed adsorbtion with concentrated cultural antigens or cross-adsorbed sera against investigated strains [4].

The cytoplasmatic activity study was performed according to common methods. Virus titers were determined in tests on cell culture based on its cytopathic activity (CPA) by the full cumulative method (offered by Reed and Muench) and expressed as lg TCD50 <sup>1</sup> /ml [26].

#### **2.4. Neuroinvasiveness**

To estimate the neuroinvasiveness of TBEV strains we determined the index of invasiveness (II) – the difference between the virus titers after intracerebral (mNic) and subcutaneous

<sup>1</sup> Tissue cytopathogenic dose

(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 the virus strain.

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‐

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

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

99

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

100

**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,

100

genotype 1 (28 strains)

 178-79 886-84 genotype 3 (10 strains) genotype 2 (14 strains) OHF

substitution level was close to the species separation border [11] (Table 2).

100

JN003206-JN003209, GU183382, GU183384. OHF – Omsk haemorrhagic fever virus.

pendent genotype.

99

0.02

100

100

#### **2.5. Thermoresistance**

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 or more than 3.1 lg – as Т50-.

#### **2.6. Rct42–feature**

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

#### **2.7. S-feature**

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