Tick-borne encephalitis virus in a highly endemic area in Kemerovo (Western Siberia, Russia)

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International Journal of Medical Microbiology 298 (2008) S1, 94–101 www.elsevier.de/ijmm

Tick-borne encephalitis virus in a highly endemic area in Kemerovo (Western Siberia, Russia) Gerhard Doblera,, Gudrun Zo¨llera, Tatyana Poponnikovab, Dieter Gnielc, Martin Pfeffera, Sandra Essbauera a

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937 Munich, Germany Department of Neurology, Neurosurgery, and Medical Genetics, Kemerovo State Medical Academy, pr. Lenina 55b-148, Kemerovo 650099, Russia c Novartis Vaccines and Diagnostics GmbH & Co. KG, Marburg, Germany b

Accepted 31 March 2008

Abstract Western Siberia is the region with the highest known incidence of tick-borne encephalitis (TBE) in the world, with 40 to 480 cases/100,000 population. Few data are available on the circulation of TBE virus (TBEV) strains in the region. In the present study, a total of 468 pooled ticks (Ixodes persulcatus) collected in 7 areas around Kemerovo, Western Siberia, were tested for the presence of TBEV RNA by real-time RT-PCR. Positive tick pools were further investigated by conventional PCR and the nucleotide sequences of the partial TBEV E protein genes were compared to known nucleotide sequences of (Siberian) TBEV strains. In 4 of the 7 areas tested, TBEV RNA-positive ticks were found. Seven out of 28 tick pools were positive in real-time RT-PCR. Assuming only one tick of each pool to be positive, the overall minimal infection rate (MIR) was 1.5% (7/468), ranging from 0% up to 4% for positive regions. Molecular characterization of the E protein of 6 of the 7 positive pools exhibited a sequence variability of 1.4–2.6% in comparison to the nucleotide (nt) sequence of the Aina strain of the Siberian subtype of TBEV. The phylogenetic analysis of the nt sequences clearly indicates that two clusters of the Siberian subtype of TBEV seem to circulate simultaneously in the Kemerovo region. The pathogenicity of the respective virus variants, however, warrants further examination. r 2008 Elsevier GmbH. All rights reserved. Keywords: TBE virus; Siberian subtype; Genetic diversity; Aina strain; Ixodes persulcatus

Introduction Tick-borne encephalitis (TBE) is the most important inflammatory infection of the central nervous system (CNS) in Europe and parts of Asia. Up to 10,000 human cases of so-called Russian spring–summer encephalitis Corresponding author. Tel.: +49 89 3168 3974; fax: +49 89 3168 3292. E-mail address: [email protected] (G. Dobler).

1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.03.011

(RSSE) are reported annually from various areas of Russia. In Russia, the RSSE incidence is reported to be up to 6.8/100,000 (Zlobin and Gorin, 1996; Vorobjeva et al., 2001; Pokrovsky et al., 2003); however, in some oblasts (districts) of Western Siberia the highest incidence rates with 4100/100,000 are found in specific rural areas of Russia in highly active years (see Fig. 1; Zhukova et al., 2002; Pakhotina et al., 2002; Poponnikova, 2006). In the Kemerovo oblast, 1300–2300 cases with a suspected RSSE are hospitalized annually

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Fig. 1. The blow-up shows the Kemerovo oblast (Russia, western Siberia) with the locations where the Ixodes persulcatus ticks were collected (ANZ: Anzhero-Sudzhenzk; CHE: Chernigovskyi; BAL: Balahonovka; AND: Andreevka; OSI: Osinovka; TEB: Tebenki; KEM: Kemerovo). These districts have had the highest incidences of Russian spring–summer encephalitis in humans (40–80/ 100,000) in the Western Siberian region.

(Poponnikova, 2006) leading to incidence rates in the total population of 20–60/100,000. Out of these, 300–600 cases of RSSE per year are confirmed by serological testing. Age groups mostly affected by that disease are young adult working people, which account for up to 50% of all human cases of RSSE (Zlobin and Gorin, 1996; Saldin et al., 2000; Pakhotina et al., 2002; Zhukova et al., 2002). In Kemerovo oblast as in other parts of Western Siberia, however, an increase of the incidence rate in children (o14 years) has been observed in recent years. In Kemerovo oblast, the annual paediatric RSSE incidence now reaches up to 90/100,000 with children o5 years of age comprising 5–7% of all RSSE cases registered (Poponnikova, 2006). Few concise data are available on the TBE virus (TBEV) circulating in the region of Kemerovo. So far, two major genetic clades of TBEV of the Siberian subtype (TBEVSib) were identified (Golovljova et al., 2004). These viruses, however, were identified in ticks collected in Irkutsk, several hundred kilometres east of the Kemerovo region. So far, it is not known whether similar virus subtypes are circulating in western Siberia. Also, no data are available on TBEV infection rates in Ixodes (I.) persulcatus ticks of the Kemerovo region. In the actual study, we investigated the presence of TBEV in I. persulcatus and analysed the obtained viral sequence data.

Materials and methods Collecting and preparation of ticks Ticks were collected by flagging in June 2005 at 7 locations around Kemerovo (Fig. 1) in typical taiga forests and at their edges, a mixture between coniferous and deciduous woods covered with dense ground vegetation, mainly ferns or grass. Sampling areas were chosen according to the occurrence of TBE cases reported to the State Medical Academy of Kemerovo. For each sampling area, collected adult ticks (I. persulcatus) were separately placed into tubes and transported alive to the laboratory and frozen at 20 1C until testing in August 2005. Ticks were thawed and pooled according to the regions where they were sampled. The size of the pools ranged from 7 to 30 ticks per pool (Table 1). Each tick pool was homogenized manually with a tight-fitting plastic mortar in a 1.5-ml Eppendorf cup in 1 ml phosphate-buffered saline (PBS; pH 7.4). The homogenized tick suspension was centrifuged at 12,000 g for 5 min at room temperature. RNA was extracted from the supernatant using two different methods. Firstly, a 140-ml aliquot of the supernatant was added to 560 ml AVL buffer (QiAmp Viral RNA Mini Kit, Qiagen, Hilden, Germany). As no

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Table 1. Results from the examination of Ixodes persulcatus ticks (collected in June 2005) for tick-borne encephalitis virus by LCRT-PCR in pools of different regions of Kemerovo oblast (Russia, Western Siberia) including the GenBank numbers of the submitted nt sequences Region (longitude, latitude)

Pools

No. of ticks (pool sizes)

AVL

Paper

MIR

Virus sample

GenBank no.

Anzhero-Sudzhensk (56140 N, 861E) Andreevka (551150 N, 86160 E) Chernigovskyi (551250 N, 86180 E) Kemerovo (551120 N, 86130 E)

1 2 2 2

33 60 (30, 30) 60 (30, 30) 60 (30, 30)

– – – 2+

– – – –

0 0 0 3%

– – – 6, 7

Balahonovka (551140 N, 86180 E) Osinovka (541590 N, 86190 E) Text1EF566815Text1 Tebenki (541550 N, 861100 E) Total

3 7

90 (30, 30, 30) 49 (4  5; 9, 2  10)

1+ 2+

1+ 2+

1% 4%

10 12, 14

– – – EF566817, EF566818 EF566813 EF566814,

11 28

116 (9, 5  10, 3  11, 2  12) 2+a 468 7+

2+a 6+

2% 1.5%

22

EF566816

Abbreviations: AVL, Qiagen sample storing buffer; MIR, minimal infection rate; N, north, E, east. a Sample 23 was positive in the real-time PCR but revealed no amplification product in the conventional RT-PCR.

cell culture for virus isolation was available in Kemerovo and transport on dry ice could not be organized during the stay, 0.3 ml of the supernatant were spotted onto Whatman filter paper (Whatman filter paper grade no. 1; VWR International, Ismaning, Germany), resulting in a spot of about 2–3 cm diameter. The spots were dried and supernatant in AVL buffer as well as spotted on Whatman paper was stored at room temperature until further investigations were carried out about 1 week later after the transport of the samples to the Bundeswehr Institute of Microbiology in Munich (Germany).

Virus propagation The filter paper spots were punched out and soaked in 1 ml cell culture medium (Minimal Essential Medium, MEM) containing double-concentrated antibiotic–antimycotic solution (Invitrogen, Karlsruhe, Germany) for 1 h at 4 1C. From the 28 samples eluted from the Whatman paper, 500 ml were incubated on confluent Vero B4 cells (African green monkey kidney cells) and PK-1 cells (pork kidney cells) (1 h, 37 1C) in 6-well plates (Nunc, Wiesbaden, Germany). After 1 h, the inoculum was removed and 2.5 ml of MEM including 3% fetal calf serum and antibiotics–antimycotics (1  concentration) were added and the cells were further incubated at 37 1C at 5% CO2. Cultures were observed daily for cytopathic effects for 10 days.

Extraction of RNA RNAs of the original tick supernatants diluted in AVL buffer were extracted using the QiAmp Viral RNA Mini Kit (Qiagen) according to the instructions of the

manufacturer. A total of 200 ml of the Whatman paper eluates was purified using the MagNaPure LC Total Nucleic Acid Isolation Kit (Roche, Mannheim, Germany) with external lysis protocol according to the instructions of the manufacturer and extracting RNA in a final volume of 50 ml. A 200-ml aliquot of the tissue culture supernatant (see above) was also extracted using the MagNaPure system accordingly.

RT-PCRs For all samples (RNA gained from tick homogenates in AVL, Whatman paper eluates and cell culture supernatant) a real-time RT-PCR specific for TBEV (Schwaiger and Cassinotti, 2003) was used for detection of viral RNA. The protocol was modified without implementing the described internal control. Briefly, 5 ml RNA were amplified with 0.2 mM of each primer, 0.160 mM hybridization probe, and Invitrogen Platinum Quantitative RT-PCR Thermoscript One-Step-System reagents according to the manufacturer’s instructions. Amplification was performed at 60 1C for 20 min, denaturation at 95 1C for 5 min, and the cDNA was afterwards amplified in 50 cycles for 15 s at 95 1C and 60 s at 60 1C in an Mx 3000 instrument (Stratagene, Heidelberg, Germany). RNA samples with a positive signal in the real-time RT-PCR were subjected to a TBEV-specific conventional RT-PCR using primers targeting the E gene (nucleotides, nt 10–1487 of RSSEV strain Irkutsk 1m1, AB049348). Briefly, 5 ml of RNA was amplified using 0.2 mM of primers (RSSE10 50 -ACACATCTGGAGAACAGG-30 and RSSEc1487 50 -GCACCCACTCCAAGTGTCATAG-30 ) and the SuperScriptIII-One-step RT-PCR system with Platinum Taq DNA polymerase

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(Invitrogen) in a final volume of 50 ml according to the manufacturer0 s instructions. Following reverse transcription at 50 1C for 45 min and denaturation at 94 1C for 5 min, cDNA was amplified in 40 cycles for 30 s at 94 1C, 30 s at 50 1C, and 2 min at 68 1C. A final extension for 10 min at 68 1C was added. Amplified products were run in 1% agarose gels, visualized with ethidium bromide and UV illumination.

Sequencing, alignments, and phylogenetic analyses In order to determine their nucleotide sequences, the amplicons were purified using the QIAquick PCR purification kit according to the manufacturer’s instructions (Qiagen). Direct sequencing of the purified PCR products was done using the above-described RSSEV E-gene-specific primers. Cycle sequencing was done with 4 ml purified PCR product, 1 ml 10 mM primer, and the BigDyeTerminator v3.1 Cycle Sequencing Kit from Applied Biosystems (Darmstadt, Germany). Sequencing cycling conditions and purification of products with CentriSep spin columns were performed as described in the manufacturer’s instructions. Resulting sequences were submitted to GenBank (Accession numbers EF566813). Alignments of nt and deduced amino acid (aa) sequences were carried out using Clustal W (BioEdit Version 7.0.2) (Hall, 1999). Phylogenetic analyses were performed using PHYLIP Version 3.5c and maximum parsimony (Felsenstein, 1989).

Results Tick collection and virus isolation In 7 regions in the Kemerovo oblast, a total of 468 adult ticks were collected, with 33–119 ticks per sampled region (see Table 1). A total of 28 pools with 5–33 ticks per pool were created according to the sampling area (1–11 pools per site, see Table 1). In none of the filter eluates inoculated into cell culture, a cytopathic effect could be detected after 2 sub-passages.

Prevalence of tick-borne encephalitis virus RNA in tick samples and minimal infection rates From the 28 direct tick homogenate samples in AVL, 7 RNAs (7/28, 25%) were positive with cycle thresholds (Cts) of 12–20 (Nos. Z6, Z7, Z10, Z12, Z14, Z22, Z23). In comparison, 5 RNAs (5/28, 17.8%) of the 28 Whatman paper eluates reacted at Cts of 26–32 (No. Z10, Z12, Z14, Z22, Z23). One tick pool from Balahonovka was positive, whereas 2 samples from Kemerovo, Tebenki, and Osinovka, respectively, were

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reactive. In 3 of the 7 investigated regions (AngeroSudgens, Andreevka, Chernigovskyi), no RT-PCRreactive tick samples could be found, neither in tick supernatants nor on the Whatman paper eluates. Two of the tick pools (Z6 and Z7) were positive for viral RNA only in tick supernatant in AVL buffer and not in Whatman paper eluate. The minimal infection rates (MIRs) of the ticks of the respective areas were calculated according to the results of real-time RT-PCR, assuming that only one of the ticks per pool was a virus carrier. The MIRs ranged from 0% to 4.1%. In total, the areas could be divided into 3 catagories: areas without infection (Angero-Sudgens, Andreevka, Chernigovskyi), locations with medium MIRs, ranging from 0.1% to 2% (Tebenki 1.8%, Balahonovka 1.2%), and regions with high MIRs 42% (Ostrovka 4.1%, Kemerovo 3.3%).

Sequence and phylogenetic analyses of tick-borne encephalitis virus from the Kemerovo oblast For the 7 positive tick tissue homogenates, amplicons of the expected size (1477 bp) were detected by the RSSE-specific RT-PCR in 6 samples (6/28, 21%; Nos. Z6, Z7, Z10, Z12, Z14, Z22) (Table 1). After direct sequencing of the 1477-bp PCR products, it was possible to utilize a 1361-nt fragment, corresponding to nt 67–1421of RSSEV strain Aina (AF091006), from the 6 samples for further cluster analysis. Alignment of the partial E-gene sequences and deduced amino acid (aa) sequences by Clustal W showed a divergence of up to 4.5% and 0.9%, respectively. None of the nt sequences was identical to sequences of this study or any published sequences. Interestingly, sequences from tick pools from Kemerovo had an nt divergence of 4.4% (aa: 0.9%) but from Osinovka only 0.1% (aa: 0%). BlastN-search revealed closest similarity to TBEV Siberian Subtype with 96–97% nt identity for the strains Aina (AF091006) and Vasilchenko (L40361). For all sequences, the level of the nt sequence divergence to previously described Siberian TBEV in small mammals (Bakhvalova et al., 2006), Irkutsk strains (AB049348-52), and Zausaev (AF527415), was 93–95%. The nt identity to TBEV strains from Estonia, Latvia, Siberia– Novosibirsk region, and Finland was 92–94% and to the Western Subtypes and LILV 84–85%. The amino acid sequence divergence of the Kemerovo oblast TBEV sequences to those from all other analysed Siberian strains ranged up to 1%, to the Far Eastern subtype up to 3%, and to the Western subtype up to 5% (data not shown). Comparison of aligned assumed amino acid sequences revealed the pattern of the Siberian subtype, e.g. a lysine at aa 206 (corresponding to aa 184 of RT-PCR product) also present in the other Siberian

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G. Dobler et al. / International Journal of Medical Microbiology 298 (2008) S1, 94–101 AF310916 Powassan AF253419 LGT AY323489 OmskHF

AF091019 Tblood AF091016 RK1424 AF091013 N132 73 X07755 Sofjin 90 AB049345 VL99-m11 39 AB049346 KH-99-m9 51 61 AB022297 KH-98/10 DQ989336 205 48 AF091008 Crimea 98 AB237187 Kik-629/97 100 100 AB022290 Oshima 5-11 77 AB062063 Oshima 5-10 DQ862460 Glybinnoe 58 AY217093 MDJ-01 99 AY182009 Senzhang 100 AY174188 Senzhang AF091015 Pan DQ401139 Toro AF091010 K23 98 DQ393776 Est3476 AF091005 Absettarov 77 AF091017 Scharl 4 30 U27495 Neudörfl 4 49 AY268437 Kumlinge 39 21 AF091020 ZZ-9 12 AF091007 AlsI 40 2 X75286 Hypr AF091011 KemI 33 DQ393778 Est3509 100 DQ393777 Est3053 71 92 12 DQ393775 Est3051 2 AJ319582 Latvia11686 86 AJ319584 Latvia8369 20 AF091012 LjubI 69 EF113081 235 99 EF113079 166 DQ266084 263 13 AF091014 N256 41 AJ414703 Lith262 DQ235151 TSE 100 X77732 GGE 54 DQ235152 SSE 99 X86784 LILV 58 M94956 Negishi AJ415565 Latvia1-96 DQ451291 Kokkola79 DQ451296 Kokkola118 16 31 DQ451292 Kokkola81 13 DQ451294 Kokkola86 83 59 77 DQ451295 Kokkola102 DQ451293 Kokkola84 25 37 DQ451290 Kokkola39 DQ451288 Kokkola25 100 78 89 DQ451289 Kokkola26 DQ451287 Kokkola9 27 DQ451286 Kokkola8 DQ393773 Est54 66 DQ486861 Ek328 100 DQ393774 Est3535 AF527415 Zausaev AB049349 IR99-1m4 100 77 AB049352 IR99-2f7 95 99 AB049351 IR99-2m7 51 DQ385498 228 89 EF467840 228 100 99 DQ394880 Russ396 60 EF469755 Russ1486 EF469761 Russ1587 27 DQ394877 Russ499 42 EF469750 Russ1189 100 EF469739 Russ766 57 68 EF469760 Russ1577 EF470577 Russ499 Z22 96 Z6 53 EF467846 Russ1937 48 97 EF469763 Russ1658 92 EF469764 Russ1675 85 AY753582 Russ1467 60 AB049348 IR99-1m1 40 AB049353 IR99-2f13 100 AB049350 IR99-2m3 45 Z10 AF069066 Vasilchenko 100 46 AF091006 Aina EF469744 Russ942 64 89 Z14 95 83 Z12 Z7

100

38

99

16

64

Far Eastern Subtype

Western European cluster

Southern cluster

Northern cluster

Siberian Subtype

Fig. 2. Phylogenetic tree of tick-borne encephalitis viruses of the Kemerovo region (Russia, western Siberia), including Far-Eastern, European (cluster Western, Southern, Northern European), and recently published Siberian (Novosibirsk) subtypes. The tree was constructed using 589 nucleotides of the E gene sequence and Powassan virus as outgroup. Accession numbers from GenBank and strains are indicated. The scale bar indicates the number of substitutions per site. Numbers at the nodes indicate the percentage bootstrap support in DNA parsimony.

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strains Irkutsk, Aina, and Vasilchenko. Interestingly, at aa position 234, sequences of tick Z6 and Z22 have an H as the strain Zausaev (AF527415) and some Irkutsk strains (AB049349/51/52). Sequences of Z7, Z10, Z12, and Z14 have a Q at position 234 as the strains Aina and Vasilchenko (data not shown). Phylogenetic analyses of a 589 nt long portion of the E gene showed that TBEV sequences from the Kemerovo area cluster in various distinct groups with other Siberian subtype TBEV: Z7/12/14 group together with TBEV strain Russia 942 (EF469744) while Z10 is more distantly related to that group as well as to the Aina strain. Z6/22 group with other Russian strains which is supported by reasonable bootstrap values (Fig. 2). Most interesting is the observation that the two sequences derived from tick pools collected in the Kemerovo region cluster with two different clades within the Siberian subtype of TBEV.

Discussion Our study first faced the problem to store TBEV and/ or viral RNA at ambient temperature for several days. We chose the stabilization buffer of the Qiagen Viral RNA Mini Kit (AVL buffer) which already gave promising results for stabilization of RNA in our laboratory. A second method for conservation of virus and viral RNA was the absorption of tick eluates on Whatman paper (Guzman et al., 2005). They could conserve viable virus and viral RNA for several weeks. However, the viable virus and also viral RNA decreased with time and were not detectable anymore after 40–90 days. In that study, however, high-titred virus stocks were used. In our study, we found that no viable virus was detected in the filter paper eluates. Filter paper eluate Z23 was not successful in the first TBEV-specific conventional RT-PCRs, but did not last for further assays. Two tick pools (Z6/7) could not be amplified at all from filter paper eluates, but the conventional RT-PCR was possible with the RNA extracted from the ground ticks using the AVL buffer. This discrepancy in tick pools Z6 and Z7 may be because of a low copy number of viral RNA which was conserved by AVL buffer of direct tick supernatant but could not be detected anymore in the filter paper eluate. The real-time RT-PCR seems to have a higher sensitivity than conventional RT-PCR, probably due to amplification of a short fragment (68 nt) by real-time RT-PCR, in contrast to amplification of a long fragment (1477 nt) by conventional RT-PCR. Further, from this brief comparison for us so far, AVL buffer seems the preferable method for conservation of RNA and storage at room temperature for up to 1 week.

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Only limited data on the infection rates of Siberian ticks are available. Although during the last years several studies were published which compared hundreds of TBEV strains from various areas of Siberia (e.g. Irkutsk, Novosibirsk) and far-eastern Asia (e.g. Khabarovsk) (Frolova et al., 1982; Gritsun et al., 1993, 1997, 2003; Hayasaka et al., 1999, 2001; Bakhvalova et al., 2006), the only data available on TBEV infection rates of I. persulcatus ticks are those given by Pakhotina et al. (2002). According to these data, tick infection rates vary between 0.5% and 10.2%, with an average of 3.0%. Although in some of the earlier publications no molecular biological techniques, but virus isolation in suckling mice had been used, these data are in accordance with the data found in our study. Reasons for the somewhat lower MIR in our study (total in all locations tested 1.5%) could be due to the relatively low number of ticks examined in our study. We also did no exact determination of the sensitivity of the real-time RT-PCR used in our study mainly because of the lack of a Siberian virus strain. The primers were designed according to nucleotide sequence data from the GenBank. Therefore, some ticks with low TBEV titres or not perfectly matching primer (and probe) binding sites may have been missed by our detection system. The number of ticks per pool tested in our study varied from 7 to 33 ticks per pool. The high number of ticks in some of our pools was due to the labourintensive manual grinding of the pools. Low virus titres in ticks of some of the pools with higher tick numbers as well as incomplete grinding may attribute to the lower MIR found in our study. Other factors, e.g. in this study overall low sample size in each region and sampling time of the year, may influence the MIR. Thus, the extremely high infection rates of up to 30%, cited in some of the older literature (Korenberg et al., 1999) were not observed in our study. All detected TBEV sequences belonged to the Siberian subtype as shown by the sequences of the E gene. None of the 6 sequences was identical to one another nor to any other TBEV sequence found in GenBank. The variability of nt sequences was 0.5–4.9%. This variation indicates a high mutational potency of the circulating TBEV strains in their respective natural vectors and/or hosts. The Siberian subtype of TBEV can be divided in at least two subgroups based on the nt-sequence divergence within their E gene (Hayasaka et al., 2001; Gritsun et al., 2003). The deduced aa sequence of the partial E protein showed the typical Siberian subtypespecific amino acid patterns (Ecker et al., 1999; Hayasaka et al., 1999; Goto et al., 2002). Comparison of the gained sequences to a recently published sequence pool from Novosibirsk (Bakhvalova et al., 2006) revealed no further change in this specific aa signature. However, the phylogenetic comparison based on the 589-nt fragment available for the Novosibirsk strains

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showed that the partial E gene seems to be quite variable and can be divided in different clusters (Fig. 2). Z22 and Z6 showed a high similarity of the envelope protein to TBEV strains from Irkutsk (Ir99-1m1, ABO49348) regarding an aa exchange in position 234 (Q/H) which is regarded as signature for the Siberian TBEV subtype (Hayasaka et al., 2001). This mutation is supposed to be responsible for a higher virulence in rodents (Hayasaka et al., 2001). Some of the sequences we found were more closely related to strain Aina while one sequence grouped more with strain IR99 (Pogodina et al., 1981; Gritsun et al., 2003). While each of the two tick pools sampled in the Osinovka area clustered within the Aina group, the virus sequences determined from the two positive tick pools found in the Kemerovo sampling area did not cluster together and belonged to the two different subgroups of the Siberian subtype. This demonstrates a co-circulation of divergent TBEV strains in one single natural focus. It is unclear whether this co-circulation itself can have an effect on the evolution of TBEV. The detection of two different subgroups of the Siberian subtype of TBEV is of interest for another reason. Evidence is increasing that viruses of different subgroups can have an impact on the outcome of clinical manifestation in human disease. While the Ainarelated TBEV strains are mainly associated with acute disease, the virus strains of the IR99 subgroup can be found in chronic TBE, which are observed in 1–1.7% of the hospitalized cases in Kemerovo (Poponnikova, 2006) and in 1.8% of the cases in the Ural region (Nadezhdina, 2001). One of our sequences (Z10 from Balahonovka) belongs to the IR99 subgroup which may support the clinical observation of chronic manifestations in children infected in the region (Poponnikova, 2006). So far, no isolates from patients with chronic TBEV infection are available from the Kemerovo region for comparative virological analyses. However, from other regions (e.g. Khabarovsk), an association between chronic TBEV infection and IR99 strains was confirmed (Gritsun et al., 2003). Although the high prevalences of TBEV of up to 4% in adult I. persulcatus will certainly contribute to the high incidence rates known for the Kemerovo oblast, it might well be that the high variability in the circulating TBE viruses is responsible for the very different clinical picture of the disease in the area ranging from subclinical forms and flu-like illness to meningitis, meningoencephalitis, poliomyelitis-like paralysis, and acute encephalitis resulting in death as well as to chronic forms of encephalitis. No matter what, TBEV infection is the leading cause of neurological disorders in western Siberia and further studies are urgently warranted to understand virus virulence as an important factor influencing the clinical manifestation of TBEV infection.

Acknowledgements We are grateful to H. Meyer (Dept. of Special Diagnostics, Bundeswehr Institute for Microbiology, Munich) for his help in designing the primers for the light cycler PCR and for the sequencing.

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