VECTOR-BORNE AND ZOONOTIC DISEASESVolume 6, Number 1, 2006© Mary Ann Liebert, Inc.

Research Paper

Natural Tick-Borne Encephalitis Virus Infection amongWild Small Mammals in the Southeastern Part of

Western Siberia, Russia

VALENTINA N. BAKHVALOVA,1 ANDREY K. DOBROTVORSKY,1,* VIKTOR V. PANOV,1VERA A. MATVEEVA,2 SERGEY E. TKACHEV,2 and OLGA V. MOROZOVA2

ABSTRACT

Infestation of small mammals, including common shrews Sorex araneus L., field mice Apodemus agrarius Pallas,and red voles Clethrionomus rutilus Schreber, with immature Ixodes persulcatus ticks and their infection with tick-borne encephalitis virus (TBEV) were studied in the forest-steppe habitat in the vicinity of Novosibirsk, Russia.Larval ticks parasitize all three host species, but virtually all nymphs were found only on field mice and red voles.Detection of the viral RNA using reverse transcription (RT) with subsequent nested polymerase chain reaction(nPCR) and of viral antigen using enzyme-linked immunosorbent assay (ELISA) revealed a high prevalence ofTBEV-positive animals in both the summer and winter. The proportion of small mammals with hemagglutina-tion inhibition (HI) antibodies was significantly lower than with ELISA-detected antibodies. Taken together, thedata suggest that small mammals may maintain TBEV as a persistent infection throughout the year. Key Words:Rodents—Insectivores—Tick infestation—TBEV infection—Immune response. Vector-Borne Zoonotic Dis. 6, 32–41.

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INTRODUCTION

ENDEMIC TICK-BORNE ENCEPHALITIS VIRUS (TBEV)is spread throughout many of the areas of

temperate forested regions of Europe and Asia,mainly within the habitats of its major arthro-pod vectors–Ixodes persulcatus Schulze and I. ricinus L. ticks (Acarina:Ixodidae) (Lvov et al.1989). The virus causes a variety of clinical man-ifestations, including severe human neurologi-cal infections, with a fatality rate of up to 30%of clinical cases (Gritsun et al. 2003). The lifecycle of the ixodid ticks lasts for 2–5 years andincludes the following stages: imago-egg-lar-vae-nymph-imago. Each stage (except for theegg) is dependent on the tick taking a single

blood meal from a vertebrate host. Larvae andnymphs parasitize a wide range of small mam-mals, including rodents, insectivores, andground-foraging birds. Adult ticks feed onlarger animals such as hares, moose, deer, cat-tle, and goats (Labzin 1985). All the vertebratesare involved in an epizootic process in whichthe phases of the virus spread and persistenceare distinguished (Litvin and Korenberg 1999).TBEV spread in natural populations is accom-plished in different ways. During feeding ofticks on animals, either viremic or nonviremictransmission (NVT) can take place (Labuda etal. 1993, 1997, Nuttall 1998, Randolph et al.1999, Gritsun et al. 2003). In addition, verticaltransstadial and sexual transmission can occur

1Institute of Systematics and Ecology of Animals, and 2Institute of Chemical Biology and Fundamental Medicine,The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

*Deceased.

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among both ticks and warm-blooded hosts(Molnarova and Mayer 1980, Chunikhin andLeonova 1985, Gerlinskaya et al., 1997). TBEVmaintenance mechanisms may also differ. Oneof them was suggested to be persistent infec-tion (Nosek et al. 1961, Kozuch et al. 1990, Nor-man et al. 1999).

In the forest-steppe region of WesternSiberia, the prevalent tick species is I. persulca-tus Schulze (Korenberg 1979). The TBEV infec-tion rate among ticks was previously shown tovary from 0.3% to 3.1% according to bioassays(Dobrotvorsky et al. 1994) and up to 46% basedon reverse transcription–polymerase chain re-action (RT-PCR) tests (Morozova et al. 2002).One should note that strains belonging to theSiberian genetic subtype prevailed among theticks studied (Bakhvalova et al. 2000).

The predominant mammalian host species inWestern Siberia are common shrews Sorex ara-neus L., field mice Apodemus agrarius Pallas, and red voles Clethrionomus rutilus Schreber(Ravkin et al. 1997). The small rodents and in-sectivores were previously shown to be com-petent reservoir hosts for TBEV and capable ofmaintaining the virus as persistent infectionsfor a long time (Kozuch et al. 1963, 1966, 1967,Naumov et al. 1984, Chunikhin and Leonova1985). The virus properties can be changed during the persistence (Smith 1994). To esti-mate the real roles of different species of smallmammals in the TBEV epizootic process, fur-ther investigations including high-sensitivemolecular tests were necessary. The aim of ourresearch was to study predominant species ofsmall mammals in Western Siberia, Russia, ashosts for both ticks and TBEV by means ofmonitoring of their natural populations usingRT–nested PCR (nPCR), enzyme-linked im-munosorbent assay (ELISA), bioassays, andhemagglutination inhibition (HI) tests.

METHODS

Animal trapping

Small mammals were trapped in aspen-birchand pine forests separated by rural areas nearNovosibirsk, Russia (54°49� N, 83°05� E). Linesof 20 live-traps situated at 10-m intervals were

set weekly and checked twice daily from Mayto August from 1995 to 2004. In addition, smallmammals were captured using permanent pit-fall lines (Narita et al. 1995), which were oper-ated from the beginning of June to the end ofAugust. The pitfalls were checked daily at 8a.m. In the winter, small mammals were cap-tured using live-traps only. The trapping ses-sions were performed once a month from Jan-uary to April 1997–2002. The captured animalswere checked for attached tick larvae andnymphs, and dissected to determine species,sex, age, and reproductive status. In total, 2,517common shrews, 536 field mice, and 868 redvoles were examined for the presence of im-mature ticks. A total of 8,455 larval andnymphal I. persulcatus ticks were collected fromthese small mammals.

Detection of TBEV RNA by RT-PCR

Total RNA was isolated from 10% suspen-sions in 0.9% NaCl of brain and/or liver of 48common shrews, 61 field mice, and 99 red volesby phenol-chloroform deproteinization withsubsequent alcohol precipitation (Sambrook etal. 1989, Bakhvalova et al. 2000). The randomhexanucleotide as well as primers correspond-ing to E gene of all TBEV isolates and strainsavailable in GenBank database (�www.ncbi.nih.gov/Genbank/index.html� (5�-AGCCAC-AGGACACGTGTA-3� (E1 [forward]) and 5�-CATCTTGACCATGGGAGA-3� (E2 (reverse))(Bakhvalova et al. 2000); 5�- TCGATGGATGT-GTGGCTTGA-3� (E3 [forward]) and 5�-CT-CATGTTCAGGCCCAACCA-3� (E4 [reverse]);5�-GCGGCCAGATGCCCAACAATGG-3� (E5[forward]) and 5�- TCCCAGGCGTGTTCTCC-TATC-3� (E6 [reverse]) were synthesized in theNovosibirsk Institute of Chemical Biology andFundamental Medicine of the Siberian Branchof the Russian Academy of Sciences. Thescheme of the primer localization is shown inFigure 1.

Reverse transcription was carried out afterpreliminary heating at 70°C for 5 min of a to-tal RNA with 0.4 nmoles of E1 and E2 primersaccording to Bakhvalova et al. (2000) or with0.8 nmoles of random hexanucleotide, incuba-tion at 4°C for 5 min, than at 42°C during 60min in the buffer containing 50 mM Tris-HCl

TBEV INFECTION OF RODENTS AND INSECTIVORES 33

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(pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mMDTT, 0.5 mM dNTP, and 100 U Moloneymurine leukemia virus (MMLV) RT (theNovosibirsk Institute of Chemical Biology andFundamental Medicine of the Siberian Branchof the Russian Academy of Sciences) and fol-lowed by the enzyme inactivation at 95°C for5 min. Final volume of each reaction was 25 �L.

PCR were performed in 20-�L reaction mix-tures containing 67 mM Tris-HCl (pH 8.9), 16.6mM (NH4)2SO4, 2 mM MgCl2, 0.01% Tween-20,200 �M of each dNTP, 5% glycerol, 0.5 �M ofthe TBEV-specific primers (E1�E2, E3�E4 orE5�E6), 2 U Taq DNA polymerase (Institute ofChemical Biology and Fundamental Medicine,Siberian Branch of the Russian Academy of Sci-ences) and 2 �L of cDNA. Amplification was per-formed in “Tercic” Thermal Cycler (DNA Tech-nology, Moscow, Russia). For the inner reactions,2 �L of the outer PCR products were added intothe reaction mixture. PCR fragments were visu-alized under ultraviolet (UV) irradiation afterelectrophoresis in 1% or 2% agarose gels con-taining ethidium bromide. Hi-Lo™ DNA Mark-ers (Minnesota Molecular Inc., USA) were usedas molecular weight markers. PCR with primersE1 and E2 resulted in a fragment of 156 bp long(Fig. 1). Nested PCR with outer primers E3 andE4 and inner primers E5 and E6 produced am-plicons of 1055 bp long (Pletnev et al. 1990) (Fig.1). Samples from 208 animals were tested.

Nucleotide sequences of PCR productswere determined using BigDye TerminatorCycle Sequencing Kit and the ABI PRISM™310 Genetic Analyzer (Applied Biosystems,USA), analyzed by BLASTN (�www.ncbi.nlm.nih.gov/BLAST/�) and aligned usingCLUSTALW (�www.ebi.ac.uk/clustalw/index.html�) (Thompson et al. 1994).

TBEV E protein detection by ELISA

Monoclonal antibody (MAb) 2H3 (0.2 mg/mL)against the TBEV (Sofjin strain) virion surfaceglycoprotein E (Matveev et al. 1989, Matveeva etal. 1998) was immobilized on nitrocellulose mem-branes followed by blocking in 0.1% casein solu-tion. After rinsing the membranes with buffer0.02 M Tris-HCl (pH 7.5), 0.15 M NaCl (TBS) con-taining 0.05% Tween-20 brain or liver suspen-sions from small mammals in dilutions 1:3, 1:9,and 1:27 were incubated with these membranesovernight at room temperature. The membraneswere washed four times with TBS/Tween-20buffer, and antigen–antibody complexes were re-vealed with mouse polyclonal antibodies againstthe TBEV proteins conjugated with alkalinephosphatase followed by membranes washingthree times with TBS/Tween-20 buffer and rins-ing once with 0.1 M Tris-HCl (pH 9.5) with sub-sequent staining using BCIP and NBT. In total,the samples from 175 animals were tested.

BAKHVALOVA ET AL.34

1400 bp

1 100 300 500 700

E gene

M 1

1) E3+E62) E5+E6 1) E1+E2

2 3 4 5 6 7 8C1+ C2+

900 1100

E6E2E5E3 E1 E4

1300 1488

1000 bp

750 bp

500 bp400 bp

300 bp

200 bp

100 bp

156 bp

1055 bp

B

A

FIG. 1. Results of reverse transcriptase–polymerasechain reaction (RT-PCR) detection of the tick-borne en-cephalitis virus (TBEV) RNA in samples from wildsmall mammals. (A) Scheme of localization of theprimers on the TBEV E gene numbered from begin-ning of the gene according to Pletnev et al. (1990). (B)Results of electrophoresis in 2% agarose gel of PCRproducts with E1�E2 primers (lanes 1–4) and nestedPCR products with outer E3�E4 and inner E5�E6primers (lanes 5–8). Lane M, molecular weight mark-ers. Lane C, negative control. Lanes C1� and C2�stand for positive controls for PCR with E5�E6 andE1�E2 primers, respectively. Lanes 1–4, samples iso-lated from brain suspensions of red voles. Lanes 5–8,samples isolated from brain suspensions of commonshrews.

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Bioassay

Detection of pathogenic TBEV was per-formed mainly using 2-week-old ICR labora-tory mice. After cyclophosfan injection as pre-viously described (Bakhvalova et al. 2003),brain and liver from animals were incubated incultural medium in vitro for 2 weeks.

Additional testing using 1–2-day-old suck-ling mice was carried out for samples from 24red voles and eight common shrews. Organsfrom the wild animals were suspended (10%w/v) in Hanks’s solution with 10% fetal bovineserum and antibiotics, and centrifuged at2,500g for 5 min. Supernatants were inoculatedboth intracerebrally and subcutaneously intofour to six laboratory mice. At 7 days post-in-fection, brain suspensions from two mice fromeach group were used for two further passages.The remaining mice were observed for 21–30days before the sera were taken for serologicalanalysis. Identification of the viral isolates wasperformed by RT-nPCR, ELISA, and HI test.

Serological analysis of antibodies of trapped wild animals

Aliquots of 100 �L of blood from dissectedchest cavity and heart (which corresponded to50 �L of serum) were loaded onto ashless fil-ter paper discs 15 mm in diameter. The discswere air-dried at room temperature and storedat �15°C for the serological tests.

HI antibodies against TBEV were detected ac-cording to Clarke and Casals (1958). For thisstudy, the blood samples were eluted from fil-ter disks in borate buffer 0.15 M NaCl, 0.05 MH3BO3, 0.02 M NaOH (pH 9.0). After treatmentwith kaolin and goose erythrocytes, dilutions ofthe studied sera were 1:10. Subsequent twofolddilutions of eluates from 1:10 to 1:640 were usedin the test with 4–8 hemagglutinating units ofTBEV antigen in borate buffer (pH 9.0). Analy-sis of HI antibodies was performed for sera from763 common shrews, 367 field mice, and 552 redvoles trapped from May to August 1995–2004.

For ELISA, both IgM and IgG antibodieswere eluted from filters in 200 �L of TBS bufferovernight.

To detect IgM antibodies, approximately 0.2mg of the TBEV glycoprotein E (kindly providedby Dr. A.S. Karavanov, Institute of Poliomielitis

and Viral Encephalitis, Moscow, Russia) was im-mobilized onto nitrocellulose filters. After block-ing in 0.1% casein, the filters were incubated withthe studied samples in dilutions 1:40, 1:120, 1:360,and 1:1080 overnight at room temperature. Anti-gen–antibody complexes were detected with aconjugate of affinity-purified rabbit antibodiesagainst mouse IgM (H) with horseradish perox-idase. Immune complexes were then stained byH2O2 with 4-chloro-L-naphthol (Aldrich Chemi-cal Company, USA). Serum from a control labo-ratory uninfected mouse was used as a negativecontrol; hyperimmune mouse ascitic fluid, con-taining antibodies against TBEV proteins, wasused as a positive control.

For IgG detection, MAb 2H3 (0.2 mg/mL)against the TBEV (Sofjin strain) E virion surfaceglycoprotein (Matveev et al. 1989) was immobi-lized on nitrocellulose membranes. After block-ing, the membranes were incubated with theTBEV proteins from inactivated purified vaccine(“Virion,” Tomsk, Russia) at a dilution of 1:10overnight at room temperature. The nitrocellu-lose membranes were then incubated with thestudied samples at room temperature for 6 h. Toreveal immune complexes, a conjugate of proteinA with horseradish peroxidase kindly providedby Dr. L.E. Matveev with subsequent staining byH2O2 with 4-chloro-1-naphthol was used. Serumfrom a healthy uninfected mouse served as a neg-ative control; MAb 14D5 against the TBEV E pro-tein (Matveev et al. 1989) was a positive control.

In total, 27 samples from adult red volestrapped during June to August 2001 were ana-lyzed by means of ELISA to detect IgM and IgG.

Statistical analysis

Tick loads were compared using the Mann-Whitney U-test (Sokal and Rohlf 1981). The percentages of animals infected with TBEV orcontaining virus-specific antibodies were com-pared by Student’s criterion (Lakin 1974). Thedifference at p � 0.05 was assumed as significant.

RESULTS

Abundance of tick larvae and nymphs

The seasonal activity of I. persulcatus ticks inSiberia was observed from the beginning of May

TBEV INFECTION OF RODENTS AND INSECTIVORES 35

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to the end of August. In this spring-summer pe-riod, 20.7% of common shrews, 41.2% of fieldmice, and 59.9% of red voles were infested withat least one tick. Larvae comprised 96.5%, 83.1%,and 93.0% of ticks collected from these mammalspecies, respectively. During 9 years of observa-tions, field mice and red voles were more heav-ily infested than common shrews with both lar-vae and nymphs (Table 1; p � 0.001). ImmatureI. persulcatus ticks were not found on small ro-dents or insectivores captured in the period fromthe beginning of September to the end of April.

Prevalence of TBEV-positive insectivores and rodents

TBEV-specific RNA was detected by RT-PCR, and the viral glycoprotein E was detected

using ELISA, in brains and livers of field-cap-tured common shrews, field mice, and redvoles. The majority of positive and negative re-sults of both RT-PCR and ELISA matched forthe samples of brains (�2 � 24.18, d.f. � 1, p � 0.001) and livers (�2 � 26.16, d.f. � 1, p � 0.001).

Based on the detection of TBEV-specific RNAin samples from brains (Fig. 1), the infectionrates were significantly higher in commonshrews and red voles than in field mice (p �0.001 and p � 0.01, respectively; Table 2). Nu-cleotide sequences of the corresponding RT-PCR products belonged to TBEV E gene frag-ment of Far Eastern genetic subtype (Ecker etal. 1999) with a few transitions compared toprototype Sofjin strain (Fig. 2). Similar but

BAKHVALOVA ET AL.36

TABLE 1. ABUNDANCE OF IXODES PERSULCATUS TICKS ON THREE SPECIES OF

SMALL MAMMALS (DATA POOLED FOR MAY–AUGUST 1995–2003)

Mean abundance(ticks per 1 animal � SD)

Species Larvae Nymphs

Common shrews Sorex araneus 0.53 � 1.96 0.02 � 0.18(2,517)

Field mice Apodemus agrarius 1.26 � 2.86 0.26 � 1.11(536)

Red voles Clethrionomys rutilus 3.46 � 6.73 0.26 � 0.77(868)

Total number of studied mammals of each species is shown in parentheses.SD, standard deviation.

TABLE 2. PREVALENCE OF THE TBEV-POSITIVE SMALL MAMMALS (% � SE) (DATA POOLED FOR 1998–2004)

RT-PCR ELISA

Brain Brainand/or and/or

Species Brain Liver liver Brain Liver liver

Common shrews, Sorex araneus1 58.3 � 7.1 62.5 � 8.6 83.3 � 6.8 44.0 � 9.9 70.0 � 10.2 69.2 � 12.8

Red voles, Clethrionomys rutilus1 44.4 � 8.4 33.3 � 10.5 61.9 � 10.8 57.1 � 9.5 6.1 � 9.4 80.0 � 9.22 42.9 � 6.3 N/A — 33.3 � 6.0 N/A —

Field mice, Apodemus agrarius2 19.7 � 5.1 31.2 � 8.2 40.6 � 8.7 16.9 � 4.9 31.2 � 8.2 43.3 � 9.0

Total 39.9 � 3.4 43.5 � 5.4 61.4 � 5.3 33.1 � 3.6 40.0 � 5.7 60.3 � 6.2

1, Winter–spring trapping.2, Summer trapping.TBEV, tick-borne encephalitis virus; SE, sampling error; N/A, not analyzed; RT-PCR, reverse transcriptase–poly-

merase chain reaction; ELISA, enzyme-linked immunosorbent assay.

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slightly lower proportions of infected animalswere found by means of ELISA detecting theTBEV E protein in brain samples. According tothe ELISA results, the difference in the per-centage of the TBEV-positive individuals be-tween common shrews and field mice was alsosignificant (p � 0.05).

TBEV infection of red voles during both thewinter and summer periods was shown to beapproximately the same (Table 2). Similar num-bers of RT-PCR and ELISA positive sampleswere found in different seasons.

Bioassays using 2-week-old ICR laboratorymice did not lead to the recovery of TBEV with-out special treatment with immunodepressant-cyclophosphan and subsequent explantation of

organs from wild animals. However, infectionof 1–2-day-old suckling mice with homo-genates of organs from wild small mammalswas more successful. In 9.3% of samples stud-ied, the clinical manifestations of TBEV infectionwere registered for mice after both first and sec-ond passages. However, the observed symp-toms differed from acute tick-borne encephali-tis. They included languor, lack of appetite orrefusal to eat, slight tremor of extremities withsubsequent recovery, or sudden death. BothTBEV RNA and antigen were detected in brainsuspensions of these experimental mice afterbioassays, but hemagglutinating antigen was ei-ther not detected in all samples, or positives hadlow titers, varying from 1:20 to 1:40. Moreover,

TBEV INFECTION OF RODENTS AND INSECTIVORES 37

FIG. 2. Alignment of the tick-borne en-cephalitis virus (TBEV) gene E fragment nu-cleotide sequences of Far Eastern strainSofjin (GenBank accession number X03870);RNA isolated from red vole and Siberianstrain Aina (GenBank accession numberAF091006). The numbers correspond to nu-cleotide positions of the TBEV E gene (Plet-nev et al. 1990). *Conservative nucleotidesin the last lane. Variable nucleotides areshaded.

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pathogenicity due to TBEV was irregularly ob-served during serial passage of the virus.

Antibodies against TBEV

The virus-specific HI antibodies were annu-ally tested in blood samples from red voles,field mice, and common shrews. The HI anti-body titers for the rodents (from 1:10 to 1:640)exceeded those for common shrews (from 1:10to 1:160). Moreover, HI antibodies were regu-larly revealed in blood from red voles (for eightfrom 10 years of observations), more rarely in

sera from field mice (for five from 10 years),whereas in samples from common shrews, forthree from 10 years only. During the study pe-riod, the annual proportion of animals with HIantibodies varied: among red voles, from 0% to10.2%; for field mice, from 0% to 4.3%; and forcommon shrews, from 0% to 2.6%. Averagepercentage of red voles with HI antibodiesagainst the TBEV (data pooled for 10-year pe-riod [1995–2004]) was significantly higher thanthat with common shrews and field mice (p �0.001, p � 0.05, respectively; Table 3). Oneshould note that the proportion of red voles

BAKHVALOVA ET AL.38

TABLE 3. PERCENTAGE (% � SE) OF SMALL MAMMALS WITH HEMAGGLUTINATION INHIBITION (HI) ANTIBODIES AGAINST

THE TBEV (DATA POOLED FOR MAY–AUGUST 1995–2004)

Demographic category

Species Overwintered Young-of-the-year Total

Common shrews, Sorex araneus 1.26 � 0.9 0.5 � 0.3 0.65 � 0.3(154) (609) (763)

Field mice, Apodemus agrarius 1.59 � 1.1 1.68 � 0.8 1.65 � 0.7(126) (241) (367)

Red voles, Clethrionomys rutilus 7.5 � 1.7 2.86 � 0.9 4.87 � 0.9(239) (313) (552)

Sample size is given in parentheses.SE, sampling error; TBEV, tick-borne encephalitis virus.

FIG. 3. Correlation between abundance of Ixodes persulcatus on different species of small mammals and percentageof the animals with hemagglutination inhibition (HI) antibodies against the tick-borne encephalitis virus (TBEV).

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with HI antibodies was significantly higher foroverwintered animals compared to young-of-the-year rodents. Based on the long-term data,the proportion of small mammals with HI an-tibodies against TBEV correlated with thespecies-specific tick infestation pattern (Fig. 3).

Taking into account high frequencies of bothTBEV RNA and antigen detection in organs ofsmall mammals, and the low proportion of an-imals with HI antibodies, it seemed reasonableto compare immune response using both HI testand ELISA. According to ELISA data, most redvoles (81.5 � 7.5%) had IgM antibodies againstthe E protein, but IgG antibodies were detectedmore rarely (17.2 � 7.0%). Titers of IgM anti-bodies varied from 1:5 to 1:90; for IgG antibod-ies, it varied from 1:5 to 1:15. HI antibodies withtiters of 1:10–1:20 were found in three sera alsopositive for IgM but not for IgG. It is interest-ing to note that, for negative samples withoutIgM, other antibodies (including those detectedby HI test or ELISA) were not revealed.

DISCUSSION

Our data on the distribution of I. pesulcatuslarvae and nymphs confirmed the previousfindings on species-specific infestation patternsof vertebrates (Labzin 1985, Okulova 1986,Talleklint and Jaenson 1997, Stanko and Mik-lisova 2000). The different tick load on a mam-mal species and the ratio of larvae and nymphscould be caused by ecological differences of thereservoir hosts and their ectoparasites, physio-logical properties and protective capacities ofdifferent species of the vertebrates (Randolf1979, Okulova 1986).

Pathogenic TBEV strains may be isolatedfrom both ticks (Naumov and Gutova 1977, Ko-renberg and Kovalevskii 1999) and from verte-brate animals (Pogodina et al. 2004) using sus-ceptible laboratory animals or cell cultures.Both TBEV RNA and E antigen were often de-tected in samples from ticks (Morozova et al.2002) and small mammals (Fig. 1, Table 2).Based on direct sequencing of the PCR prod-ucts of E gene fragments, the viral RNA iso-lated from small mammals could be belongedto Far Eastern (Figs. 1 and 2) and Siberian ge-netic subtypes (Pogodina et al. 2004).

TBEV-induced antibodies were detected inthe majority of sera from the red voles collectedduring different seasons. Previously, the TBEVneutralization test had also revealed virus-neu-tralizing antibodies in 3.5–78.0% of small mam-mals in natural populations (Nikiforov et al.1961, Okulova 1986). However, the percentageof summer-captured small mammals with HIantibodies against TBEV was below 10% (Table3), which corresponded to data for other en-demic regions: 12.3–14.7% in European Russia(Kucheruk et al. 1965) and 4.1–25.8% in CentralSiberia (Gutova and Naumov 1987, Kislenko etal. 1994). Moreover, experimental infection ofnarrow-sculled voles Microtus gregalis with 5.3lgLD50/0.1 mL of strain Sofjin has shown thatthree out of 11 animals failed to produce HI an-tibodies and complement-binding antibodieswithin 45 days (Pchelkina et al. 1969). There-fore, the immune response against TBEV in nat-ural reservoir hosts does not necessarily in-volve the formation of HI antibodies.

The detection of TBEV-infected small mam-mals in winter and early spring suggests thepossibility of long-term persistence of thepathogen in wild rodents. This is consistentwith previous finding for TBEV in the formerCzechoslovakia (Kozuch et al. 1967) and the ob-servation that experimental subcutaneous in-oculation of bank voles Clethrionomys glareoluswith TBEV isolated from I. persulcatus ticks re-sulted in the viral antigen persisting in thebrain of the animals for up to 10 months post-infection (Okulova 1986). Long-term TBEV sur-vival in the presence of virus-specific antibod-ies might be explained by sub-neutralizingconcentrations of polyclonal antibodies en-hancing virus penetration into host monocytesvia Fc-receptors (Thomas 1993) and/or induc-ing the selection of escape mutants (Steinhauerand Holland 1987). Moreover, since antibodiescannot penetrate infected cells, neutralizationof intracellular TBEV might be incomplete.

Prevalence of TBEV-infected ticks is knownto increase during development from the lar-val to imago stage and it can be hardly ex-plained by viraemic transmission since wild rodents do not develop prolonged viraemia(Korenberg and Kovalevskyi 1977, Naumov etal. 1984). However, small rodents and insec-tivores are known to support NVT of fla-

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viviruses between co-feeding ticks (Jones et al.1987, Alekseev and Chunikhin, 1990, Labuda etal. 1993) even if warm-blooded animals are im-mune (Jones et al. 1987, Labuda et al. 1997).Based on these data, including a high preva-lence of the TBEV-positive small mammals, dif-ficulty to recover pathogenic TBEV from itsnatural mammal hosts in comparison withstrains from ixodid ticks, and the weak virus-specific immune response, one might concludethe existence of TBEV persistent infections formajority of wild animals in endemic regions.

ACKNOWLEDGMENTS

The work was supported in part by SiberianBranch of the Russian Academy of Sciences forthe Integration of Basic Research (grant N 51)and Russian Foundation of the FundamentalSciences (REFI) (grants N 01-04-49535 and N 04-04-48545).

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Address reprint requests to:Dr. Olga Morozova

Institute of Chemical Biology and Fundamental Medicine

Siberian Branch of the Russian Academy of Sciences

Lavrentyev’s Avenue 8630090 Novosibirsk, Russia

E-mail: mov@niboch.nsc.ru

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