ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2009, Vol. 24, No. 4, pp. 183–188. © Allerton Press, Inc., 2009.Original Russian Text © N.V. Fomenko, O.V. Stronin, M.N. Khasnatinov, G.A. Danchinova, J. Bataa, N.A. Gol’tsova, 2009, published in Molekulyarnaya Genetika, Mikrobi-ologiya i Virusologiya, 2009, No. 4, pp. 18–22.

183

INTRODUCTIONLyme borrelios (LB), a natural transmissive disease

in terms of the extent of its distribution and frequency,occupies a significant position among natural infections[1]. The causative agents of LB, spirochetal complex

Borrelia burgdorferi

sensu lato

(s.l.)

, are transmitted tohumans by ticks of the genus

Ixodes.

Two species

Bor-relia afzelii

and

Borrelia garinii

exist in the territoriesof the Russian Federation [1]; they are widespread, cir-culate in different ecosystems, and have a great numberof reserve hosts and carriers. The

B. garinii

populationis characterized by genetic heterogeneity [2, 15].

During its life cycle, the antigen component of theBorrelia complex

B. burgdorferi s.l.

is subject to signif-icant variations. Even among the same species of Bor-relia, variability was observed in surface protein com-ponents, especially in OspA and OspC. OspA protein isthe main surface protein in the Borrelia complex

B. burgdorferi s.l.

[11]. The

ospA

gene that codes OspAprotein is located in the linear plasmid lp54 and isfound within the same reading frame with the

ospB

gene. For some isolates of the

B. garinii

species, thehigh degree of homology of the

ospA

and

ospB

geneshas been shown [6]. The most variable region of the

ospA

gene located at the 3'-end of gene differs in termsof its length and the number and position of nucleotidesubstitutes [20, 21].

It has been shown that the degree of homology forOspA protein from different species of the complex

B. burgdorferi s.l.

is 83.3% [22, 24]. Currently, thesecondary structure of the OspA has been determined;the protein has an unusual structure that consists of21 repeated antiparallel

β

-sheets followed by a single

α

-helix. The carboxyl end domain forms 14–21

β

-sheetsthat include the highest hypervariable protein segment[13, 20, 21]. OspA protein plays a significant role inadhesion, colonization, and the survival of borrelia inthe midguts of the tick [27]. The TROSPA receptor(tick receptor for OspA) for OspA protein, whichenables the attachment of spirochetes to the inner sur-face of an midgut, has been identified [17]. It has beenshown that OspA protein aids adhesion to endotheliumand neurons. Meanwhile, the interaction with surfaceproteins of neurons is considered to be one of the pos-sible mechanisms of pathogenesis during neuroborre-liosis [19].

The

ospA

gene of

B. garinii

is considered to have themost heterogenic sequence. Based on nucleotidesequence analyses of the

ospA

gene of the borrelia com-plex

B. burgdorferi s.l.

, three groups of

B. garinii

havebeen isolated [15]. Previously, based on the genetic andimmunological heterogeneity of the OspA protein fromEuropean strains, seven sereotypes were isolated, fiveof which (the 3rd–7th types) were

B. garinii

[25]. How-

EXPERIMENTAL WORKS

Heterogeneity of the

ospA

Gene Structure from Isolates of

Borrelia garinii

and

Borrelia afzelii

from Western Siberia and Mongolia

N. V. Fomenko

a

, O. V. Stronin

b

, M. N. Khasnatinov

c

, G. A. Danchinova

c

, J. Bataa

d

, and N. A. Gol’tsova

b

a

Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Prospect Lavrent’eva 8, Novosibirsk, 630090, Russia

b

Virion Scientific Production Association (NPO Microgen), Tomsk, Russia

c

Institute of Epidemiology and Microbiology, Irkutsk Medical University, Irkutsk, Russia

d

National Center for Communicable Disease, Ulan Bator, Mongoliae-mail: fom@niboch.nsc.ru; nataliyaf@ngs.ru

Received March 12, 2009

Abstract

—In this work, identification and analyses of 48 full-length sequences of the

ospA

gene isolates of

Bor-relia garinii

and

Borrelia afzelii

from Western Siberia and Mongolia has been made. It was shown that

B. garinii

isolates was of its high genetic heterogeneity of the

ospA

gene. Four genetic groups of the

ospA

gene from the

Ixodes persulcatus

tick collected in of Western Siberia and Mongolia were defined. The basic differences in thegenetic variants of the

ospA

gene considered are seen in regions which code for antibody determinants of thheOspA protein.

Key words

: heterogeneity,

ospA

gene,

Borrellia garinii

,

Borrellia afzelii

, Western Siberia, Mongolia

DOI:

10.3103/S0891416809040041

184

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 24

No. 4

2009

FOMENKO et al.

ever, during analyses of

B. garinii

isolated from the FarEast and Japan, nine serotypes were determined, whichindicates the possibility of a larger genetic variety ofthe OspA from

B. garinii

(14). Based on the high vari-ability of the OspA protein, the study of regional fea-tures of heterogeneity of the gene that codes OspA pro-tein is highly significant.

The aim of this work was to study the genetic heter-ogeneity of the

ospA

gene of isolates

B. afzelii

and

B.garinii

circulating in the territories of western Siberiaand Mongolia.

MATERIALS AND METHODS

In this work, 22 isolates of

B. afzelii

and 26 isolatesof

B. garinii

(see table) were used; samples wereobtained from the internal organs of the tick

Ixodes per-sulcatus

collected from the territories of Novosibirskand the Tomsk region, as well as from the territories ofthe Selenge Aimag in northern Mongolia. Dependingon the region where the ticks were captured, each iso-late was given the following designation for easy iden-tification: Nov refers to isolates from Novosibirskregion, Tom refers to isolates from Tomsk, and Mngrefers to isolates from Mongolia (see table). Bacterio-logical culturing was conducted as described previ-ously by A. Barbour [5].

The isolation of Borrelia DNA was carried out usingthe standard method with guanidinium thiocynate fol-lowed by phenol-chroloform extraction or using com-mercial kits (Vector-best, Novosibirsk, Russia).

For the amplification and determination of the com-plete nucleotide sequence of the

ospA

gene, we designedthe following primers: forward pA3 5'-ctatttgttatttgt-taatcttatac-3', reverse pA4 5'-gcaaatcctagtaaatattgtttc-3',and pA6 5'-attggctaatgccttgcaggatg-3'. Nucleotidesequences corresponding to these primers are located innontranslated regions, which enables one to determinethe complete nucleotide sequence of the coding regionof the

ospA

gene. The amplification of the gene wascarried out in the following condition of 35 cycles:

94

°

C for 10 s, 50

°

C for 15 s, and 72

°

C for 50 s in thecondition stated previously [4].

The complete nucleotide sequence of the

ospA

gene(819–828 bp) was determined using primers that corre-spond to the fragments analyzed by PCR and the BigDye

TM

Terminator Cycle Sequencing Kit (Applied Bio-systems, USA) DNA Sequencing Center of the Sibe-rian Branch of the Russian Academy of Sciences(http://www.sequest.niboch.nsc.ru).

A comparison and analyses of the determined nucle-otide sequence of

B. burgdorferi s.l.

was performed usingthe BlastN (http://www.ncbi.nlm.nih.gov/BLAST) andCLUSTALW (http://www.ebi.ac.uk/clustalw) programs.Using the method of joining neighbors, a dendrogrammwas constructed using the MEGA 4.0 program [12].The reliability of the constructed dendrogram was eval-uated using a butstrae analyzer. The butstrae index wascalculated for a total number of 1000 repeats.

The determined sequences of the

ospA

gene wereentered into the GenBank database with the followingnumbers: DQ479275-DQ479312 and EU635987-EU635992. The numbers of known GenBank sequencesused for the comparison are presented in Fig. 1.

RESULTS AND DISCUSSION

In this work, 48 complete nucleotide sequences ofthe

ospA

gene were determined, of which 22 sequencesbelonged to

B. afzelii

strains and 26 belonged to

B. garinii

strains. Previously, based on analyses of the5S-23S RNA intergenic spacer sequence, we showedthat 11 isolates of

B. garinii

belonged to the subgroup20047, nine belonged to

B. garinii

subgroup NT29,while the group 5S-23S RNA intergenic spacersequence of six were not determined (see table) [4]. Ithas been shown that isolates Nov9906 and Tom1003have an insertion, while isolates Nov7006, 14506,15506, and 54306 have a deletion in the 5S-23S RNAintergenic spacer sequence. Based on analyses of the16S gene of rRNA, these isolates are attached to

B. garinii

[3, 4].

Group membership of isolates from

B. garinii

and

B. afzelii

specie

Species Group Isolates

B. afzelii

VS461 Mng702, Mng4302, Mng6702, Tom3401, Tom603, Tom703, Tom1503, Tom2303, Tom2403, Tom2803, Tom3703, Tom4703, Tom5403, Tom6303, Nov1105, Nov3005

NT28 Mng3602, Tom1303, Nov11506, Nov12406

NT28 with extra TTAA sites Tom1103, Tom2504

B. garinii

NT29 Mng1602, BgVir-1, Tom5102, Tom3803, Nov405, Nov2005, Nov2006, Nov1006, Nov14606

20047 Mng4702, Tom3101, Tom5202, Tom2903, Nov105

20047 with an insertion Tom203, Tom303, Tom1805, Tom3005, Tom7105, Nov3305

not determined Tom1003, Nov9906, Nov7006, Nov15506, Nov14506, Nov54306

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY

Vol. 24

No. 4

2009

HETEROGENEITY OF THE

ospA

GENE STRUCTURE FROM ISOLATES 185

To determine the nucleotide sequences of the

ospA

gene, primers pA1/pA2 were designed. However, whenPCR was carried out with these primers, DNA isolatesTom1805 and Tom7105 yielded negative results. Forthese isolates, the amplification of the inner fragment ofthe gene was carried out using primers pA1 5'-gggaatag-gtctaatattagcc-3' and pA2 5'-cactaattgttaaagtygaagt-3';data were published previously [18]. Sequences fromisolates Tom1805 and Tom7105 were identical to eachother. When compared with sequences of the

ospA

genedeposited in the GenBank database, they were verysimilar to sequences of

B. garinii

Jem4 isolated inJapan from skin biopsies of LB patients [23]. The3' nontranslated region of the

ospA

gene of this straindiffers from the sequences typical of the

B. garinii

strain. Based on the sequence of the Jem 4 strain,primer pA6 was designed. Analyses were conducted ofisolates, Tom1805, and Tom7105 using the pair ofprimers pA3 and pA6; furthermore, the obtained PCRfragments were of the expected length and thesequences of the

ospA

gene from these two isolateswere determined.

The determination of the complete nucleotidesequences of the

ospA

gene from 26 strains of

B. garinii

indicated that the length varies from 819 to 828 bp The

ospA

gene sequence of

B. garinii

is characterized by, notonly a large number of nucleotide substitutions, but alsothe presence of the deletion and insertion of 3–6-bplengths. It is worth noting that, in spite of the presence ofthe high heterogenicity of the

ospA

gene sequence, some

Khab-2119 AY260462

Nov1006Nov2005

B. garinii

K241 AB007107

B.garinii

JEM5 U93708

Mn g 4702Tom 5102, 3803, Nov 14606

B. garinii

JEM2 U93708

B.garinii

U047 AB007106K hab-24 AY339605

Mng 1602

Khab-448 AY260454Tom 1003, Nov 9906Khab-560 AY260461

Nov405

B. garinii

K269 AB007109

Tom 3101

B. garinii JEM6 U93709B. garinii F518 AB007101

Khab-506 AY339611Nov2006BgVir-1

B. garinii T25 X80254B. garinii JEM4 AB001041Tom 1805, Tom 7105B. garinii pBr X80256

Nov 105Khab-616 AY502601Tom 2903

Nov 7006, 14505, 15506, 54306B. garinii pBi X80257B. garinii TRO X65598

Nov 3305, Tom 3005Tom 203, 303B. garinii WABSou X85441B. garinii PHei X80251

Khab-489 AY339609Tom 5202B. garinii DK29 X63412B. garinii K48 X62624

B. garinii Goe 2 X60300B. garinii N34 Y10894

B. garinii B29 M88764B. valasiana VS116 AF095940

B. turdi Ya501 AB016975B. bissetii 25015 AF230516B. burgdorferi B-31 X14407B. burgdorferi 297 X85442

B. spielmanii AF102057B. afzelii VS461 Z29081Nov 3005, 11506, Tom 603, 703, 1503, 2303Tom 3401, 2403, 2803, 3703, 4703, 5403B. afzelii XJ23 U78301Khab-625 AY502599Nov 1105, 12406, Mng 3602, Tom 1103, 1303Mng 702, 4302, 6702, Tom 6303, 2504

B. japonica H014 (Y10863)B. lustitaniiae PotiB2 Y10838

8962

93

8986

100

98

375279

79

99

73

33100

24

88

88

55

80

51

9988

9957

100

99

100

5591

5658 67

100

100

97

88

100

7731

49

97

3679

99100

100

100

I

II

III

IV

70

1367

0.02

Fig. 1. Dendrogram constructed based on complete nucleotide sequence of the ospA gene of borrelia complex B. burgdorferi s.l.using NJ method.

186

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 24 No. 4 2009

FOMENKO et al.

isolates have a 100% degree of homology. It was shownthat the level of homology of the ospA gene of complexB. burgdorferi s.l. from different species is 83.3–100%[22, 24]; meanwhile, the 3'-end of gene is different, espe-cially in terms of its high variability. The degree ofhomology of the ospA gene from our strains was 83.1–100% for the B. garinii species and 99.2–100% for theB. afzelii species.

The construction of dendrograms based on nucle-otide sequences of the ospA gene showed that dendro-grams of different topologies are generated using fourmethods of analysis (UPMGF, NJ, MP, and ME) [8]. Inthis work, we chose the dendrogram constructed usingthe NJ method because it does not need the change forconstant speed along the taxons; hence, in our view, thisenables one to correctly reconstruct the tree topology.We analyzed 108 full-length sequences of the ospAgene available in the GenBank database. For a visualdemonstration of dendrogram construction (see Fig. 1),previously determined ospA gene sequences that weremost similar to the sequences under study were chosen.For comparison, we used sequences of the ospA genefrom five strains of B. garinii that belogned to differentserotypes (3rd–7th types), including strain pBr form the3rd serotype, TRO and pBi from the 4th serotype, pHeifrom the 5th serotype, N34 from the 6th serotype, andT25 from the 7th serotype (see Fig. 1)[25].

It was shown that B. garinii isolates form four easilydistinguishable subclusters (support index is more than 95)(see Fig. 1). Subcluster I, which includes five isolatesfrom western Siberia and two isolates from Mongolia isfarther from the other subclusters formed by sequencesof the B. garinii species (see Fig. 1). Six isolates of thissubcluster are isolates from the B. garinii subgroupNT29 and one B. garinii subgroup 20047 (see table).Together with our isolates, strains obtained from theKhabarovsk krai, Japan, and China preferentiallybelong to subcluster I. Not even a strain from the fivepreviously isolated OspA serotypes belonged to thissubcluster. Apparently borrelia with this ospA genestructure is endemic in Asian parts of the continent andis not found in Europe. This is possibly due to the pecu-liarity of the TROSPA receptor of tick OspA as themain surface protein responsible for the adaptation ofborrelia to the tick organism [16, 17].

Isolates belonging to subcluster II are also groupedwith isolates from Khabarovsk krai, Japan and China(see Fig. 1). Six of our strains, two of which (Nov9906and Tom1003) have insertions in their intergenic spacersequences of 5S–23S rRNA belongs to subcluster II(see table) [4]. The degree of homology of the ospAgene sequences of isolates Nov9906 and Tom1003 was100%. Comparing them to sequences previously pub-lished in GenBank, showed that with a high degree ofhomology (99%), they corresponds to the sequences ofKhab-448 strains, isolated from I. persulcatus fromKhabarovsk krai. This subcluster did not contain strainsbelonging to the five types of the OspA serotypes.

Subcluster III has the least number of isolates fromwestern Siberia; it includes only three (11.5%) of theexperimental isolates that belong to B. garinii subgroup20047 (See Fig. 1); isolates from Japan and Europe alsobelong to this subcluster. Isolates Tom1805 andTom7105 from LB patients in Japan and Germany,respectively, were similar to pathogenic strains B. gari-nii pBr and B. garinii JEM4, correspondingly [20, 23].Furthermore, strains of T25 B. garinii from I. ricinustick isolated in Germany also belong to this subcluster.pBr and T25 strains have previously been identified asthe 3rd and 7th serotypes of B. garinii, respectively[23]. Isolates with the type-III ospA sequence were notfound in the territories of the Khabarovsk krai [14],which explains the rare occurrence of borrelia with thisospA gene sequence [20,21].

Subcluster IV with the highest numbers containsisolates from Europe, Khabarovsk krai, and westernSiberia, but does not contain a single isolate from Japanor China. Ten (20.1%) of isolates obtained in this workbelong to this subcluster, all of which belong to theB. garinii subgroup 20047 (see Fig. 1). Strains pBi,TRO, pHei, and N34, which represent the 4th, 5th, and6th B. garinii serotypes, respectively, belong to sub-cluster IV [26]; four isolates, i.e., Nov7006, 14506,15506, and 54306, with deletions in the intergenicspacer sequences of 5S–23S rRNA (see table) alsobelong to subcluster IV [3, 4]. The degree of homologyof the ospA gene sequences for these isolates was100%. Comparative analyses of sequences for thesefour isolates with the remainder showed a high degreeof homology (98.2%) with isolate sequence Khab-616from the Khabarovski krai. The determined heteroge-neity of B. garinii ospA gene sequences correspondswith the previous demonstration of its existence inB. garinii serotypes in the territories of western Siberia,the Far East, and Japan [14].

The determination of 22 complete nucleotidesequences of the ospA gene of borrelia B. afzeliishowed that the length of all analyzed sequences was822 bp The degree of homology of the obtainedsequences is 99.2–100%, 11 isolates of which haveidentical ospA genes. For nine sequences, two nucle-otide substitutions at positions 114 and 150 bp wereidentified with only the substitution at position114 leading to the substitution of the amino acidmethionine for isoleucin (M I). A comparison ofthe nucleotide sequence of the ospA gene fromB. afzelii isolates with previously published sequencesshowed that 11 of them were identical to the ospA genesequence of B. afzelii Khab-625 and XJ23 isolatedfrom I. persulcatus in Khabarovsk krai and China,respectively (see Fig. 1). B. afzelii isolates were moresimilar to isolates from Japan and Khabarovsk krai thanto isolates from Europe (see Fig.1, B. afzelii VS461).An interesting peculiarity of Asian isolates of B. afzeliiis their geographical homogeneity. Strains 2–3000 kmapart have identical ospA gene sequences; this struc-tural stability of the ospA gene in B. afzelii as compared

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 24 No. 4 2009

HETEROGENEITY OF THE ospA GENE STRUCTURE FROM ISOLATES 187

to B. garinii was previously indicated for borrelia iso-lates from Europe [23].

The amino-acid sequence of the OspA protein wasdetermined based on the 48 nucleotide sequences of theidentified ospA gene. It was shown that the length of theprotein of B. afzelii is 274 aa and varies from 273 to276 aa for B. garinii. A comparison of the determinedand previously known amino-acid sequence of theOspA protein showed that the amino terminus of theprotein is more conservative than the carboxyl terminus(data not shown). Previously, it was shown that the pro-tein segments, 85–103 aa and 229–248 aa are importantfor the attachment of borrelia to the intestines of ticks [16].Analyses of our OspA protein sequence at 85–103 aa seg-ment yielded seven hypervariable positions. Most ofthe substitutions did not lead to changes in the class ofamino acid; only a few isolates had substitutions thatled to a change in the charge (data not shown).

The most variable segment was 229–248 aa with onlyeight conservative positions for all analyzed sequences(Fig. 2). Interestingly, some positions in the segment(229–248 aa) have conservative amino-acid residueswithin one or a few subclusters, but differ from othersubclusters. For example, isolates belonging to subclusterI have isoleucin (I) at position 232 and phenylalanine (F)at position 235, while all other isolates considered had atthreonine (T) and leucin (L) these positions, respectively(see Fig. 2). For other positions, variability is onlyobserved within one particular subcluster with the rest ofthe sequences being conservative, e.g., position 229 ofsubcluster II (see Fig. 2). It is possible that the heteroge-neity of the OspA protein at this functionally importantsegment is significant for the adaptation of borrelia to theinternal environment of ticks.

We compared the amino-acid sequences of theOspA protein of B. garinii species isolated from west-ern Siberia and Mongolia with sequences of B. burg-dorferi s.s. strains B31 (see Fig. 2). For comparison, wechose the region 180–273 aa with previously deter-mined localized antigen determinants for monoclonalantibody LA-2 [7, 9]. Also, a comparison was con-ducted with B. afzelii strains P/Gau strain based onwhich monoclonal antibody mAb336 was obtained [10].Previously, the differences in the reaction of LA-2 andmAb336 monoclonal antibodies with OspA protein of

15

Nov1006

Nov2005

Tom

5102

Mng4702

Mng1602

Tom

3101

BgV

ir-1

Nov405

Nov2006

Tom

1003

Nov105

Tom

7105

Tom

3005

Tom

2903

Tom

203

Tom

5202

Nov7006

B31

P/G

au

181

191

201

211

221

231

241

251

261

271

III

IVIII

16

17

18

19

20

21

Fig. 2. Comparison of amino-acid sequences of the OspAprotein segment that forms carboxy terminal barrel domainspecific to OspA protein from B31 strain of B. burgdorferi s.s.Arrows below show β sheets, below are correspondingnumbers of β sheet. Curly brackets on the left indicate theB. garinii subclusters of the isolates studied. One star indi-cates OspA protein sequence of strains B. burgdorferi s.s,two stares indicates P/Gau B. afzelii. Light-grey back-ground indicates epitopes for monoclonal antibodies LA-2,Dark-grey background indicates epitopes for monoclonalantibodies mAb336. Stars below indicate the position of allconservative sequences studied

* **

188

MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY Vol. 24 No. 4 2009

FOMENKO et al.

the borrelia species B. afzelii and B. burgdorferi s.s., aswell as with some strains of B. garinii, were shown.

Analyses of 17 variants of amino-acid sequencesshowed the presence of a greater number of substitu-tions at the location of LA-2 antigen determinants, aswell as mAb336. In addition to the substitution, a dele-tion of 1 aa was observed at position 209 for 13 of theanalyzed sequences (sequence of B. burgdorfero s.s.strain B31). The insertion of 1 aa at position 252 hasbeen determined for three variants. Most of the substi-tutions lead to a change in the class of amino acids; insome cases, there is a change in the charge of aminoacids. Many of these substitutes are found in the firstepitop (231–236 aa) of the mAb336 antibody. Also, itwas shown that positions 258 and 266 aa are importantfor the specific interaction with mAb336; when leucinis present at this position, the interaction does notoccur. This was demonstrated with two EuropeanB. garinii strains K48 and DK29 [9]. In our sequences,leucin observed at position 258 or 266 in three of theisolates enabled us to assume that there are differencesin the immunological properties of the OspA protein inthese isolates. The results of comparing sequences ofB. garinii species at epitope locations confirms the pre-vious assumption on the impossibility of obtaining awider protective effect from vaccines based on a singlestrain [24].

Therefore, based on the analyses, a considerabledegree of heterogeneity has been shown for the ospAgene sequence. Four genetic groups of B. garinii wereisolated. Most differences in ospA gene sequences inthe four groups are at determinant locations in the anti-gen. The sequences of B. afzelii species have higherdegrees of homology. Since the most variable segmentcodes protein regions that include antigen determi-nants, when OspA protein is used for diagnostics, it isimportant to consider the possible differences in itsimmunoreative properties.

ACKNOWLEDGMENTS

This study was supported by the integrated projectof the Siberian Branch of the Russian Academy of Sci-ences no. 10, “Dynamics of the Ecosystem of Novosi-birsk Akademgorodok: Results of 50 Years ofResearch,” integrated interdisciplinary grant no. 24Siberian Branch of the Russian Academy of Sciences(2006–2008), “Role of Microorganisms in Functioningof Living Systems: Fundamental Problems andBioengineering Supplements.”

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