Effect of Glycosaminoglycans on Pathogenic Properties Far-Eastern Tick-Borne Encephalitis Virus.

We studied the effect of sulfated glycosaminoglycan on the infection properties of high-virulence Dal'negorsk strain and low-virulence Primorye-437 of tick-borne encephalitis virus. Differences in reproductive activity of these strains and their tropism to the target cells were revealed. Glycosaminoglycan reduced pathogenetic activity of high-virulence strain in vitro, but had no effect on low-virulence strain. The interaction of imperfect virus particles of non-pathogen strain with the glycosaminoglycan led to their accumulation in cell, but in the culture medium of SPEV cells infected with experimental and control samples, accumulation of virus particles did not differ. The results on activity of glycosaminoglycan binding with strains differing by their biological and molecular genetic characteristics can be used to assess their pathogenic potential.

[Genetic diversity of cyanophages of the Myoviridae family as a constituent of the associated community of the Baikal sponge Lubomirskia baicalensis].

Molecular-genetic analysis of cyanophages of the family Myoviridae from the associated community of the endemic Baikal sponge Lubomirskia baicalensis was carried out based on the g20 gene fragment. A large cyanophage diversity according to the g20 marker gene was found in the sponge. The Baikal sponge cyanophages were shown to be similar to those inhabiting plankton. Moreover, specific cyanophage groups that are significantly different from all of the known groups inhabiting the Lake Baikal were revealed.

[Investigation of nuclear and mitochondrial DNA polymorphism in closely related species of endemic Baikal sponges].

In order to investigate the phylogenetic relationships among endemic Baikal sponges of the Lubomirskiidae family and to clarify their taxonomy, the sequence variation at the silicatein-encoding gene and the mtDNA intergenic region was examined. Phylogenetic analysis of the silicatein α1 gene exonic regions in six freshwater sponge species revealed considerable interspecific variability of this region. The analysis performed did not support the monophyly of the Lubomirskia and Baikalospongia genera. The mDNA region between the COX2 and ATP6 genes was examined in five species from the Lubomirskiidae family, including multiple samples for analyzing intraspecific variations. According to the data obtained, the Baikalospongia genus was monophyletic with respect to Lubomirskia, while B. bacilifera and B. recta did not form monophyletic groups. Molecular data indicate that taxonomy of Lubomirskiidae should be revised. It was demonstrated that, in endemic Baikal sponges, the accelerated evolution was accompanied by an increase in the length of the noncoding regions in both mitochondrial and nuclear genes.

[Freshwater sponge silicateins: comparison of sequences and exon-intron structure of genes].

Siliceous sponge spicules contain silicateins--proteins taking part in biogenic silica precipitation and determination of the spicule morphological features. The exon-intron structure of four silicatein-alpha isoforms: -alpha1,-alpha2, -alpha3 and -alpha4 from endemic baikalian sponge Lubomirskia baicalensis was studied. For eight sponge species, including both cosmopolitan (Spongilla lacustris, Ephydatia muelleri, E. fluviatilis) and Baikal endemic (L. baicalensis, L. incrustans, Baikalospongia intermedia, B. fungiformis, Sw. papyracea) species, seventeen gene fragment sequences of different silicatein isoforms were determined. It was shown that cosmopolitan and endemic Baikalian sponges differ from each other by gene structure (have different length ofintrons). Among Baikalian sponges silicatein-alpha1 has the most variable intron length, and silicatein-alpha4 is the most conservative. Phylogenetic analysis of amino-acid silicatein sequences allow identify different silicatein isoforms, which authentically differ form four clusters on phylogenetic tree. Phylogenetic analysis of exon-intron sequences gives the possibility to separate different sponge species in the clusters.

[Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection].

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tickborne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 --> Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 --> Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.

[Phylogenetic position of sponges from Chagytai and Tore-Khol' lakes].

Morphological and molecular genetic data for freshwater sponges from the lakes of Tuva Depression, Baikalospongia dzhegatajensis (Rezvo, 1936), forms Dzh05 and Dzh06, from Chagatai Lake, as well as forms TKhl and TKh2, from the Lake Tore-Khol, were obtained and examined. In the sponges examined, which on phylogenetic tree clustered together with the Ephydatia fluviatilis (Linneaus, 1758) sponge from the family Spongillidae, the ITS rDNA regions were sequenced. Comparison of highly variable interal spacer regions of the mitochondrial genome was performed using corresponding sequences of three sponges from the family Spongillidae (E. fluviatilis, E. muelleri and Spongilla lacustris), sponges from the Chagatai and Tore-Khol lakes (Dzh06 and TKh2) with an unknown status, and sponges from the Baikalian family Lubomirskiidae. Minimum genetic differences were observed between E. fluviatilis, Dzh06, and TKh2 (from 0.003 to 0.01% of nucleotide substitutions), while maximum differences were found between the species of Lubomirskiidae and Spongillidae (from 0.928 to 2.06%). The data obtained indicated that sponges from Chagatai and Tore-Khol lakes were most close to E. fluviatilis.

[Phylogenetic analysis and distribution of far eastern tick-borne encephalitis virus subtype (Flaviridae, Flavirus, TBEV-FE) from Asia.]

To date, a lot of data on molecular genetic characteristics of different tick-borne encephalitis virus strains has appeared. Only on the basis of the E protein genome, sequences of about 1,500 TBEV strains were registered in GenBank. PURPOSE: The purpose of the work - revision and comparative analysis of data on complete genomes sequences of the Far Eastern subtype of TBE virus strains distributed in the Asian part of Eurasian continent. MATERIAL AND METHODS: The data on the complete genomes of 84 strains of TBEV isolated in Asia were used; phylogenetic analysis was performed. RESULTS AND DISCUSSION: it was shown that variants of the TBEV of the Far Eastern subtype are circulating here and form three separate clusters (Sofjin, Senzhang- и Shkotovo-like strains). Sofjin strain (Sofjin-1953, Sofjin-Chumakov, Sofjin-KSY) was considered to be the reference for Far Eastern TBE virus subtype strains and a cluster of Sofjin-like strains. Sofjin-like strains were not found in China and Japan, but widely distributed throughout the area of Primorsky and Khabarovsk krai. The group of Senzhang-like strains was distributed in China, Eastern Siberia, Khabarovsk krai and northern Primorsky krai, but was not found in Japan (Hokkaido). According to molecular genetic characteristics the youngest and more genetically homogeneous group was the Shkotovo-like strains, isolated in the southern part of Primorsky krai, however not found on Hokkaido Island (Japan). CONCLUSION: revision of the complete genome characteristics of TBEV strains revealed the features of micro-evolutionary process of viral populations in the Asian part of Eurasia, show the individual affection of strains to certain territories, as well as detect random finds of such strains in the territories of other natural foci.

[Silicateins identification of the freshwater sponge L. baicalensis].

Siliceous spicules of the freshwater Baikal sponge Lubomirskia baicalensis contain several proteins including silicateins. Existences of four different genes of silicatein alpha (alpha1, alpha2, alpha3, alpha4) which are related to silicatein alpha from the sea sponges were found when cDNA library analysis was made. The intron-exon structure of the full-size silicatein alpha1 gene was determined. This gene has total length of 1988 bp and includes 6 introns (1007 bp) and 7 exons (981 bp). With use of mass-spectrometric analysis of the spicule proteins tryptic digest, two silicateins alpha were authentically found.

[Biological and molecular genetic characteristics of a Far-Eastern tick-borne encephalitis virus population and its pathogenetic implication].

The authors have got an idea of the structure of the tick-borne encephalitis (TBE) virus population forming in the human body after tick bite in the south of the Far East. A hundred and forty-five antigen-positive samples were virologically studied in enzyme immunoassays. Human blood leukocytic virus isolation on the first day of tick suction testified to the capacity of the virus to adsorb and multiply just in the peripheral blood immunocompetent cells. The bulk (as high as 70%) of the TBE virus population was non-neuroinvasive strains, most of which could rapidly eliminate from man and albino mice. The neuroinvasive strains (as high as 30%) caused encephalitis in albino mice and different TBE forms (inapparent, feverish, focal). The sequences of 160 bp fragment of glycoprotein E gene of 24 strains have shown that they belong to one Far Eastern subtype of TVE virus.

[Molecular epidemiology of tick-borne encephalitis].

The review presents information on the development of studies into the molecular epidemiology of tick-borne encephalitis (TBE) in Russia and foreign countries. The existence of three major virus genotypes has been established by various techniques, such as genomic fragment sequencing, molecular hybridization using genotype-specific probes, and restriction fragment length polymorphism test. Each of the genotypes prevails in different parts of a natural habitat; the Ural-Siberian genotype (a Siberian subtype) is most commonly encountered. The genetic differences between the strains belonging to different genotypes are great and comparable with differences between some mammalian flaviviruses transmitted by ticks (viruses of a TBE complex). Further studies of the molecular epidemiology of TBE are of importance in understanding the evolution of the causative agent, improving the taxonomy and the classification of flavivuruses, and designing highly effective methods for the specific diagnosis, prevention, and treatment of the disease.

[Analysis of chromosome deletions of TbDl, RD6 and pks15/1 in clinical strains of Mycobacterium tuberculosis].

Deletions are very important sources of the variability among members of the mycobacterial tuberculosis complex (MTC). Deletion analysis of MTC clinical isolates was performed to clarify phylogenetic relationships and help to identify epidemiologically significant groups of the MTC. In this study, the variability of the TbDl, RD6 and pks15/1 chromosome loci in clinical MTC strains and comparison of those results with IS6110-RFLP (restriction fragment length polymorphism), sSNP (synonymous single nucleotide polymorphism), PGG (Principal Genetic Group) typing data were used to determine if these chromosome regions constitute good molecular markers for some of the epidemiologically important groups of the MTC. In the present study, 122, 61 and 294 clinical isolates were tested for the TbDl, RD6 and pks15/1 deletions, respectively. Specific probes were designed and used in RFLP analysis as well as sequencing techniques were applied. We found that all strains with intact TbDl region belonged to the sSNP cluster I, PGG 1 (katG463Leu and gyrA95Thr). The RD6 deletion was not determined to be a strict characteristic feature of any specific genetic group of the tested M.tb strains, but presence of this deletion is presumed for strains of high virulence, and associated with principal genetic groups 2 or 3. The genetic event that led to this deletion likely occurred in the strain that belongs to PGG 1. Identification of strains with an intact pksl5/1 gene cluster provided a potential marker for virulence. An intact pks15/1 gene cluster is required for the biosynthesis of the phenolic glycolipids (PGL-tb), production of which by clinical isolates was correlated with virulence.

[Clinical and laboratory characterization of Ixodes tick-borne borreliosis in the Baikal area].

Materials on Ixodes tick-borne borreliosis (ITB) for the period of 2000 - 2003 (i.e. 1,222 blood serum samples from patients with acute ITB and 629 patients with chronic ITB) were analyzed. The proportion of seropositive samples among those obtained from patients with acute and chronic ITB was determined with the use of the indirect immunofluorescence test. In addition, data on different clinical forms of the disease were presented. A high percentage of neurological manifestations in the early period of ITB (31%) and in the late period of the disease (72.8%) was noted. The conclusion was made concerning the necessity of using several diagnostic methods for more exact determination of the structure of the disease.

Characterisation of morbilliviruses isolated from Lake Baikal seals (Phoca sibirica).

Sequence analysis of the haemagglutinin protein (H) gene of the morbillivirus (PDV-2) isolated from a Siberian seal (Phoca sibirica) during the 1987/1988 epizootic in Lake Baikal revealed that it was most closely related to two recent isolates of canine distemper virus (CDV) from Germany and different from CDV vaccines currently in use in that region. The virus continued to circulate in seals in Lake Baikal after the 1987/1988 epizootic since sera collected from culled seals in the spring of 1992 were positive in morbillivirus ELISA tests, reacting most strongly with the CDV antigen.

Canine distemper virus in Lake Baikal seals (Phoca sibirica).

The virus epizootic which resulted in significant mortality in Siberian seals (Phoca sibirica) in Lake Baikal during 1987/88 was caused by canine distemper virus. Sequence analysis of the virus glycoprotein genes revealed that it was most closely related to recent European field isolates of canine distemper virus. This paper presents evidence that the same virus continued to circulate in seals in Lake Baikal after the initial epizootic. Three out of 45 brain tissue samples collected from seals culled in the spring of 1992 were positive for canine distemper virus-specific nucleic acid by the reverse transcription/polymerase chain reaction and the sequences were closely related to that of the original virus isolated in 1988.

[Cloning and sequencing the cDNA for the beta-subunit of Baikal omul gonadotropin]. / Klonirovanie i sekvenirovaniie kDNK beta-sub''edinitsy gonadotropinov Baikal'skogo omulia.

Two types of cDNA encoding gonadotropin beta subunits (GTH beta) were isolated from a cDNA library prepared from pituitary gland of Baikal omul (Coregonus autumnalis migratorius Georgi). The nucleotide sequences of cDNA were determined. The CTHI beta and GTHII beta cDNAs code for polypeptides of 137 and 142 amino acids, respectively. Both of them include a putative signal peptide of 24 amino acids. The predicted amino acid structures of omul gonadotropins were compared with those of other vertebrate species.

[Phylogenetic relationships between 16 species of Lake Baikal sculpin fish based on a nucleotide sequence analysis of a mitochondrial DNA fragment of the cytochrome b gene]. / Filogeneticheskie vzaimootnosheniia sredi 16 vidov podkamenshchikovykh ryb oz. Baikal na osnove analiza nukleotidnoi posledovatel'nosti fragmenta gena tsitokhroma b mitokhondrial'noi DNK.

The fragment of mitochondrial cytochrome b gene from 16 species of Baikalian sculpins belonging to three families--Cottidae, Comephoridae and Abyssocottidae--was amplified by polymerase chain reaction and sequenced. Phylogenetic trees were built from data obtained. Some disconformity were noted between systematical and phylogenetic notions of today and obtained results. For example, branch lengths among some Cottidae species are larger than among any different family species. Moreover, schemes indicate separate position of Limnocottus euristomus from Limnocottus genus species. Phylogenetic trees confirm earlier conclusions, about recent--in geological scales of times--origin of baikalian Cottoidei (2-2.5 Myr).

[BspMII-family of tandemly organized DNA sequences of the Baikalian cottoid fish (Cottodei)]. / BspMII-semeistvo tandemno organizovannykh posledovatel'nostei DNK baikal'skikh kottoidnykh ryb (Cottoidei).

A new family of highly repetitive tandemly arrayed DNA sequences was revealed by electrophoretic analysis of BspMII (T'CCGGA) digest of genomic DNA from Baikalian cottoid fishes. Three such repeats from two species B. nikolskii and P. kneri were cloned and sequenced. The sizes of repeat elements are 238, 154, and 123 bp. All three repeats have highly homologous flanking regions 53 and 63 bp in length. It was shown that chains of DNA repeats had an ability to form stable secondary structures in vitro. It is supposed that shorter repeats (154 and 123 bp) originated from the 238 bp repeat by deletion its central fragments. An element having a the promoter for RNA polymerase III was found within the 238 bp repeat. The origin of this element from one of the tRNA genes is suggested. The possibility of using BspMII repeats as an evolutionary marker for studying the genesis of Cottoidei is discussed.

[Nucleotide sequence of a fragment of the Cottocomephorus grewingki phosphofructokinase gene]. / Nukleotidnaia posledovatel'nost' fragmenta gena fosfofruktokinazy Cottocomephorus grewingki.

The nucleotide sequence of a 208-bp fragment of the pfk gene encoding phosphofructokinase from Baikalian fish Cottocomephorus grewingki (Cottidae family) was determined. The fragment shows the codes of a sequence of 38 amino acid residues and contains a 94-bp intron. The nucleotide sequence of the C. grewingki phosphofructokinase gene and the corresponding amino acid sequence display the maximum homology to phosphofructokinases from rabbit muscles and a rat liver.

[Molecular typing of the tick-borne encephalitis virus isolated from patients with different-infection severities in the south of Russia's Far East]. / Molekuliarnoe tipirovanie shtammov virusa kleshchevogo éntsefalita, vydelennykh ot liudei s razlichnoi stepen'iu tiazhesti infektsii na territorii iuga Dal'nego Vostoka Rossii.

The thick-borne encephalitis virus (TBEV), which is widespread in the Eurasian continent, belongs to the Flaviviridae family, Flavirus genus, and comprises the Far Eastern, Siberian and West European subtypes. It was for the first time that the gene part of the E 24 strain envelope glycoprotein of TBEV, which caused infection in residents of the South of Russia's Far East, was analyzed. It was established that the TBEV Far-Eastern subtype causes different-severity disease cases ranging from the focal ones with the lethal outcome to latent infection forms. On the basis of the phylogenetic analysis, the Far-Eastern subtype was shared between 4 sub-clusters, 2 of which constitute a majority of the analyzed TBEV strains.

[Molecular-genetic comparison of morbilliviruses which caused epizooty in Baikal (Phoca siberica) and Caspian (Phoca caspica) seals]. / Molekuliarno-geneticheskoe sravnenie morbillivirusov, vyzvavshikh épizootii baikal'skikh (Phoca sibirica) i kaspiiskikh (Phoca caspica) tiulenei.

The nucleotide sequences were determined for a phosphoprotein gene fragment of canine distemper virus (CDV) by using the RT-PCR method with the subsequent sequencing of amplicons from total RNA isolated from 2 samples of Caspian seals, 15 samples of Baikal seals and from samples of dog's and sea-lion's brains. The above materials were phylogenetically analyzed. The heterogeneity of the virus circulating in the Baikal-seal population was demonstrated. Morbillivirus, that caused epizooty in Caspian seals, was shown to be a CDV variant, whose phosphoprotein gene structure was not different, within the analyzed stretch, from the corresponding gene of the most widespread variant of the Baikal seal virus. The data obtained suggest that morbillivirus could be transmitted by birds during their seasonal migrations.