COMPARISON OF INFLUENZA A VIRUS INHIBITION IN VITRO BY SIRNA COMPLEXES WITH CHITOSAN DERIVATIVES, POLYETHYLENEIMINE AND HYBRID POLYARGININE-INORGANIC MICROCAPSULES.

Anti-influenza drugs and vaccines have a limited effect due to the high mutation rate of virus genome. The direct impact on the conservative virus genome regions should significantly improve therapeutic effectiveness. The RNA interference mechanism (RNAi) is one of the modern approaches used to solve this problem. In this work, we have investigated the antiviral activity of small interfering RNA (siRNA) against the influenza A/PR/8/34 (H1N1), targeting conserved regions of NP and PA. Polycations were used for intracellular siRNA delivery: chitosan's derivatives (methylglycol and quaternized chitosan), polyethyleneimine, lipofectamine, and hybrid organic/non-organic microcapsules. A comparative study of these delivery systems with fluorescent labeled siRNA was conducted. The antiviral activity of three small interfering RNAs targeting the NP (NP-717, NP-1496) and PA (PA-1630) influenza A viruses genes was demonstrated, depending on the chosen carrier. The most effective intracellular delivery and antiviral activity were observed for hybrid microcapsules.

[Analysis of complete sequence of cryptic plasmid pTP33 from Yersinia pestis isolated in Tuva natural focus of plague].

This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin­antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms­endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).

[Maldi-tof ms analysis for yersinia pestis, vibrio cholera, and francisella tularensis identification].

Numerous studies showed that a new technology for the clinical microbiology laboratories, Matrix-Assisted Laser Desorption Ionization--Time of Flight Mass Spectrometry (MALDI-ToF MS), allows fast, accurate, and effective identification of most clinically relevant microorganisms to be implemented. In the present review, we discuss applications of this approach for identification and typing of extremely dangerous pathogens--Yersinia pestis, Vibrio cholera, and Francisella tularensis, including the advantages and disadvantages of the method, sample preparation and biosafety problems.

[Multilocus sequence-typing of vibrio cholerae strains with various epidemic importance].

The allele polymorphism of the housekeeping genes (dnaE, lap, recA, pgm, gyrB, cat, chi, gmd) from the Vibrio cholerae strains with different epidemic importance (n = 41) isolated in Siberia and at the Far East during the cholera pandemic VII was tested. All toxigenic strains isolated at the period of epidemic complications irrespective of time and source of isolation were characterized by the identical allele profile and belonged to the same sequence-type. Nine sequence types were detected in non-epidemic isolates. The dendrogram clustering was associated with the serogroup and in some cases with the territory and time of isolation. The structure heterogeneity of the non-toxigenic V. cholerae housekeeping genes was in most cases caused by the synonymous nucleotide replacements (Dn/Ds < 1) indicating the prevalence of the negative V. cholerae at the analyzed genome sites. The revealed distinctions in the structure of housekeeping genes of the V. cholerae with different epidemic importance can be regarded as evidence of various evolutional directions in these strain groups.

[MALDI-TOF mass-spectrometric analysis of cell proteins during identification of leptospira genus members].

AIM: Development of methodological approaches for identification of leptospira by using MALDI-TOF direct protein profiling technology. MATERIALS AND METHODS: Analysis of cell proteins of 34 leptospira strains was carried out in Microflex LT by using "MALDI Biotyper 3.0 for identification and classification of microorganisms" program. RESULTS: 19 reference spectra of reference leptospira strains from 7 species were generated and imported into MALDI Biotyper 3.0 database. Identification of 6 strains with undetermined taxonomic position was carried out. CONCLUSION: The approved method allows determination of leptospira species with accuracy that depends on their adaptation to nutrient media, preparation approach and sample storage conditions for mass-spectrometry.

[Comparative multilocus VNTR- and SNP-analysis of Bacillus anthracis vaccine strains].

Comparative VNTR- and SNP-genotype analysis of four Bacillus anthracis strains (three vaccinal and one virulent) was carried out using modern molecular-genetic typing methods. It is established that these strains formed four SNP patterns completely corresponding to the VNTR-profiles. It was demonstrated that all strains tested except vaccinal B. anthracis STI-1 could not belong by SNP-profile to three global genetic lines of the anthrax agent extended in the world.

[The approbation of technique of mass spectrometry with matrix-activated laser desorption/ionization for identification of plague agent].

The study of sampling of strains of Y. pestis of main and altaic subspecies was implemented. The modern technique of identification of microorganisms was applied using MALDI-TOF mass spectrometry analysis. The evaluation of biological safety of method of sampling preparation was implemented. To supplement the identification base "BioTyper" the spectrum of typical strains of Y. pestis were obtained. The enhanced identification base was used to evaluate possibilities of application of MALDI-TOF technology for identification and taxonomic differentiation of Y. pestis from other representatives of genus of Yersinia. In the process of study a complete concordance of results of mass spectrometry identification and classic cultural method was observed. On the basis of mass spectrometry characteristic of analyzed sampling the differentiation between strains of Y. pestis of subspecies pestis and strains of subspecies altaica was implemented. The study results testify the effectiveness of application of mass spectrometry analysis for reliable interspecies and intraspecific differentiation of plague agent. The simplicity and velocity of sampling preparation and implementation of analysis and low cost of active storage allow considering the MALDI-TOF technology of mass spectrometry identification as highly perspective method for laboratory diagnostic of plague agent.

[The development and implementation of polymerase chain reaction to detect in real-time operation mode yersinia pestis in field material].

The article presents the results of development and practical implementation of system of polymerase chain reaction testing in real-time operation mode to detect agent of plague infield material. In laboratory conditions the system demonstrated good results and hence it was applied in conditions of field laboratory of epidemiologic team during planned epizootologic examination of Gorno-Altaisk hot spot of plague. The sampling consisted of more than 1400 objects. It was demonstrated that high sensitivity and specificity is immanent to proposed system. The adaptation of the system to the real time amplifier "Smart Cycler" (Cephid, USA) having some specific technical characteristics makes it possible to consider the proposed test-system as an effective sensitive and precise instrument for screening studies in the process of regular epizootologic examinations of hot spots of plague.

[Molecular-genetic analysis of the epidemical strains of the Vibrio cholerae El Tor isolated from the Siberian and maritime regions of Russia].

The detection of the biotype-specificity, pathogenicity determinants, and sequencing of the ctxB gene and the ctxAB promoter was carried out for analysis of the Vibrio cholerae El Tor strains genome structure. The strains (n = 90) were isolated during cholera epidemic outbreaks in Siberia and the Far East. All toxigenic Vibrio cholerae El Tor strains were divided into two groups: the first group included strains isolated during 1970s: they had the genotype ctxB3+rstREl+rstRCl-rstC+TLC+tbr4. All epidemic dangerous El Tor biotype strains isolated in 1990s belonged to the second group. The strains were characterized as atypical variants because of classical genotype (ctxB1) ctxB gene harboring. The second group fell into three genotypes according to the set of genetic markers (ctxB, rstR, rstC, TLC, tbr). It was suggested that the set of genetic determinants could be used as a marker for epidemiological analysis of spreading of atypical ET strains. The comparative analysis of genome structure enables to suggest possible ways of pathogen evolution.

[Detection of "hybrid" Vibrio cholerae eltor strains during epidemic complications in Syberia and Far East].

AIM: Biotyping of Vibrio cholerae eltor isolated during epidemic complications of cholera in Syberia and Far East by phenotypic and genotypic properties complex. MATERIALS AND METHODS: 45 strains of V. cholerae were studied. Phenotypic analysis was performed by using a complex ofbiovar determining tests. Genotyping was performed by detecting ctxAB, tcpA, toxR, rstRgenes, and ctxB gene structure analysis. RESULTS: All the V. cholerae during epidemiologic complications in Syberia in the 1970s belong to eltor biovar by phenotypic properties and have eltor specific alleles of tcpA and rstR genes, and ctxB of the third genotype in the genome. In the 1990s the strains were phenotypically matching eltor biovar, but had genetical determinants of both eltor(tcpAE1, rstRE1) and classical (ctxB1, rstR(Cl) biovar. CONCLUSION: The cause of epidemic complications of cholera in Syberia in the 1970s was V. cholerae eltor with typical eltor biovarphenotypical and genotypical properties. In the 1990s cases of introduction into the region of "hybrid: V. cholerae eltor strain were ascertained, developing into acute cholera outbreaks in several cases.

Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation.

The present study investigates epidemiological diversity and multidrug resistance spreading among Mycobacterium tuberculosis strains circulating in Moscow, Russian Federation. Among 115 M. tuberculosis strains selected randomly from the sputum of epidemiologically unrelated tuberculosis (TB) patients, multidrug-resistant (MDR) strains predominated. Mutations in the RRDR of the rpoB gene were detected in 64 (83.1%) of 77 rifampicin (RIF)-resistant strains. The Ser531→Leu substitution was prevalent among them (76.5%). Aberrations in the Ser315 codon of katG and/or in the inhA promoter region were found in 79 (84.0%) of 94 isoniazid (INH)-resistant strains. Strains belonging to the Beijing family prevailed. Seventy-one different patterns were identified using the 24-VNTR loci typing scheme. Three main 24-loci VNTR clusters included 34 strains which belonged to the Beijing family. The spoligotyping and 24-loci VNTR typing combination demonstrated maximal discriminatory power. Among the Beijing strains, the MDR phenotype was revealed more frequently than among the others. High genetic heterogeneity of the studied population was shown by the assessment of VNTR loci variability in the analyzed group and in the strains from other parts of Russia. Comparison of the 24-VNTR locus typing and spoligotyping data with revealed resistance-associated mutation allows us to make a suggestion that the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy occurred in the studied population simultaneously.

[Molecular characteristic of methicillin-resistant Staphylococcus aureus strains isolated in Moscow clinics].

UNLABELLED: OBJECTIVES; Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen of nosocomial infection. The goal of this work was to evaluate the clonality of hospital-acquired MRSA (HA-MRSA) circulating in Russian Federation and to compare different multiplex PCR techniques with SNP-based approach for MRSA typing. METHODS: Epidemiologically unrelated MRSA isolates (n = 62) from Moscow hospitals were selected for typing. Genomic DNA from clinical isolates was purified using the DNA express kit (Lytech Ltd, Russia). Staphylococcus chromosomal cassette mec (SCCmec) typing was performed by PCR using the previously described methods. Seven loci from five housekeeping genes (arcC162, arcC210, aroE132, gmk123, tpi241, tpi243 and yqiL333) were used for SNP-typing. Detection of particular nucleotides in selected loci was carried out in the thermocyclic primer extension reaction, followed by mass spectrometry of the products. Standard MLST procedure was performed as reference method. RESULTS: The majority of the MRSA isolates (93.6%) belong to world-wide disseminated clonal complex (CC) 8. Three isolates (4.8%) belong to CC 1. All ST 239 isolates were found to carry SCCmec type III; ST 8 isolates, SCCmec type IV. CONCLUSION: Among Russian MRSA CC 8 isolates carrying SCCmec IV type are predominant. SNP-typing is powerful toll for studies of molecular epidemiology of MRSA.

[Detection of mutations in codon 306 of the embB gene for molecular genetic characterization of clinical Mycobacterium tuberculosis strains].

A total of 254 Mycobacterium tuberculosis strains were used in the study. Among them, there were 183 ethambutol (EMB)-resistant strains, 13 multidrug resistant ones, but EMB-sensitive, and 39 strains sensitive to rifampicin (RIF), isoniazid (INZ), and EMB. All the strains were analyzed for genetic changes in three loci: embB306, rpoB, and katG/inhA promoter, which were associated with the formation of resistance to EMB, RIF, and INZ, respectively. The Mycobacterium tuberculosis strains were obtained from pulmonary tuberculosis patients living in the Central Region of the Russian Federation. Resistance to RIF, INZ, and EMB was revealed by the absolute concentration test. The inhibitory concentration (IC) of EMB was determined for all the strains. Genetic changes in the above loci were estimated by mini-sequencing, followed by mass-spectrometry recording MALDI-TOF products. The relative low frequency of embB306 mutations was observed among the EMB-resistant strains (about 41.5%). Mutations in codon 306 were detected only in strains with EMB IC > or = 2 mg/L. A statistical significant association was found between the frequency of embB306 mutations and the multidrug resistant phenotype. A combination of these mutations with the traditional genetic markers of multidrug resistance may be used for the more effective detection of multidrug-resistant strains.

[Gene frequencies and heterozygosity of the AB0 and RH blood group alleles in the populations of two cities of the Donetsk region, Ukraine]. / Chastoty genov i geterozigotnost' po alleliam sistem grupp krovi ABO i RH naseleniia dvukh gorodov Donetskoi oblasti Ukrainy.

The frequencies of the AB0 and RH blood group alleles and heterozygosity indices were determined for the populations of two large industrial cities of Gorlovka and Mariupol. In the population of Gorlovka the gene frequencies were as follows: AB0*0 = 0.576, AB0*A = 0.266, AB0*B = 0.158, and RH*D = 0.592, in Mariupol the frequencies were AB0*0 = 0.584, AB0*A = 0.265, AB0*B = 0.151, and RH*D = 0.604. In Gorlovka the heterozygosity indices in respect to the AB0 and RH alleles were 0.572 and 0.483, respectively; in Mariupol, 0.566 and 0.478, respectively. There were no statistically significant differences between the two populations in respect to the genetic markers analyzed. However, the heterozygosity values obtained were more similar to the corresponding estimates for some populations of Russia, than for the total population of the Ukraine.