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Abstract We have studied the antimicrobial and hemolytic activity of synthetic antimicrobial peptides (SAMPs), i.e., Arg9Phe2 (P1-Arg), Lys9Phe2 (P2-Lys), and His9Phe2 (P3-His), which have a “linear” type of amphipathicity and contain the cationic amino acid residues of arginine, lysine, or histidine. In this study, we have used various pathogenic microorganism strains of gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica), gram-positive bacteria (Staphylococcus aureus), and the conditionally pathogenic yeast fungus (Candida albicans). It has been shown that the replacement of the arginine residues by lysine or histidine residues in the tested SAMPs significantly degrades their antibacterial properties in the series: P1-Arg > P2-Lys $$ \gg $$P3-His. The cationic analog of SAMP, P1-Arg, has the highest antibacterial activity (MIC50 = 43–76 μM), while peptide P3-His does not exhibit this activity (MIC50 > 100 μM). The P1-Arg and P2-Lys peptides were 6–10 times more active against the opportunistic fungus C. albicans (MIC50 6.7 and 10.9 μM, respectively) and the P3-His peptide has 100-times increased antimycotic activity (MIC50 0.6 μM) compared with their effect on bacterial cells. All of the tested peptides with the linear type of amphipathicity and low hydrophobicity, i.e., P1-Arg, P2-Lys, and P3-His, that contain only two Phe residues regardless of the presence of cationic amino acids (Arg, Lys, or His) exhibit a relatively low hemolytic activity (not more than 4% hemolysis at 1000 μM peptide concentration). Thus, considering the same synthesis efficiency (56–63%) and approximately the same low toxicity of the tested SAMPs with a linear type of amphipathicity, it is recommended to use those that contain the cationic arginine or histidine residues to create antibacterial or antifungal peptide agents, respectively.

The conjugates of RNase P guiding oligo(2'-O-methylribo)- and oligodeoxyribonucleotides (EGS oligonucleotides) with oligo(N-methylpyrrole) have been synthesized for the first time. The ability of RNase P to hydrolyze RNA in the presence of EGS oligonucleotides and their conjugates with oligo(N-methylpyrrole) has been demonstrated in model systems using fluorescently labeled chemically synthesized oligoribonucleotides corresponding to the fragments of mRNA of the ftsZ and gyrA genes of Acinetobacter baumannii. It has been shown that hydrolysis of RNA by RNase P occurs more efficiently in the presence of the conjugates than in the case of unmodified oligodeoxyribonucleotides. The introduction of oligo(N-methylpyrrole) in EGS oligo(2'-O-methylribonucleotides) insignificantly changes the effectiveness of hydrolysis. It has been demonstrated that the cell-penetrating ability of oligonucleotides is enhanced because of the presence of oligo(N-methylpyrrole) at the 5'-end.

The increasing prevalence of bacterial pathogens with multiple antibiotic resistance requires development of new approaches to control infections. Phage therapy is one of the most promising approaches. In recent years, research organizations and a number of pharmaceutical companies have intensified investigations aimed at developing bacteriophage-based therapeutics. In the United States and European countries, special centers have been established that experimentally apply phage therapy to treat patients who do not respond to antibiotic therapy. This review describes the features of bacteriophages as therapeutic tools, critically discusses the results of clinical trials of bacteriophage preparations, and assesses the prospects for using phage therapy to treat certain types of infectious diseases.

One of the promising strategies in the development of efficient antibacterial drugs is the use of antisense oligonucleotides specifically interacting with mRNAs of genes controlling the key functions of the bacterial cell such as survival and reproduction. The present paper reports the development of an algorithm for searching and testing the antibacterial activity of antisense oligonucleotides using mRNA of the rpoD gene encoding the sigma factor of the Staphylococcus aureus RNA polymerase. Based on the calculated thermodynamic parameters and the rpoD mRNA 5'-fragment (1–377 nt region) secondary structure model, 11 antisense oligonucleotides 18–20 nt in length complementary to the rpoD mRNA regions with unstable secondary structure have been selected. Hybridization of the selected oligonucleotides with the total S. aureus RNA followed by selective in vitro digestion of antisense oligonucleotide/mRNA rpoD heteroduplexes with RNase H and assessment of rpoD mRNA digestion rate using PCR revealed six candidate sequences which reduced the rpoD mRNA level by 2.2–4 times. Phosphorothioate oligonucleotides targeted to the regions 1–20, 30–47, and 82–101 of the rpoD mRNA synthesized on the basis of these sequences efficiently suppressed the growth of S. aureus bacteria in vitro. It has been demonstrated that the efficiency of penetration of these oligonucleotides into the bacterial cell may be significantly increased by introducing arginine-rich peptide into the oligonucleotide structure. As a result of this work, we have developed a simple and efficient algorithm for selection of antisense oligonucleotides against bacterial RNAs encompassing all stages starting from the binding stage and ending with the analysis of biological activity.

The antimicrobial and hemolytic activities of R9F2С2 (P1ss), (KFF)3KС2 (P2ss), and (RAhaR)4AhaβAС2 (P3ss) (where Aha is 6-aminohexanoic acid and βA is beta-alanine) synthetic antimicrobial peptides (SAMP) with different amphipathic properties and containing the cysteine residues with free sulfhydryl groups were studied. The introduction of cysteine residues into the composition of SAMP was shown to increase their antimicrobial activity 3–7 times, while their hemolytic activity increased 2–12 times in relation to human erythrocytes for different peptides in different ways, which determined their different selectivity. The P1ss peptide with a linear type of amphipathicity showed the highest antimicrobial activity and high selectivity against the fungus Candida albicans and bacterium Staphylococcus aureus (MIC 0.5 μM; TI 52 μM). The P1ss peptide possessed not only greater antimicrobial activity against pathogenic fungus C. albicans in comparison to the P2ss peptide (MIC 3.9 μM) with the classical helical amphipathicity, but also more than three times lower hemolytic activity (MHC 26 and 8 μM, respectively). Therefore, TI for the P2ss peptide (TI 2.1) turned out to be more than 20 times lower than that for the P1ss peptide. Thus, the P1ss peptide is the most promising antimicrobial preparation among the studied SAMP.

A novel thermophilic bacteriophage AP45 and its host strain Aeribacillus sp. CEMTC656 were isolated from the Valley of Geysers, Kamchatka Peninsula, Russia. Bacteriophage AP45 was identified as a member of the Siphoviridae family by electron microscopy. It showed high thermostability and had a slow cycle of reproduction. The AP45 genome had 51,606 base pairs (bp) and contained 71 open reading frames (ORFs), 40 of them encoding proteins of predicted function. Genes encoding DNA and RNA polymerases were not identified, indicating that AP45 used host polymerases. Based on the ORF65 encoding putative endolysin, the recombinant protein rAP45Lys was developed and its peptidoglycan-hydrolyzing activity was demonstrated. The AP45 genome exhibited limited identity to other phage sequences; the highest identity, 36%, was with the genome of the thermophilic Geobacillus myovirus D6E. The majority of putative proteins encoded by the AP45 genome had higher similarity to proteins from bacteria belonging to the Bacillaceae family, than to bacteriophages. In addition, more than half of the putative ORFs in the AP45 genome were highly similar to prophage sequences of A. pallidus strain 8m3, which was isolated in north-east China. The AP45 phage and revealed prophages might be members of a new genus belonging to the Siphoviridae family.

Despite the elimination of smallpox, orthopoxviruses continue to be a source of biological danger for humans, as cowpox and monkey pox viruses circulate in nature and the last virus can cause both sporadic cases of human diseases and outbreaks of smallpox-like infection. In addition, periodic vaccination is necessary for representatives of some professions (scientists studying pathogenic orthopoxviruses, medical personnel, etc.). Vaccination against smallpox virus with live vaccinia virus, which was widely used during the elimination of smallpox, induces the formation of long-term immunity in vaccinated people. However, providing a high level of protection, the vaccination is often accompanied by serious post-vaccination complications, the probability of which is particularly great for individuals with compromised immunity. In this regard, the development of preparations for the prevention and treatment of infections caused by orthopoxviruses remains important today. The aim of this study was to assess the immunogenicity in the mouse model of recombinant protein р35Δ12, designed previously on the base of the cowpox virus protein p35. It was previously shown that the protein р35Δ12 was recognized by fully human neutralizing anti-orthopoxviral antibody with high affinity. In this work, recombinant protein р35Δ12 produced in E. coli cells XL1-blue and purified by chromatography was used for two-time immunization of mice. Two weeks after the second immunization, blood samples were taken from mice and serum antibodies were analyzed. It was shown by ELISA and Western-blot analysis that immunized mice sera contained IgG antibodies specific to recombinant protein р35Δ12. Confocal microscopy showed that antibodies induced by the р35Δ12 protein were able to recognize Vero E6 cells infected with the LIVP-GFP vaccinia virus. In addition, the antibodies in the serum of immunized mice were able to neutralize the infectivity of the vaccinia virus LIVP-GFP in the plaque reduction neutralization test in vitro. These experiments have demonstrated promising properties of the р35Δ12 protein if it were used as a component of vaccine for prophylaxis of orthopoxvirus infections.

Currently, a therapeutic drug based on recombinant antibodies for the prevention and treatment of tick-borne encephalitis virus (TBEV) is developed in ICBFM SB RAS, and the chimeric antibody ch14D5 is considered as one of the key components of this drug. It was previously shown that this antibody is directed to the domain D3 of the glycoprotein E of TBEV. It was previously shown that this antibody is able to protect mice from the European subtype of TBEV, strain “Absettarov”, and the presence of virus-neutralizing activity against the Far Eastern subtype of TBEV, strain 205 was also shown for this antibody. However, it remains unclear whether this antibody exhibits selectivity for different subtypes of TBEV. The aim of this study was to investigate the effect of amino acid sequence differences of recombinant D3 domains derived from the glycoprotein E of TBEV of the Far Eastern, Siberian and European subtypes on the binding of the protective antibody ch14D5 to these proteins. Using Western blot analysis and surface plasmon resonance, it was shown that ch14D5 antibody has the highest affinity (KD= 1.7±0.5 nM) for the D3 domain of the TBEV of the “Sofjin-Ru” strain belonging to the Far Eastern subtype of the virus. At the same time, the affinity of ch14D5 antibody for similar D3 proteins derived from “Zausaev”, “1528-99” and “Absettarov” strains of the Siberian and European subtypes of TBEV was noticeably lower (KD= 25±4, 300±50, 250±50 nM, respectively). In addition, information about the spatial arrangement of amino acid residues that are different for the studied recombinant proteins indicates that the epitope recognized by the ch14D5 antibody is in close proximity to the lateral ridge of D3 domain of E glycoprotein.

Tick-borne encephalitis virus (TBEV), belonging to the Flaviviridae family, is the most significant pathogen transmitted by Ixodes ticks and causing one of the most severe human neuroinfections. In Russia, serum immunoglobulin produced from the donor blood is currently used for post-exposure prophylactic and therapy of tick-borne encephalitis virus. However, it is known that preparations obtained from donated blood have certain disadvantages, and therefore development of novel preparations for post exposure prophylaxis and therapy of tick-borne encephalitis is required. To develop an alternative preparation, which does not include donor blood, a chimeric antibody ch14D5 against glycoprotein E of TBEV was constructed.This study was aimed to investigate protective efficacy of the chimeric antibody ch14D5 against the Far-Eastern, Siberian, and European subtypes of TBEV in in vivo experiments.A peripheral mouse model of tick-borne encephalitis was used in this study: the chimeric antibody ch14D5 was administrated intravenously in mice one day after their intraperitoneal infection with TBEV strains Sofjin, Vasilchenko, and Absettarov. Anti-TBEV serum immunoglobulin was used as a control preparation, which was administered in the same way. Protective efficacy of the chimeric antibodies 14D5 was assessed using the log-rank test. In the study, the presence or absence of antibody-dependent enhancement of infection (ADE) was examined when mice, infected with different subtypes of the TBEV, got the antibody ch14d5.Obtained results demonstrated high efficacy of the ch14D5 antibody in post-exposure prophylaxis of the disease in mice infected with any of the used TBEV strains, as well as the absence of ADE.It was shown that protective efficacy of antibody ch14D5 is higher than that of the anti-TBEV serum immunoglobulin, and antibody ch14D5 could be used for development of a therapeutic preparation for post-exposure prophylaxis.

Tick-borne encephalitis virus (TBEV) causes 5−7 thousand cases of human meningitis and encephalitis annually. The neutralizing and protective antibody ch14D5 is a potential therapeutic agent. This antibody exhibits a high affinity for binding with the D3 domain of the glycoprotein E of the Far Eastern subtype of the virus, but a lower affinity for the D3 domains of the Siberian and European subtypes. In this study, a 2.2-fold increase in the affinity of single-chain antibody sc14D5 to D3 proteins of the Siberian and European subtypes of the virus was achieved using rational design and computational modeling. This improvement can be further enhanced in the case of the bivalent binding of the full-length chimeric antibody containing the identified mutation.

A novel Enterobacter cloacae phage, EC151, was isolated and characterized. Electron microscopy revealed that EC151 has a siphovirus-like virion morphology. The EC151 nucleotide sequence shows limited similarity to other phage genomes deposited in the NCBI GenBank database. The size of the EC151 genome is 60,753 bp and contains 58 putative genes. Thirty-nine of them encode proteins of predicted function, 18 are defined as hypothetical proteins, and one ORF identifies as the tRNA-Ser-GCT-encoding gene. Six ORFs were predicted to be members of the deazaguanine DNA modification pathway, including the preQ0 transporter. Comparative proteomic phylogenetic analysis revealed that phage EC151 represents a distinct branch within a group of sequences containing clades formed by members of the Seuratvirus, Nonagvirus, and Vidquintavirus genera. In addition, the EC151 genome showed gene synteny typical of the Seuratvirus, Nonagvirus, and Nipunavirus phages. The average genetic distances of EC151/Seuratvirus, EC151/Nonagvirus, and EC151/Vidquintavirus are approximately equal to those between the Seuratvirus, Nonagvirus, and Vidquintavirus genera (~0.7 substitutions per site). Therefore, EC151 may represent a novel genus within the Siphoviridae family. The origin of the deazaguanine DNA modification pathway in the EC151 genome can be traced to Escherichia phages from the Seuratvirus genus.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Antimicrobial peptides, including synthetic ones, are becoming increasingly important as a promising tool to fight multidrug-resistant bacteria. We examined the effect of cationic peptides H2N-Arg9-Phe2-C(O)NH2 and H2N-(Lys-Phe-Phe)3-Lys-C(O)NH2 on Staphylococcus aureus, which remains one of the most harmful pathogens. Antiseptic chlorhexidine served as reference preparation. We studied viability of S. aureus and examined its ultrastructure under treatment with 100 &micro, M of R9F2 or (KFF)3K peptides or chlorhexidine using transmission electron microscopy of ultrathin sections. Bacterial cells were sampled as kinetic series starting from 1 min up to 4 h of treatment with preparations. Both peptides caused clearly visible damage of bacteria cell membrane within 1 min. Incubation of S. aureus with R9F2 or (KFF)3K peptides led to cell wall thinning, loss of cytoplasm structure, formation of mesosome-derived multimembrane structures and &ldquo, decorated fibers&rdquo, derived from DNA chains. The effect of R9F2 peptides on S. aureus was more severe than the effect of (KFF)3K peptides. Chlorhexidine heavily damaged the bacteria cell wall, in particular in areas of septa formation, while cytoplasm kept its structure within the observation time. Our study showed that cell membrane damage is critical for S. aureus viability, however, we believe that cell wall disorders should also be taken into account when analyzing the effects of the mechanisms of action of antimicrobial peptides (AMPs).

The genus Anaplasma (family Anaplasmataceae, order Rickettsiales) includes obligate intracellular alphaproteobacteria that multiply within membrane-bound vacuoles and are transmitted by Ixodidae ticks to vertebrate hosts. Since the last reclassification of Anaplasmataceae twenty years ago, two new Anaplasma species have been identified. To date, the genus includes eight Anaplasma species (A. phagocytophilum, A. marginale, A. centrale, A. ovis, A. bovis, A. platys, A. odocoilei, and A. capra) and a large number of unclassified genovariants that cannot be assigned to known species. Members of the genus can cause infection in humans and a wide range of domestic animals with different degrees of severity. Long-term persistence which, in some cases, is manifested as cyclic bacteremia has been demonstrated for several Anaplasma species. Zoonotic potential has been shown for A. phagocytophilum, the agent of human granulocytic anaplasmosis, and for some other Anaplasma spp. that suggests a broader medical relevance of this genus. Genetic diversity of Anaplasma spp. has been intensively studied in recent years, and it has been shown that some Anaplasma spp. can be considered as a complex of genetically distinct lineages differing by geography, vectors, and host tropism. The aim of this review was to summarize the current knowledge concerning the natural history, pathogenic properties, and genetic diversity of Anaplasma spp. and some unclassified genovariants with particular attention to their genetic characteristics. The high genetic variability of Anaplasma spp. prompted us to conduct a detailed phylogenetic analysis for different Anaplasma species and unclassified genovariants, which were included in this review. The genotyping of unclassified genovariants has led to the identification of at least four distinct clades that might be considered in future as new candidate species.

A new crAss-like genome encoding diversity-generating retroelement (DGR) was found in the fecal virome of a healthy volunteer. The genome of the phage referred to as the crAssphage LMMB, belonged to the candidate genus I of the AlphacrAssvirinae subfamily. The DGR-cassette of the crAssphage LMMB contained all the essential elements: the gene encoding reverse transcriptase (RT), the target gene (TG) encoding the tail-collar fiber protein, and variable and template repeats (VR and TR) with IMH (initiation of mutagenic homing) and IMH* sequences at the 3&prime, end of the VR and TR, respectively. Architecture of the DGR-cassette was TG-VR(IMH)-TR(IMH*)-RT and an accessory variable determinant (avd) was absent from the cassette. Analysis of 91 genomes and genome fragments from genus I of the AlphacrAssvirinae showed that 15 (16%) of the genomes had DGRs with the same architecture as the crAssphage LMMB, while 66 of the genomes contained incomplete DGR-cassettes or some elements of the DGR.

A total of 705 rodents from Myodes, Microtus, and Apodemus genera, 396 adult questing Ixodes persulcatus, and 115 Ixodes larvae and nymphs taken from rodents (and then molted under laboratory conditions to nymphs and adults) were collected in 2013-2018 in Omsk province, Russian Siberia, and examined for the presence of Anaplasmataceae. DNA of Anaplasma phagocytophilum was detected in 29.5 % rodents, 3.8 % questing I. persulcatus, two molted adult I. persulcatus, and one molted adult Ixodes trianguliceps. Ehrlichia muris DNA was found in specimens from 12.1 % rodents, 3.0 % questing I. persulcatus, 14 % molted adult I. persulcatus, and one molted adult I. trianguliceps. Neoehrlichia mikurensis DNA was found in 0.6 % blood samples. It was suggested that in the studied area A. phagocytophilum and E. muris are mainly transmitted to small rodents by I. trianguliceps and I. persulcatus, respectively. Based on groEL gene sequence analysis, three phylogenetic clusters of A. phagocytophilum (clusters 4, 5, 6, according to Jaarsma et al., 2019) were identified. Most of genotyped A. phagocytophilum isolates obtained from rodents (87.6 %) and a single isolate found in a molted adult I. trianguliceps belonged to cluster 5. Cluster 6 contained 11.8 % genotyped specimens from rodents, and one questing and two molted adult I. persulcatus, while cluster 4 included specimens from 93 % genotyped questing I. persulcatus and one vole. The finding of A. phagocytophilum from clusters 5 and 6 in voles from the same sampling area indicated that clusters 5 and 6 segregate according to the tick-carriers, but not the geography. Most of the genotyped specimens of E. muris and N. mikurensis corresponded to typical genotypes detected in Asian Russia previously. In addition, new genetic variants of E. muris and N. mikurensis, which significantly differed from other known isolates and formed separate branches on phylogenetic trees, were identified in several voles.

Comparative antimicrobial properties of three artificial cationic synthetic antimicrobial peptides (SAMP): (RAhaR)4AhaβA (where R is Arg, Aha is 6-aminohexanoic acid, βA is beta-alanine), (KFF)3K and R9F2 with various amphiphilic properties have been studied relative to pathogenic strains of microorganisms: Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Salmonella enterica, Gram-positive bacteria Staphylococcus aureus, and pathogenic yeast fungus Candida albicans. The selectivity index (SI) values of the peptide preparations were calculated as the ratio of the 50% cytotoxic concentration (TC50) towards eukaryotic host cells to the MIC50 values of the testing antimicrobial peptides. The studied SAMPs appeared to be the most active against the pathogenic yeast fungus C. albicans and the bacterial strains St. aureus and P. aeruginosa. The SI values in these cases exceed 40. Some assumed molecular interactions of the studied SAMPs on the microbial cells have been considered, and possible pathways to increase their antimicrobial activity have been suggested. The proposed SAMPs can serve as a basis for the design and synthesis of new promising synthetic antimicrobial agents.

Four lytic Proteus bacteriophages, PM75, PM85, PM93, and PM116, which are active against multi-drug-resistant strains of P. mirabilis, were isolated from cattle and poultry samples. According to electron microscopy data, all of the investigated phages belonged to the family Podoviridae. They all demonstrated lytic activity against sensitive strains of P. mirabilis, and three of the phages, PM85, PM93, and PM116, are potential candidates for use in antibacterial treatment. The genomes and putative proteins of bacteriophages PM85, PM93, and PM116 were similar to those of Proteus phage vB_PmiP_Pm5460 [KP890822], and the investigated phages formed a distinct clade within the genus Sp6virus, subfamily Autographivirinae. The genome sequence of phage PM75 was similar to that of a previously described Proteus phage, PM16 [KF319020], and both of them demonstrated low nucleotide sequence identity to the genomes of the other most similar phages, namely, Vibrio phage VP93, Pantoea phage LIMElight, and KP34-like bacteriophages. According to cluster analysis of the complete genome sequences and phylogenetic analysis of the proteins essential for their life cycle, phages PM75 and PM16 are distinct from other similar phages from the phiKMV supergroup and should be recognized as constituting a new genus, "Pm16virus", within the subfamily Autographivirinae.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is a causative agent of a severe neurological disease. There are three main TBEV subtypes: the European (TBEV-Eu), Far Eastern (TBEV-FE), and Siberian (TBEV-Sib). Currently, three lineages within TBEV-Sib have been recorded. In this study, the genetic and biological characteristics of a new original strain, TBEV-2871, isolated in the Novosibirsk province of Western Siberia, Russia were investigated. The strain has low neuroinvasiveness in mice. Phylogenetic analysis demonstrated that TBEV-2871 belongs to TBEV-Sib, but does not cluster with any of the TBEV-Sib lineages. The TBEV-2871 strain has 88-89% nucleotide sequence identity with the other TBEV-Sib strains, 84-86% nucleotide sequence identity with the TBEV-FE and TBEV-Eu subtypes and is genetically close to the subtype division border. The TBEV-2871 polyprotein sequence includes 43 unique amino acid substitutions, 30 of which are recorded at positions that are conserved among all TBEV subtypes. Strain TBEV-2871 and two similar but not identical isolates found in Kemerovo province, Western Siberia are separated into a new lineage tentatively named Obskaya after the name of Ob riber, in the vicinity of which the TBEV-2871 was first found. A molecular evolution investigation demonstrated that within TBEV-Sib, the Obskaya lineage likely separated 1535years ago, which is even earlier than the Baltic lineage.

Tick-borne encephalitis virus (TBEV) is divided into three subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE) subtypes. The geographical range of TBEV-Eu dominates in Europe, but this subtype is present focally across the whole non-tropical forested Eurasian belt, through Russia to South Korea. However, the TBEV-Eu strains isolated outside Europe remain poorly characterized. In this study, full-genome sequences of eight TBEV-Eu isolates were determined. These strains were isolated from Ixodes persulcatus ticks, long-tailed ground squirrel ( Spermophilus undulatus ), and human blood in the natural foci of Western and Eastern Siberia, Russia. A phylogenetic analysis of all available TBEV-Eu genomic sequences revealed that strains from Siberia were closely related to other strains from Europe and South Korea. The closest relation was identified between the Siberian strains and strains from Zmeinogorsk (Western Siberia, Russia) and strain Absettarov (Karelia, Russia), and were most divergent from strains from the Czech Republic and Norway. TBEV-Eu strains isolated in Eastern Siberia were more closely related phylogenetically to strains from South Korea, but strains from Western Siberia grouped together with the strains from Europe, suggesting two genetic TBEV-Eu lineages present in Siberia.

Background The Ixodes pavlovskyi tick species, a member of the I. persulcatus/I. ricinus group, was discovered in the middle of the 20th century in the Russian Far East. Limited data have been reported on the detection of infectious agents in this tick species. The aim of this study was to investigate the prevalence and genetic variability of a wide range of infectious agents in I. pavlovskyi ticks collected in their traditional and recently invaded habitats, the Altai Mountains and Novosibirsk Province, respectively, which are both located within the Western Siberian part of the I. pavlovskyi distribution area. Results This study reports the novel discovery of Borrelia bavariensis, Rickettsia helvetica, R. heilongjiangensis, R. raoultii, “Candidatus Rickettsia tarasevichiae”, Anaplasma phagocytophilum, Ehrlichia muris, “Candidatus Neoehrlichia mikurensis” and Babesia microti in I. pavlovskyi ticks. In addition, we confirmed the previous identification of B. afzelii, B. garinii and B. miyamotoi, as well as tick-borne encephalitis and Kemerovo viruses in this tick species. The prevalence and some genetic characteristics of all of the tested agents were compared with those found in I. persulcatus ticks that were collected at the same time in the same locations, where these tick species occur in sympatry. It was shown that the prevalence and genotypes of many of the identified pathogens did not significantly differ between I. pavlovskyi and I. persulcatus ticks. However, I. pavlovskyi ticks were significantly more often infected by B. garinii and less often by B. bavariensis, B. afzelii, “Ca. R. tarasevichiae”, and E. muris than I. persulcatus ticks in both studied regions. Moreover, new genetic variants of B. burgdorferi (sensu lato) and Rickettsia spp. as well as tick-borne encephalitis and Kemerovo viruses were found in both I. pavlovskyi and I. persulcatus ticks. Conclusion Almost all pathogens that were previously detected in I. persulcatus ticks were identified in I. pavlovskyi ticks; however, the distribution of species belonging to the B. burgdorferi (sensu lato) complex, the genus Rickettsia, and the family Anaplasmataceae was different between the two tick species. Several new genetic variants of viral and bacterial agents were identified in I. pavlovskyi and I. persulcatus ticks.

Klebsiella pneumoniae is a common pathogen, associated with a wide spectrum of infections, and clinical isolates of K. pneumoniae often possess multiple antibiotic resistances. Here, we describe a novel lytic N4-like bacteriophage KP8, specific to K. pneumoniae, including its genome, partial structural proteome, biological properties, and proposed taxonomy. Electron microscopy revealed that KP8 belongs to the Podoviridae family. The size of the KP8 genome was 73,679 bp, and it comprised 97 putative open reading frames. Comparative genome analysis revealed that the KP8 genome possessed the highest similarity to the genomes of Enquatrovirus and Gamaleyavirus phages, which are N4-like podoviruses. In addition, the KP8 genome showed gene synteny typical of the N4-like podoviruses and contained the gene encoding a large virion-encapsulated RNA polymerase. Phylogenetic analysis of the KP8 genome revealed that the KP8 genome formed a distinct branch within the clade, which included the members of Enquatrovirus and Gamaleyavirus genera besides KP8. The average evolutionary divergences KP8/Enquatrovirus and KP8/Gamaleyavirus were 0.466 and 0.447 substitutions per site (substitutes/site), respectively, similar to that between Enquatrovirus and Gamaleyavirus genera (0.468 substitutes/site). The obtained data suggested that Klebsiella phage KP8 differs from other similar phages and may represent a new genus within the N4-like phages.

Parechovirus (the Picornaviridae family), recently classified as Parechovirus A and formerly known as Human parechovirus (HPeV), can cause a wide range of human diseases. Over the past decade, several studies have reported HPeV epidemiology in different regions; however, information from Russia is limited. A total of 632 stool samples collected in Novosibirsk, Russia during January–March 2012 were screened for HPeV by RT-PCR. The study cohort comprised 572 patients with acute gastroenteritis and 60 healthy children. Seven of 572 (1.2%) gastroenteritis cases were HPeV-positive, including one co-infection with rotavirus and astrovirus. All positive patients were ≤1 year old, and five of them were younger than 3 months. None of the healthy controls provided an HPeV-positive sample. Six HPeV isolates were classified as HPeV-1 and one as HPeV-5 using phylogenetic analysis. Two complete genome sequences of HPeV-1 and one of HPeV-5 were determined and analyzed. Phylogenetic analysis showed that the studied Russian strains are probably recombinants. P1 region sequences of two Russian HPeV-1 strains clustered with rare contemporary HPeV-1A strains, whereas their P3 regions were phylogenetically closer to the archival Harris strain. The Russian HPeV-5 strain formed a common cluster with other HPeV-5 strains only for the P1 region, while the P3 region grouped with the German HPeV-2 strain. In the Russian HPeV-5 strain, the lack of the arginine-glycine-aspartic acid (RGD) motif at the C-terminus of VP1 was observed. This is the first complete genome characterization of the Russian HPeV strains detected in sporadic cases of pediatric acute gastroenteritis.

International audience; β-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-β-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-β-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing β-glucans with different lengths of oligo-β-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a β-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-β-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these β-glucan-specific mAbs could be useful in combinatorial antifungal therapy.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.

The glycosylation profile of the chimeric antibody ch14D5a against the tick-borne encephalitis virus has been analyzed. It has been found that the ch14D5a antibody is completely N-glycosylated at the asparagine 297 residue of both heavy chains, and the major glycoforms correspond supposedly to glycoforms G0F, G1F, and G2F, which are most typical for human immunoglobulins IgG and for antibodies secreted by CHO cells.

The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites.

Infectious diseases caused by bacterial or viral agents represent the major cause of human pathogenesis and mortality worldwide. A development of novel antibacterial therapeutics and diagnostic tools is a very acute task. The use of DNA and RNA aptamers targeted to certain bacteria could be a promising solution to this problem. Here, we propose a new protocol of selection of 2′-fluoro RNA aptamers capable to internalize into bacterial cells. Using whole-cell SELEX against Pseudomonas aeruginosa, enriched 2′-fluoro RNA library was obtained, and its sequencing and data analysis were fulfilled. It was found that the central region of predominating aptamer sequence is identical to the fragment of P. aeruginosa rRNA. A possibility of internalizing of this aptamer into bacterial cells is shown. It is hypothesized that aptamers could be internalized more effectively as heterodimeric complexes.

The aim of the study was to obtain the complex characteristics of tick-borne encephalitis virus (TBEV) of European subtype circulating in Western and Eastern Siberia. Using full-genome sequencing approach it was demonstrated that TBEV strains of European subtype isolated in Siberia are genetically similar to the strains from European part of its habitat range, and with the representatives from South Korea. It was confirmed that the homology of TBEV strains of European subtype isolated in different parts of the virus habitat area from Scandinavian countries in the west to the eastern borders of the area (South Korea) is much higher than the homology level of TBEV strains of Far Eastern and Siberian subtypes. The Siberian population of TBEV of European subtype is presented with two groups of strains called as Eastern Siberian and Western Siberian variants, which differ in the combinations of amino acid substitutions in all proteins except NS2B protein. It is found that TBEV strains of European subtype from Siberia possess high neurovirulence, but some of them, like strains from Europe, demonstrate low invasiveness. It is shown that TBEV strains of European subtype have good adaptive capacity, and therefore, can easily adapt to the circulation in various biocenoses in the territory of different landscape-geographical zones. It was found that the circulation of TBEV of European subtype is fixed in Siberia territory for over 40 years. It was emphasized that in spite of circulation of TBEV of European subtype in the significantly different areas by climatic conditions, topography, landscape, habitat characteristics it possesses a high degree of genome stability.

Ixodiae ticks have been counted and sampled in six distant locations within the Altai-Sayan Mountain Area and northern forest steppe in 2012. In total, 1829 individuals of I. persulcatus and 719 of I. pavlovskyi ticks were collected by flagging. I. persulcatus is found in all six examined locations and I. pavlovskyi is found in Western Altai, Northeastern Altai, and Northern Altai, as well as in forest parks near Akademgorodok of Novosibirsk. In total, 16S rRNA and COI sequences have been determined in 144 and 164 I. persulcatus individuals, as well as in 60 and 55 I. pavlovskyi. Selectively equivalent mutant forms are present in I. pavlovskyi populations of Northeastern Altai and Northern Altai. The hydrothermal regime in the forest parks near Novosibirsk is optimal for the I. pavlovskyi ticks. These ticks have recently colonized the forest parks near Novosibirsk and therefore are genetically close to I. pavloskyi from the Altai–Sayan Mountain Area populations.

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2 h. Furthermore, compounds 1a–c with aromatic linkers and compound 1e showed the highest antiviral activity.

Rickettsia spp. are intracellular Gram-negative bacteria transmitted by arthropods. Two potentially pathogenic rickettsiae, Candidatus Rickettsia tarasevichiae and Rickettsia helvetica, have been found in unfed adult Ixodes persulcatus ticks. The aim of this study was to assess the prevalence and genetic variability of Rickettsia spp. in I. persulcatus ticks collected from different locations in the Russian Far East. In total, 604 adult I. persulcatus ticks collected from four sites in the Khabarovsk Territory (continental area) and one site in Sakhalin Island were examined for the presence of Rickettsia spp. by real-time PCR. Nested PCR with species-specific primers and sequencing were used for genotyping of revealed rickettsiae. The overall prevalence of Rickettsia spp. in ticks collected in different sites varied from 67.9 to 90.7%. However, the proportion of different Rickettsia species observed in ticks from Sakhalin Island significantly differed from that in ticks from the Khabarovsk Territory. In Sakhalin Island, R. helvetica prevailed in examined ticks, while Candidatus R. tarasevichiae was predominant in the Khabarovsk Territory. For gltA and ompB gene fragments, the sequences obtained for Candidatus R. tarasevichiae from all studied sites were identical to each other and to the known sequences of this species. According to sequence analysis of gltA, оmpB and sca4 genes, R. helvetica isolates from Sakhalin Island and the Khabarovsk Territory were identical to each other, but they differed from R. helvetica from other regions and from those found in other tick species. For the first time, DNA of pathogenic Rickettsia heilongjiangensis was detected in I. persulcatus ticks in two sites from the Khabarovsk Territory. The gltA, ompA and оmpB gene sequences of R. heilongjiangensis were identical to or had solitary mismatches with the corresponding sequences of R. heilongjiangensis found in other tick species.

Rotaviruses C (RVC) cause sporadic cases and outbreaks of diarrhea in humans and animals worldwide. The aim of this study was to monitor RVC during a surveillance study of sporadic cases of viral gastroenteritis in the Novosibirsk and Omsk regions of Russia from 2006 to 2011. A total of 2144 stool samples from children and adults hospitalized with acute gastroenteritis were tested for RVC by RT-PCR. Sixteen RVC-positive stool samples were detected at a rate of 0.6% (13/2037) in children and 2.8% (3/107) in adults. The low detection rate suggested that RVC infection was an uncommon cause of hospitalization in Russia. The complete VP7, VP4, VP6, and NSP4 gene sequences were determined. It was found that RVCs with at least two different genome backgrounds circulated in Siberia. VP4, VP6, and NSP4 gene sequences of most Russian RVC strains clustered with South Asian strains, while the VP7 gene showed a closer relationship to European strains. Meanwhile, only VP4 and NSP4 sequences of the strain Omsk08-386 clustered with South Asian strains, while its VP6 and VP7 sequences clustered with European strains. This is the first genetic characterization of Russian RVC strains and the first report on the prevalence of RVC in the Asian part of Russia.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

Candida albicans is becoming increasingly harmful for humans, which determines the need for new effective antifungal preparations. Currently, when testing antifungals, various morphological methods are used, among which transmission electron microscopy (TEM) is not the leading one. In this work, we used TEM to study the submicroscopic changes in C. albicans cells induced by cationic peptides R9F2 and (KFF)3K. Studies were performed on C. albicans-34 strain from the Collection of EMTC of ICBFM SB RAS in logarithmic phase. R9F2 and (KFF)3K showed an antifungal effect (MIC 10 and 20 &mu, M) and suppressed fungal hyphal growth. Semithin and ultrathin sections of fungal suspensions incubated with 10 &mu, M of peptides were studied at regular intervals from 15 min to 24 h. The first target of both peptides was plasmalemma, and its &ldquo, alignment&rdquo, was the only common morphological manifestation of their effect. Other changes in the plasmalemma and alteration of the vacuole and cell wall ultrastructure distinctly differed in cells treated with R9F2 and (KFF)3K peptides. In general, our work has shown pronounced differences of the temporal and morphologic characteristics of the effect of peptides, evidently related to their physicochemical properties. The benefit of TEM studies of ultrathin sections for understanding the mechanisms of action of antifungal drugs is shown.

The tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is currently subdivided into three main subtypes-the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberian (TBEV-Sib). The TBEV-Sib is the most common subtype and found in all regions where TBEV was detected, except for Central and Western Europe. Currently, four genetic lineages have been described within TBEV-Sib. In this study, detailed analysis of TBEV-Sib genetic diversity, geographic distribution, phylogeography and divergence time of different TBEV-Sib genetic lineages based on E gene fragments, complete genome sequences, and all currently available data in the GenBank database was performed. As a result, a novel Bosnia lineage within the TBEV-Sib was identified. It was demonstrated that the Zausaev lineage is the most widely distributed among the TBEV-Sib lineages, and was detected in all studied regions except the Far East. The Vasilchenko lineage was found from Western Siberia to the Far East. The Baltic lineage is presented from Europe to Western Siberia. The Obskaya lineage was found only in Western Siberia. TBEV strains from a newly described Bosnia lineage were detected in Bosnia, the Crimean peninsula, Kyrgyzstan and Kazakhstan. The greatest divergence of the TBEV-Sib genetic variants was observed in Western Siberia. Within the TBEV-Sib, the Obskaya lineage diverged from the common ancestor the earliest, after that the Bosnia lineage was separated, then the Baltic lineage, and the Zausaev and Vasilchenko lineages diverged most recently.

North Asian tick-typhus (NATT), also known as Siberian tick typhus, is the main tick-borne rickettsiosis in Siberia, Russia. Recently, a fatal infection in a four-year-old girl with typical tick-borne rickettsiosis symptoms (fever, rash, eschar at the site of the tick bite, myalgia) and meningeal syndrome was registered. In order to identify the etiology of this infection, blood and brain samples from the patient were examined for the presence of a wide range of tick-transmitted agents and enteric viruses by polymerase chain reaction with subsequent sequencing. Rickettsia sibirica subsp. sibirica and "Candidatus Rickettsia tarasevichiae" DNA was identified in both blood and brain samples. Rickettsia sibirica was characterized by the gltA, ompA and ompB genes, and "Candidatus R. tarasevichiae" was characterized by the gltA and ompB genes. We report the first case of mixed R. sibirica and "Candidatus R. tarasevichiae" human infection with a fatal outcome in Russia.

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatus and I. pavlovskyi ticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatus and I. pavlovskyi ticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis", and Babesia microti. The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas.

Background Species of Canidae in Russia can be infested with up to 24 different tick species; however, the frequency of different tick species infesting domestic dogs across Russia is not known. In addition, tick-borne disease risks for domestic dogs in Russia are not well quantified. The goal of this study was to conduct a nationwide survey of ticks collected from infested dogs admitted to veterinary clinics in Russian cities and to identify pathogens found in these ticks. Methods Ticks feeding on dogs admitted to 32 veterinary clinics in 27 major cities across Russia were preserved in ethanol and submitted to a central facility for examination. After identification, each tick was evaluated for infection with known tick-borne pathogens using PCR. Results There were 990 individual ticks collected from 636 dogs. All collected ticks belonged to the Ixodidae (hard ticks) and represented 11 species of four genera, Dermacentor, Ixodes, Rhipicephalus and Haemaphysalis. Four most common tick species were D. reticulatus, followed by I. persulcatus, I. ricinus and R. sanguineus. Ixodes persulcatus ticks were found to be infected with 10 different pathogens, and ticks of this species were more frequently infected than either D. reticulatus or I. ricinus. Ixodes persulcatus females were also more frequently co-infected with two or more pathogens than any other tick. Pathogenic species of five genera were detected in ticks: Anaplasma centrale, A. phagocytophilum and A. marginale; Babesia canis, B. microti, B. venatorum, B. divergens, B. crassa and B. vogeli; Borrelia miyamotoi, B. afzelii and B. garinii; Ehrlichia muris, E. canis and E. ruminantum; and Theileria cervi. Anaplasma marginale, E. canis, B. crassa, B. vogeli and T. cervi were detected in I. persulcatus, and Babesia canis in D. marginatum, for the first time in Russia. Conclusions Multiple ticks from four genera and 11 species of the family Ixodidae were collected from domestic dogs across Russia. These ticks commonly carry pathogens and act as disease vectors. Ixodes persulcatus ticks present the greatest risk for transmission of multiple arthropod-borne pathogens.

Genetic variability of I. apronophorus from Western Siberia, Russia was examined using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes and compared to those of Ixodes persulcatus and Ixodes trianguliceps from the same site. The I. apronophorus sequences demonstrated the highest nucleotide and haplotype diversity for both mitochondrial genes, whereas I. persulcatus was more variable in the nuclear ITS2. Phylogenetic analysis of the molecular sequence data showed that I. apronophorus differed from other Ixodes species, including Romanian I. apronophorus. The level of identity between 16S rRNA gene sequences of Siberian and Romanian I. apronophorus was only 91%; these sequences did not form a monophyletic group, indicating that I. apronophorus from Siberia and Romania could be different tick species. The analysis of morphological features of the Siberian I. apronophorus confirmed their consistency with those for the previously described I. apronophorus species. Based on the 16S rRNA and ITS2 sequences, Siberian I. apronophorus clustered together with Ixodes kazakstani and Ixodes scapularis, which are the recognized members of the Ixodes ricinus-I. persulcatus species complex within the subgenus Ixodes, and can be assigned to this complex.

In the recent years, multidrug-resistant bacteria have become a global threat, and phage therapy may to be used as an alternative to antibiotics or, at least, as a supplementary approach to treatment of some bacterial infections. Here, we describe the results of bacteriophage application in clinical practice for the treatment of localized infections in wounds, burns, and trophic ulcers, including diabetic foot ulcers. This mini-review includes data from various studies available in English, as well as serial case reports published in Russian scientific literature (with, at least, abstracts accessible in English). Since, it would be impossible to describe all historical Russian publications; we focused on publications included clear data on dosage and rout of phage administration.

Tick-borne encephalitis (TBE) is an illness caused by tick-borne encephalitis virus (TBEV) infection which is often limited to a febrile illness, but may lead to very aggressive downstream neurological manifestations. The disease is prevalent in forested areas of Europe and northeastern Asia, and is typically caused by infection involving one of three TBEV subtypes, namely the European (TBEV-Eu), the Siberian (TBEV-Sib), or the Far Eastern (TBEV-FE) subtypes. In addition to the three main TBEV subtypes, two other subtypes; i.e., the Baikalian (TBEV-Bkl) and the Himalayan subtype (TBEV-Him), have been described recently. In Europe, TBEV-Eu infection usually results in only mild TBE associated with a mortality rate of2%. TBEV-Sib infection also results in a generally mild TBE associated with a non-paralytic febrile form of encephalitis, although there is a tendency towards persistent TBE caused by chronic viral infection. TBE-FE infection is considered to induce the most severe forms of TBE. Importantly though, viral subtype is not the sole determinant of TBE severity; both mild and severe cases of TBE are in fact associated with infection by any of the subtypes. In keeping with this observation, the overall TBE mortality rate in Russia is ∼2%, in spite of the fact that TBEV-Sib and TBEV-FE subtypes appear to be inducers of more severe TBE than TBEV-Eu. On the other hand, TBEV-Sib and TBEV-FE subtype infections in Russia are associated with essentially unique forms of TBE rarely seen elsewhere if at all, such as the hemorrhagic and chronic (progressive) forms of the disease. For post-exposure prophylaxis and TBE treatment in Russia and Kazakhstan, a specific anti-TBEV immunoglobulin is currently used with well-documented efficacy, but the use of specific TBEV immunoglobulins has been discontinued in Europe due to concerns regarding antibody-enhanced disease in naïve individuals. Therefore, new treatments are essential. This review summarizes available data on the pathogenesis and clinical features of TBE, plus different vaccine preparations available in Europe and Russia. In addition, new treatment possibilities, including small molecule drugs and experimental immunotherapies are reviewed. The authors caution that their descriptions of approved or experimental therapies should not be considered to be recommendations for patient care.

This hospital-based surveillance study was carried out in Novosibirsk, Asian Russia from September 2009 to December 2012. Stool samples from 5486 children with diarrhea and from 339 healthy controls were screened for rotavirus, norovirus, astrovirus, and bocavirus by RT-PCR. At least one enteric virus was found in 2075 (37.8%) cases with diarrhea and 8 (2.4%) controls. In the diarrhea cases, rotavirus was the most commonly detected virus (24.9%), followed by norovirus (13.4%), astrovirus (2.8%) and bocavirus (1.1%). Mixed viral infections were identified in 4.3% cases. The prevalence of enteric viruses varied every season. Rotavirus infection was distributed in a typical seasonal pattern with a significant annual increase from November to May, while infections caused by other viruses showed no apparent seasonality. The most common rotavirus was G4P[8] (56%), followed by G1P[8] (20.1%), G3P[8] (5.5%), G9P[8], G2P[4] (each 1.3%), six unusual (1.2%), and five mixed strains (0.5%). Norovirus GII.3 (66.5%) was predominant, followed by GII.4 (27.3%), GII.6 (3.7%), GII.1 (1.6%), and four rare genotypes (totally, 0.9%). Re-infection with noroviruses of different genotypes was observed in four children. The classic human astrovirus belonged to HAstV-1 (82%), HAstV-5 (8%), HAstV-4 (4.7%), HAstV-3 (4%) and HAstV-2 (1.3%). Consecutive episodes of HAstV-1 and HAstV-4 infections were detected in one child with an 8-month interval. Bocavirus strains were genotyped as HBoV2 (56.5%), HBoV1 (38.7%), HBoV4 (3.2%) and HBoV3 (1.6%). In the controls, norovirus strains belonged to GII.4 (n = 4), GII.1, GII.3, and GII.6, and HBoV2 strain were detected. Most of the detected virus isolates were characterized by a partial sequencing of the genomes. The genotype distribution of most common enteric viruses found in the Asian part of Russia did not differ considerably from their distribution in European Russia in 2009–2012.

The Russian Far East is an endemic region for tick-borne rickettsioses. However, the prevalence and genetic variability of Rickettsia species in this region have not been extensively investigated. In this study, 188 Dermacentor silvarum, 439 Haemaphysalis concinna, and 374 Haemaphysalis japonica adult ticks were collected from four locations in Khabarovsk Province and three locations in Amur Province in the Russian Far East. These ticks were examined for the presence of Rickettsia spp. by amplifying a fragment of the gltA gene. Identified rickettsiae were genotyped by sequencing of the gltA, 16S rRNA, ompA, ompB, and sca4 genes. In the examined ticks, Rickettsia heilongjiangensis, the causative agent of Far-Eastern tick-borne rickettsiosis, was found in 10.5% of H. concinna and in 1.9% of H. japonica ticks, while Rickettsia sibirica, the agent of Siberian tick typhus, was detected in only one H. concinna tick. In addition, Rickettsia raoultii was found predominantly in D. silvarum (>70%) and significantly less frequently in Haemaphysalis ticks (

In southern regions of Western Siberia, Ixodes persulcatus ticks co-exist with Ixodes pavlovskyi. Both tick species have similar morphology, and natural hybridization of I. persulcatus and I. pavlovskyi ticks has been observed. To investigate the role of I. pavlovskyi ticks and I. persulcatus/ I. pavlovskyi hybrids as vectors, correct identification of tick species is necessary. The nuclear crt gene might be useful in distinguishing I. persulcatus from I. pavlovskyi ticks. Genetic variability in this gene has been studied and substantial differences between crt gene sequences of I. persulcatus and I. pavlovskyi ticks has been demonstrated. In this study, maximum-likelihood analysis showed that sequences of I. persulcatus and I. pavlovskyi ticks were identical or highly homologous among themselves. These results were confirmed by a Bayesian phylogeny. We conclude that crt gene sequences of I. persulcatus and I. pavlovskyi cannot be used for distinguishing these tick species and that the conflicting results of prior studies reflect samples from incorrectly identified ticks rather than real genetic differences between I. persulcatus and I. pavlovskyi.

We investigated whether levels and repertoires of anti-interleukin-18 (IL-18) autoantibodies (auto-Abs) differ in multiple sclerosis (MS) patients and healthy donors (HDs). IL-18 concentration in MS patients' sera was higher than in HD, but the level of anti-IL-18 auto-Abs was lower in MS patients. Correlation patterns of IL-18/anti-IL-18 auto-Abs system differed in HD and MS patients, so we have compared segment composition of the anti-IL-18 single-chain variable fragments (scFvs) selected from MS and naïve phage display libraries. Considerable differences between anti-IL-18 auto-Abs of these libraries were found. MS panel contained auto-Abs displaying both signs of "fetal" and somatically hypermutated repertoires. Naïve panel mainly contained the naïve antibodies. These variations from the norm are possible results of abnormal regulation of the repertoire perhaps determined by remodeling of the molecular mechanisms involved in the V(D)J recombination and/or abnormal selection by antigen in MS pathogenesis.

The seasonality of epidemic and epizootic processes of tick-borne encephalitis (TBE) and borreliosis among the populations of Ixodes persulcatus and Ixodes pavlovskyi ticks was studied. The study was carried out in 2011–2012 on the flatland part of Toguchin and in the vicinities of Akademgorodok in Novosibirsk, on the border between the forest and forest-steppe zones of the Ob region. The results obtained for I. persulcatus did not contradict the previously published studies. The starting point and peak of mass activity of I. pavlovskyi ticks were registered later (the last ten days of May) than that of I. persulcatus ticks. Seasonal variations in the physiological age of I. pavlovskyi ticks were similar to that of I. persulcatus ticks. The only exception to this was detected in the last ten days of April when young specimens prevailed in the population of I. pavlovsky ticks in contrast to the I. persulcatus population. The large influence of I. persulcatus on the circulation of TBE was shown, as well as the influence of I. pavlovskyi on the spreading of Borrelia burgdorferi s.l.

Diabetic foot ulcers occur as a common complication of diabetes. Healing of the ulcers is largely delayed by the concomitant infection. Antibiotic treatment of infected ulcers is complicated by formation of microbial biofilms , which are often heterogeneous and resistant to antibiotics. Bacteriophage therapy is considered as an additional approach to the treatment of infected wounds. Here, we describe the basic method of application of bacteriophages for treatment of infected diabetic foot ulcers, including ones that are very large.

BACKGROUND This is a prospective, multicentered study conducted in 9 large urban areas in Russia, in order to determine the burden of rotavirus gastroenteritis in children

Kemerovo virus (KEMV), a member of the Reoviridae family, Orbivirus genus, is transmitted by Ixodes ticks and can cause aseptic meningitis and meningoencephalitis. Recently, this virus was observed in certain provinces of European part of Russia, Ural, and Western and Eastern Siberia. However, the occurrence and genetic diversity of KEMV in Western Siberia remain poorly studied. Therefore, the aim of this work was to investigate the prevalence and genetic variability of KEMV in Ixodes ticks from Western Siberia. A total of 1958 Ixodes persulcatus, I. pavlovskyi ticks and their hybrids from Novosibirsk and Omsk provinces, Altai Republic (Russia) and East Kazakhstan province (Kazakhstan) were analyzed for the presence of KEMV and tick-borne encephalitis virus (TBEV) RNA. It was observed that the KEMV distribution area in Western Siberia was wider than originally thought and included Northern and Northeastern Altai in addition to the Omsk and Novosibirsk provinces. For the first time, this virus was found in Kazakhstan. The occurrence of KEMV was statistically lower than TBEV in most locations in Western Siberia. KEMV was found both in I. persulcatus and I. pavlovskyi ticks and in their hybrids. Notably, KEMV variants observed in the 2010s were genetically different from those isolated in the 1960s, which indicated the ongoing process of evolution of the Kemerovo virus group. Moreover, the possibility of reassortment for KEMV was demonstrated for the first time.