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Wild animals are reservoir hosts for a number of tick-transmitted agents, and long-term persistence of the agents is a key factor for their effective transmission from animal hosts to ticks. To study the persistence of Anaplasmataceae in rodents, 59 adult Myodes spp. voles (M. rutilus, M. rufocanus, and M. glareolus) were captured in Omsk Province, Asian Russia, freed from all ectoparasites, and kept in individual cages. Their blood samples were regularly analyzed for the presence of DNA of Anaplasma phagocytophilum, Ehrlichia muris, and "Candidatus Neoehrlichia mikurensis". Anaplasma phagocytophilum, E. muris, and mixed infections were found in 29 (49 %), 17 (29 %), and 14 (24 %) voles, respectively. DNA of "Candidatus N. mikurensis" was not identified. Long-term persistence of A. phagocytophilum and E. muris in naturally infected Myodes spp. was shown for the first time. Thus, 12 animals with A. phagocytophilum were found infected for more than three months and four of them carried the bacterium throughout 22-55 weeks of observation. Four voles were infected with E. muris for 26-40 weeks. Fragments of groESL operon were sequenced for genotyping A. phagocytophilum and E. muris. In voles with A. phagocytophilum, the determined sequences belonged to two phylogenetic clusters. Most sequences were identical or closely related to those found in small mammals and Ixodes trianguliceps ticks from Western Siberia and European countries; one sequence was previously identified in rodents and Ixodes persulcatus and Ixodes pavlovskyi ticks from Asian Russia. Sequences of groESL fragments from E. muris were close to those determined previously in small mammals and I. persulcatus ticks from Siberia. In addition, a new groESL sequence of E. muris was identified in one vole and the sequence substantially differed from all known corresponding E. muris sequences (≥ 14 mismatches).

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.

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.

Blood samples were taken from 541 small mammal captured in 2013–2015 in Znamensky district of Omsk region from Ixodes persulcatus and Ixodes trianguliceps sympatric area and examined for the Babesia spp. presence by nested PCR with subsequent sequencing of positive samples. Babesia microti DNA was found in 31,1 % of positive samples; a proportion of infected mammals varied from 5,3 % to 61,6 % in different sampling periods. B. microti DNA was found in samples from three prevailing Myodes species as well as from a root vole (Microtus oeconomus), field voles (Microtus argestis) and Siberian chipmunks (Tamias sibiricus). It was shown that identified B. microti samples belong to two genetic groups: B. microti ‘US’-type and B. microti ‘Munich’-type; notably that > 90 % infected mammals contained DNA of nonpathogenic for human B. microti ‘Munich’-type. We suppose that I. trianguliceps tick is the most probable vector of B. microti ‘Munich’-type.

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.

Implant-associated infection (IAI) remains a significant issue in orthopaedic surgery. Bacteriophages are naturally occurring viruses that kill bacteria. Bacteriophages are being considered as a reliable alternative and/or adjunct to antibiotics, especially with the rise of multidrug-resistant bacterial strains. The isolation of bacteriophages from the environment against major bacterial pathogens may be easier than searching for new antibiotics against these virulent bacterial stains. Although bacteriophages are not pathogenic to mammalian cells, they induce a humoral immune response in a mammalian host resulting in the formation of anti-phage antibodies that can influence the effect of phage therapy. We detail the experiences of several centers in Poland, the Republic of Georgia, the Russian Federation, and France that have extensive experience with phage therapy for IAI. Phage therapy is an emerging new paradigm for the treatment of IAI as multidrug-resistant bacteria become more common and national governments as well as global health agencies push to improve antibiotic stewardship in clinical practice.

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.

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.

The most epidemiologically significant tick species in Siberia involved in transmission of a large number of pathogens causing human infectious diseases is Ixodes persulcatus. Ixodes pavlovskyi, being more active, also poses epidemiological threats. These tick species share morphology, activity seasons and geographic distribution range. In this paper, we characterize the geographic and genetic structures of I. persulcatus and I. pavlovskyi populations inhabiting the southern part of Western Siberia (Russia and Kazakhstan)—the western part of I. pavlovskyi distribution range. The data are based on six distinct Ixodes tick populations. Analysis of the concatenated mitochondrial marker sequences (16S rRNA and COI) and the nuclear sequence (ITS2) showed genetic polymorphisms in both I. persulcatus and I. pavlovskyi ticks inhabiting the sympatric zone. We could not determine the phylogeographic structure of I. pavlovskyi populations whereas for I. persulcatus significant within-region variance was shown. Notably, the abundance of I. persulcatus ticks negatively correlates with nucleotide and haplotype diversity in the concatenated sequence of mitochondrial gene (16S rRNA and COI) fragments. This is the first description of the genetic polymorphism of I. persulcatus and I. pavlovskyi ticks coexisting in a sympatric zone based on analysis of mitochondrial and nuclear markers.

Recently, a new Ehrlichia genetic variant, Ehrlichia sp. Khabarovsk, was identified in tissue samples of small mammals captured in the Russian Far East. To further characterize Ehrlichia sp. Khabarovsk, tissue homogenate from a naturally infected gray red-backed vole ( Myodes rufocanus ) was passaged three times in newborn laboratory mice. Using nested PCR Ehrlichia sp. Khabarovsk DNA was detected in tissue samples from infected mice at 1–4 weeks post inoculation. Electron microscopic examination revealed morulae containing gram-negative bacterial cells in monocytes of mouse spleen and liver. The size and ultrastructure of these cells corresponded to those described previously and allowed us to identify the bacteria as Ehrlichia sp. The comparison of ehrlichial 16S rRNA, groEL and gltA genes and putative GroEL and GltA amino acid sequences has demonstrated that Ehrlichia sp. Khabarovsk, like Ehrlichia ruminantium , is more distant from all other Ehrlichia species than these species are between themselves. Phylogenetic analysis has shown that Ehrlichia sp. Khabarovsk belongs to the clade formed by Ehrlichia spp. but clusters separately from other Ehrlichia species and genetic variants. These data indicate that Ehrlichia sp. Khabarovsk can be considered as a new candidate species. We propose to designate it as ‘ Candidatus Ehrlichia khabarensis’ according to the territory where this species was found.

Antibacterial activity of tetracationic compounds based on 1,4-diazabicyclo[2.2.2]octane (DABCO) bis-quaternary salts with dodecyl substituents bound by linker groups containing four carbon atoms was studied. All the compounds exhibited activity against the test-strains under study. The gram-positive culture S. aureus was found to be the most sensitive to the influence of compounds in question. The highest activity against S. aureus was exhibited by the compounds containing a but-2-yne or a butane linker; their efficiency exceeded that of the reference compound, antibiotic Ciprofloxacin. For the gram-negative culture P. aeruginosa, the highest activity was exhibited by the compound containing a but-2-yne linker. The compounds with a but-2-yne or a butane linker are stable in the presence of nitrogen-containing nucleophiles, which makes them promising for further studies.

A series of new quaternary 1,4-diazabicyclo[2.2.2]octane derivatives was synthesized and evaluated for activity against several strains of both Gram positive and Gram negative bacteria and one strain of fungus under different inoculum size. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six species of microorganisms were tested. Results show a clear structure–activity relationship between alkyl chain length of substitutions of 1,4-diazabicyclo[2.2.2]octane tertiary amine sites and antimicrobial activity. In the case of compounds 4a–4k, MIC was found to decrease with the increase of the alkyl chain length from ethyl to dodecyl and then to increase at higher chain length (n > 14). The MIC values were found to be low for the compounds 4f and 4g with alkyl chains ranging from 10 to 12 carbons in length (1.6 μg/ml) and were comparable to the reference drug Ciprofloxacin. Also, time-kill assay was performed to examine the bactericidal kinetics. Results indicated that 4f and 4g had rapid killing effects against Staphylococcus aureus, and eliminated 100% of the initial inoculum of bacteria in 2.5 h at the concentration of 10 μg/ml. In addition, compound 4g eliminate more than 99.9% of the initial inoculum of Ps. aeruginosa after 2.5 h of interaction but the activity of compound 4f against this species seems to be weak. Thus, 4g had strong bactericidal activity and could rapidly kill Gram positive S. aureus, as well as Gram negative Ps. aeruginosa at low and high inoculum size.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.

The majority of human group A rotaviruses possess the P[8] VP4 genotype. Recently, a genetically distinct subtype of the P[8] genotype, also known as OP354-like P[8] or lineage P[8]-4, emerged in several countries. However, it is unclear for how long the OP354-like P[8] gene has been circulating in humans and how it has spread. In a global collaborative effort 98 (near-)complete OP354-like P[8] VP4 sequences were obtained and used for phylogeographic analysis to determine the viral migration patterns. During the sampling period, 1988-2012, we found that South and East Asia acted as a source from which strains with the OP354-like P[8] gene were seeded to Africa, Europe, and North America. The time to the most recent common ancestor (TMRCA) of all OP354-like P[8] genes was estimated at 1987. However, most OP354-like P[8] strains were found in three main clusters with TMRCAs estimated between 1996 and 2001. The VP7 gene segment of OP354-like P[8] strains showed evidence of frequent reassortment, even in localized epidemics, suggesting that OP354-like P[8] genes behave in a similar manner on the evolutionary level as other P[8] subtypes. The results of this study suggest that OP354-like P[8] strains have been able to disperse globally in a relatively short time period. This, in combination with a relatively large genetic distance to other P[8] subtypes, might result in a lower vaccine effectiveness, underscoring the need for a continued surveillance of OP354-like P[8] strains, especially in countries where rotavirus vaccination programs are in place. ispartof: Molecular Biology and Evolution vol:32 issue:8 pages:2060-71 ispartof: location:United States status: published

This study used geological, geochemical, and microbiological data to examine the Uzon oils and conditions within the Uzon caldera. The trace-element compositions of crude oils and solutions from boreholes and hydrothermal springs were determined by ICP-MS. The majority of hydrothermal manifestations within the Uzon caldera are controlled by three trends of faults. The major fault zone, trending nearly E–W, is located between Kikhpinych and Taunshits volcanoes. It acts as a magma conduit and hosts numerous oval-shaped hydrothermal vents. The Oil site is situated on the periphery of the hottest area of highest hydrothermal activity within the Eastern thermal field. On the Eh–pH diagram, most solutions of the Uzon caldera define distinct fields and trends which correlate with the stability fields for sulfur and iron. The Oil site is characterized by very wide variations in temperature and Eh–pH parameters of pore solutions. The geochemical signatures of the solutions at this site are broadly similar to those from other areas of the Uzon caldera, but differ in their sulfide ion and sulfate ion concentrations. These differences can be explained by mixing of deep chloride-sodium hydrothermal solutions and solutions produced during surface oxidation of sulfide-containing material. With respect to the average continental crust, the Uzon oil is enriched in S, As, and Hg. The crude oil and solutions have broadly similar concentrations of B, S, Cl, As, Se, Br, Cd, I, Hg, and Pb and highly variable concentrations of Ti, V, Cr, Co, Ni, Cu, Cd, Nb, and Sn. The community structure of archaeal assemblages in springs and test pits at the Eastern thermal field was analyzed by 16S rRNA library and pyrosequencing methods. It was found that the proportion of archaea in the microbial communities of the Uzon caldera ranges from 2 to over 70% of the total sequences identified. Crenarchaeota were found in large proportions in all samples except one. The majority of the sequences in four samples were affiliated with Euryarchaeota, which comprise methanogenic archaea, extreme halophiles, and some extreme thermophiles. The results of geological, mineralogical-geochemical, microbiological and physicochemical studies of oil seeps in the Uzon caldera reveal distinctive geochemical characteristics of crude oil and the complexity of oil formation.

The complete genomes of two human bocavirus 4 (HBoV4) isolates recovered in 2011 in Novosibirsk, Russia have been determined. A set of primers was designed based on the determined and previously published HBoV sequences; this primer pair was able to detect all possible HBoV replicative intermediates. This primer set was used to assay all HBoV genotypes and detected only those structures that correspond to an episomal form of this viral genome. Also, for the first time, head-to-tail nucleotide sequences have been determined for HBoV4. Secondary structures of the terminal noncoding regions (NCRs) of episomal forms have been computed for all HBoV genotypes, as well as for the canine bocavirus. Conserved secondary structures in episomal NCRs, which are likely to play an important part in the replication of bocaviruses, were found. NCR heterogeneity in the genomes of individual HBoV isolates has been shown for the first time.

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.

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.

The regulatory region of the Escherichia coli yfiA gene was reconstructed by using the SITECON web resource and mathematical modeling; the complexity of its expression under oxidative stress was assessed. Simulation of the response of E. coli cells transformed with the pYfi-gfp plasmid to oxidative stress indicated that the maximum agreement with experimental data was achieved in a model implying complex action, apparently mediated by several transcription factors (TFs). The regulatory region of the yfiA gene was inspected for potential TF binding sites, and highly reliable predictions were made for TFs MarA, IscR, MetJ, PurR, and SoxS, which directly or indirectly participate in the response of the gene to oxidative stress, and for CRP, a global regulator of carbohydrate catabolism. The presence of binding sites for CRP, MarA, and SoxS in the E. coli yfiA promoter was confirmed by a electrophoretic mobility-shift assay with purified recombinant TFs. This fact explains the sensitivity of yfiA to mitomycin and radical-forming agents.

Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8-10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 10⁷ CFU), or S. aureus (8.96 × 10⁷ CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of "empty" cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.

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.

Babesia microti is a genetically diverse group of protozoan parasites whose life cycle is associated with both small mammals and Ixodes spp. ticks. In this study, the prevalence of different B. microti genetic groups in ticks and small rodents in an area with Ixodes persulcatus and Ixodes trianguliceps occurring in sympatry was examined. A total of 541 small mammals were captured during eight sampling periods between 2013 and 2015 at a site in the Omsk region of Russia and tested for the presence of B. microti using nested PCR with subsequent sequencing of positive samples. B. microti DNA was found in 31.6 % of examined samples, and prevalence rates ranged from 5.3 to 61.6 % in different sampling periods. The sequenced B. microti samples belonged to two genetic groups: enzootic B. microti ‘Munich’ type and zoonotic B. microti ‘US’ type. B. microti ‘Munich’ type was more common across all sampling periods, with greater than 80 % prevalence in infected animals. Despite the high B. microti ‘Munich’-type prevalence in voles, B. microti was not found in any of 394 adult I. persulcatus ticks collected by flagging or in the 84 I. persulcatus or 20 I. trianguliceps ticks taken from voles and molted under laboratory conditions. It was demonstrated that B. microti ‘Munich’-type DNA can be detected in the blood of naturally infected voles for at least 20 weeks after capture. Thus, the high prevalence of B. microti ‘Munich’ type in small mammals may be explained by the prolonged persistence of B. microti in the blood of wild voles.

The growing body of knowledge concerning the molecular biology of viruses and virus-cell interactions provides possibilities to use viruses as a tool in an effort to treat malignant tumors. As a rule, tumor cells are highly sensitive to viruses, which can be used in cancer therapy. At the same time, the application of viral oncolysis in cancer treatment requires that the highest possible safety be ensured for both the patient and environment. Human enteroviruses are a convenient source for obtaining oncolytic virus strains, since many of them are nonpathogenic for humans or cause mild disease. The current progress in genetic engineering enables the development of attenuated enterovirus variants characterized with high safety and selectivity. This review focuses on the main members of the Enterovirus genus, such as ECHO, coxsackievirus, and vaccine strains of poliovirus as a promising source for the development of oncolytic agents applicable for cancer therapy. We have summarized the data concerning recently developed and tested oncolytic variants of enteroviruses and discusses the perspectives of their application in cancer therapy, as well as problems associated with their improvement and practical use.

Human astrovirus is one of the etiological agents of acute gastroenteritis in humans, mostly in young children and elderly people. Complete genome sequencing of four human astrovirus strains isolated in Novosibirsk, Russia was performed. Analysis of these sequences and the sequences available in GenBank database has detected numerous potential recombination breakpoints. For the first time the rate of human astrovirus evolution was estimated based on the genome fragments without recombination breakpoints; the determined rate is typical of the RNA viruses with high evolutionary rate, amounting to approximately 3.7 × 10(-3) nucleotide substitutions per site per year, and for the synonymous changes, 2.8 × 10(-3) nucleotide substitutions per site per year.

Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.

Six unique phage antibodies against human tumor necrosis factor (TNF) were selected from the earlier constructed naive combinatorial library of single-chain antibodies by affinity selection. The TNF binding of these antibodies was examined by enzyme immunoassay and Western blot analysis. The specificity of the selected antibodies was determined from their binding to interferons alpha and gamma, bovine serum albumin, ovalbumin, and ubiquitin. Two antibodies (sA1 and sB3) were converted into a soluble single-chain antibodies as individual molecules. Their affinity proved to be 2.5 and 13.7 nM, respectively.

Twenty unique phage antibodies to human tumor necrosis factor alpha were selected from a naive combinatorial library of human single chain variable fragments. Analysis of gene segments encoding selected antibodies showed that the repertoire of variable domains of heavy and light chains included variable domains of both naive autoantibodies and antibodies produced through somatic hypermutation.

Four unique phage single-chain variable fragments (scFvs) to recombinant human interleukin 18 (IL-18) has been selected from a naïve combinatorial library of human scFvs. Binding of unique phage antibodies with IL-18 was tested by ELISA and Western-blotting. No cross reactivity with tumor necrosis factor α, interferons α and γ was shown for the selected antibodies. The gene segments encoding V(D)J regions of selected antibodies exhibited a high degree of homology to germline genes, therefore we suggest that the selected scFv's belong to repertoire of naïve autoantibodies.

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Galf-(1→5)-[β-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

Objectives The main causative agent of tick-borne rickettsioses in Siberia is considered to be Rickettsia sibirica ; however, only a few cases have been genetically confirmed. Other pathogenic species of Rickettsia have been detected in ixodid ticks in Western Siberia. The aim of this study was to detect the aetiological agents of tick-borne rickettsioses in Western Siberia and compare their clinical manifestations. Methods A total of 273 blood and 44 cerebrospinal fluid (CSF) samples from 273 patients hospitalized because of tick-transmitted infection in April–September 2016 were examined for the presence of Rickettsia spp., using nested PCR with subsequent sequencing. Results DNA of Rickettsia spp. was found in samples from 10 patients. The gltA gene fragment sequence analysis revealed R. sibirica DNA in seven patients (blood samples) and Rickettsia raoultii DNA in three patients (two blood and one CSF sample). Most patients infected with R. sibirica showed typical clinical symptoms, including high-grade fever (38.9–39.5°С), myalgia, rash, eschar at the site of the tick bite, and elevated levels of serum aminotransferases. In contrast, patients infected with R. raoultii showed nonspecific symptoms with short-term fever (37.2–37.7°С); one patient had a short episode of meningeal syndrome. Conclusions We report the first finding of R. raoultii DNA in clinical samples from Russian patients. The clinical manifestations of this rickettsiosis were nonspecific and differed from those caused by R. sibirica.

We describe key stages in the development of a genosensor-based bioanalytical complex capable of functioning in a micro/nanofluidic system (MNFS), which is intended for detecting damaging agents in liquid media. Using the LIGA technology, we have developed an MNFS with a channel width of a few microns or more. Methods of (i) processing microfluid channels for positioning bacterial cells-genosensors and (ii) detecting fluorescent signals in a microfluid module are developed. A search for promoters ensuring the response of genosensor structures to a broad range of damaging factors of the external environment is carried out using a bioinformatics approach (based on GenSensor database). An experimental genosensor has been constructed based on the dps gene promoter of E. coli and the pRS-GFPvaa vector plasmid. It is shown that an E. coli/pDps-gfp genosensor in a microfluid module responds by GFPvaa protein synthesis to the presence of oxidative stress agents and agents capable of penetrating membranes in the same range of concentrations as those outside the module. The results of our investigation show that the proposed interdisciplinary approach can be used for obtaining bacterial cells-genosensors capable of functioning in MNFSs and detecting damaging factors of the external environment.