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Since the onset of the COVID-19 pandemic, numerous publications have appeared describing autoimmune pathologies developing after a coronavirus infection, with several papers reporting autoantibody production during the acute period of the disease. Several viral diseases are known to trigger autoimmune processes, and the appearance of catalytic antibodies with DNase activity is one of the earliest markers of several autoimmune pathologies. Therefore, we analyzed whether IgG antibodies from blood plasma of SARS-CoV-2 patients after recovery could bind and hydrolyze DNA. We analyzed how vaccination of patients with adenovirus Sputnik V vaccine influences the production of abzymes with DNase activity. Four groups were selected for the analysis, each containing 25 patients according to their relative titers of antibodies to S-protein: with high and median titers, vaccinated with Sputnik V with high titers, and a control group of donors with negative titers. The relative titers of antibodies against DNA and the relative DNase activity of IgGs depended very much on the individual patient and the donor, and no significant correlation was found between the relative values of antibodies titers and their DNase activity. Our results indicate that COVID-19 disease and vaccination with adenoviral Sputnik V vaccine do not result in the development or enhancement of strong autoimmune reactions as in the typical autoimmune diseases associated with the production of anti-DNA and DNA hydrolyzing antibodies.

Infectious complications after primary implantation of the hip joint are 0.5–3 %, and in the case of re-endoprosthetics, the risk of periprosthetic infection can reach 30 %. Also, we should not forget about the high percentage (16–20 %) of recurrence of periprosthetic infection of the hip joint, which leads to an unsatisfactory result of treatment up to amputation of a limb or even death of the patient. The reasons for the recurrence of the infectious process can be antibiotic resistance and antibiotic tolerance of microorganisms, as well as the ability of microorganisms to form biofilms on implants. In this regard, there is a constant need to search for alternative means of antimicrobial therapy, as well as to select the optimal ways of their delivery and deposition, which is of practical importance when performing surgical interventions in traumatology and orthopedics to protect the implantable structure from possible infection of the surgical site. One of the methods currently available to combat bacterial infections acquired antibiotic resistance and antibiotic tolerance is the use of natural viruses that infect bacterial bacteriophages. The above suggests a more effective suppression of periprosthetic infection, including persisters that deviate from antibiotics. It is, as a rule, associated with biofilms if used in conjunction with antibiotics and phages, when the use of bacteriophages predetermines the effectiveness of treatment. With the use of sensitive bacteriophages in the treatment of periprosthetic infections, a significant (p = 0.030) reduction in the rate of recurrence of infection (from 31 to 4.5 %) was observed. The use of lytic bacteriophages in traumatology and orthopedics is of great interest for phagotherapy of infections caused by antibiotic-resistant and biofilm-forming strains of bacteria. A clinical study using a single-stage surgical revision with simultaneous application of antibiotics and phages in the treatment of deep periprosthesis infection of the hip joint endoprosthesis, followed by 12 months follow-up for periprosthetic infection recurrence, demonstrated the effectiveness of the use of combined antibiotic and bacteriophages treatment.

β-(1→3)-d-Glucan is a branched polysaccharide present in the inner layer of the cell wall of various representatives of the fungal kingdom. Therefore, this polysaccharide is considered as a common marker for the laboratory diagnosis of mycoses of various etiologies. However, low concentration of β-(1→3)-d-glucan in biological fluids (∼40 pg mL−1) requires the development of highly sensitive methods for its determination. One of the most convenient types of bioassays is based on immunochemical approaches, in which the most important stage is selection and characterization of monoclonal antibodies (mAb). In this communication, the binding constant of mAb 3G11 and the synthetic oligosaccharide β-d-Glcp-[(1→3)-Glcp]8, which is related to the linear moiety of fungal glucan, was determined in solution for the first time using fluorescence polarization immunoassay (FPIA) method. The obtained information will be used subsequently for the development of analytical systems for FPIA detection of β-(1→3)-d-glucan.

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.

Data on the number and diversity of psychrotolerant yeast isolated from high-altitude and surface samples of atmospheric aerosol from southwestern Siberia are derived from a microbiological study. According to certain phenotypic and genomic characteristics, the yeast isolates are referred to the Saccharomyces, Candida, Sporidiobolus, Aureobasidium, Sporobolomyces, Cryptococcus, Rhodotorula, and some other genera. Both saprophytic and pathogenic microorganisms are detected. Aureobasidium yeast strains are revealed, which actively produce exopolysaccharides and melanin and are promising for biotechnological developments.

Noroviruses (the Caliciviridae family) are a common cause of acute gastroenteritis in all age groups. These small non-envelope viruses with a single-stranded (+)RNA genome are characterized by high genetic variability. Continuous changes in the genetic diversity of co-circulating noroviruses and the emergence of new recombinant variants are observed worldwide. Recently, new recombinant noroviruses with a novel GII.P16 polymerase associated with different capsid proteins VP1 were reported. As a part of the surveillance study of sporadic cases of acute gastroenteritis in Novosibirsk, a total of 46 clinical samples from children with diarrhea were screened in 2016. Norovirus was detected in six samples from hospitalized children by RT-PCR. The identified noroviruses were classified as recombinant variants GII.P21/GII.3, GII. Pe/GII.4_Sydney_2012, and GII.P16/GII.4_Sydney_2012 by sequencing of the ORF1/ORF2 junction. In Novosibirsk, the first appearance of the new recombinant genotype GII.P16/ GII.4_Sydney_2012 was recorded in spring 2016. Before this study, only four complete genome sequences of the Russian GII.P16/GII.3 norovirus strains were available in the GenBank database. In this work, the complete genome sequence of the Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 (GenBank KY210980) was determined. A comparison of the nucleotide and the deduced amino acid sequences showed a high homology of the Russian strain with GII.P16/GII.4_Sydney_2012 strains from other parts of the world. A comparative analysis showed that several unique substitutions occurred in the GII.P16 polymerase, N-terminal p48 protein, and minor capsid protein VP2 genes, while no unique changes in the capsid VP1 gene were observed. A functional significance of these changes suggests that a wide distribution of the strains with the novel GII.P16 polymerase may be associated both with several amino acid substitutions in the polymerase active center and with the insertion of glutamic acid or glycine in an N-terminal p48 protein that blocks the secretory immunity of intestinal epithelial cells. Further monitoring of genotypes will allow determining the distribution of norovirus recombinants with the polymerase GII.P16 in Russia.Норовирусы (сем. Caliciviridae) считаются частой причиной острого гастроэнтерита у людей всех возрастов. Эти небольшие безоболочечные вирусы с одноцепочечным (+)РНК-геномом характеризуются высокой генетической вариабельностью. По всему миру наблюдается постоянное изменение генетического разнообразия циркулирующих норовирусов и появление новых рекомбинантных вариантов. Недавно опубликованы данные о распространении рекомбинантных штаммов норовируса, в которых новая полимераза генотипа GII.P16 сочеталась с капсидными белками VP1 разных генотипов. В рамках мониторинга спорадических случаев острых гастроэнтеритов в Новосибирске в 2016 г. было протестировано 46 клинических образцов от детей с диареей. Методом ОТ-ПЦР норовирус детектирован в шести клинических образ- цах от госпитализированных детей. Выявленные норовирусы путем секвенирования региона перекрывания ORF1/ORF2 были классифицированы как рекомбинантные варианты GII.P21/GII.3, GII.Pe/GII.4_Sydney_2012 и GII. P16/ GII.4_Sydney_2012. Появление нового рекомбинантного генотипа GII. P16/GII.4_Sydney_2012 впервые зафиксировано в Новосибирске весной 2016 г. До этого исследования в базе данных GenBank было доступно всего четыре полногеномные последовательности российских штаммов норовируса генотипа GII.P16/ GII.3. В настоящей работе была определена полная последовательность генома российского штамма Hu/GII.P16- GII.4/RUS/Novosibirsk/NS16-C38/2016 (GenBank KY210980). Сравнение нуклеотидной и выведенной амино- кислотной последовательностей показало высокую гомологию этого российского штамма со штаммами ге- нотипа GII.P16/GII.4_Sydney_2012 из других регионов мира. Сравнительный анализ показал, что уникальные замены произошли в последовательностях генов полимеразы генотипа GII.P16, N-терминального белка p48 и минорного капсидного белка VP2, при этом существенных изменений в гене основного капсидного белка VP1 не наблюдалось. Анализ функциональной значимости этих изменений позволил предположить, что ши- рокое распространение штаммов с новой полимеразой GII.P16, возможно, связано как с несколькими ами- нокислотными заменами в активном центре полимеразы, так и со вставкой остатка глутаминовой кислоты или глицина в N-терминальном белке p48, который блокирует секреторный иммунитет эпителиальных кле- ток кишечника. Дальнейший мониторинг генотипов позволит оценить распространение рекомбинантных норовирусов с полимеразой GII.P16 на территории России.

Galactomannan (GM) is a specific polysaccharide antigen of opportunistic mold fungi of the genus Aspergillus. The detection of GM in patients’ biological fluids is a reliable criterion for the diagnosis of invasive pulmonary aspergillosis, a severe disease. In this report, we evaluated the analytical capabilities of the fluorescence polarization immunoassay (FPIA) for detection of GM. Using FPIA, the high affinity of the monoclonal antibody 7B8 to the synthetic oligosaccharide β-d-Galf-[(1→5)-β-d-Galf]3-(1→6)-α-d-Manp related to the galactomannan fragment was shown. The binding constant in this interaction was found to be equal to (1.02±0.01) • 108 L mol-1. The detection limit determined for this oligosaccharide with the competitive FPIA method is 3 ng mL-1. The data obtained indicate the possibility of using FPIA for the diagnosis of invasive aspergillosis.

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) 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.

Tick-borne encephalitis virus (TBEV) causes about 5-6 thousand cases annually, while there is still no effective treatment for this virus. To fill this gap, a high-affinity chimeric anti-TBEV antibody ch14D5 has previously been constructed, and high protective activity in a murine TBEV model has been shown for this antibody. However, the mechanism of action of this antibody and the recognized epitope have not been known yet. In this study, it is shown by X-ray crystallography that this antibody recognizes a unique epitope on the lateral ridge of the D3 domain of glycoprotein E, which is readily accessible for binding. The orientation of this antibody relative to the virion surface makes bivalent binding possible, which facilitates the cross-linking of glycoprotein E molecules and thus blocking of surface rearrangements required for infection. Since the antibody tightly binds to this protein even at pH ∼ 5.0, it locks the virion in an acidic environment inside the late endosomes or phagosomes and, therefore, effectively blocks the fusion of the viral and endosomal/phagosomal membranes. We believe that this is why the ch14D5 antibody does not induce an antibody-dependent enhancement of infectionin vivo, which is critical in the development of antibody-based therapeutic agents. In addition, the structure of the antibody-glycoprotein E interface can be used for the rational design of this antibody for enhancing its properties.

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.

Equine piroplasmosis is a natural tick-borne infection caused by hemoprotozoan parasites of the order Piroplasmida, Babesia caballi and Theileria equi. Animals that recover from piroplasmosis remain persistently infected carriers and can transmit pathogens to vector ticks. Cases of equine piroplasmosis are periodically observed in Siberia, however, no agent of equine piroplasmosis has yet been genetically characterized in Russia. The aim of this work was studying the prevalence of the infectious agents of piroplasmosis in horses from Siberia and genotyping the detected agents. Blood samples from 155 horses were examined for the presence of Babesia and Theileria DNA by nested PCR with the subsequent sequencing of positive samples. DNA of T. equi was found in blood samples from 57.9 %, 38.5 % and 65.0 % of horses from Novosibirsk province, Irkutsk province, and the Republic of Altai, respectively. T. equi DNA was found in the samples from almost all sampling sites included in this study, indicating that most of the studied sites are endemic for equine theileriosis. Surprisingly, DNA of B. caballi was not found in any of the samples examined, even though this agent had previously been detected in many regions in Russia, including Altai. The analysis of the determined 18S rRNA gene sequences demonstrated that T. equi samples belonged to two genetic groups, which differed significantly by the sequences of the variable (V4) region of the gene. All T. equi sequences from group B were identical and corresponded to T. equi sequences found in the blood of horses from China and Korea, while T. equi sequences from group A differed by 1–5 nucleotide substitutions and were identical to the sequences from the blood of horses from India and Brazil or differed from them by single mismatches. Notably, in this study the presence of etiological agent of piroplasmosis in blood samples from horses in Russia was genetically confirmed for the first time.

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.

The aim of the study was to determine the ecology features of tick-borne encephalitis virus (TBEV) of European subtype circulating in Siberia. The strains of European subtype TBEV from Siberia were isolated in areas with different terrain types - from the plains to the midlands. The strains isolation areas differed in variety of landscapes, flora and fauna and were characterized by the presence of sharp-continental, or in some places - quite harsh climate. The composition of the main vectors and reservoir hosts of TBEV of European subtype in Siberia has its own features and is significantly different from that one in Europe. However, in spite of this, the homology of TBEV strains of European subtype isolated in different parts of habitat range of the virus from the Scandinavian countries in the West to its Eastern borders is much higher than the homology level degree of TBEV strains of FarEastern and Siberian subtypes.

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.