Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein.

INTRODUCTION: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. OBJECTIVE: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. MATERIALS AND METHODS: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. RESULTS: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. CONCLUSION: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

[Latex Agglutination as an Alternative to the Hemagglutination Reaction of Influenza Viruses].

As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus.

Dataset on safety and protective efficacy studies of COVID-19 vaccine candidates based on structurally modified plant virus in female hamsters.

This data article is related to the previous research, which addressed the development of a COVID-19 recombinant vaccine candidate. Here, we present the additional data in support of the safety and protective efficacy evaluation of two COVID-19 vaccine candidates based on the coronaviruses' S protein fragments and a structurally modified plant virus - spherical particles. The effectiveness of the experimental vaccines was studied against the SARS-CoV-2 virus in an in vivo infection model in female Syrian hamsters. The body weight of vaccinated laboratory animals was monitored. The histological assessment data of the infected with the SARS-CoV-2 virus hamsters' lungs are provided.

Osteogenic and Angiogenic Properties of Heparin as a System for Delivery of Biomolecules for Bone Bioengineering: a Brief Critical Review.

The review considers complex, controversial, and individual effects of heparin and its derivatives on the bone and circulatory systems in dependence of the dose, the state of the cells and tissues of the recipient. General data on the anticoagulant activity of heparin and its derivatives are presented; special attention is paid to the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis. We also discuss the ability of heparin to bind osteogenic and angiogenic biomolecules in the context of the development of systems for their delivery and sustained controlled release and propose a schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering.

First evidence of virus-like particles in the bacterial symbionts of Bryozoa.

Bacteriophage communities associated with humans and vertebrate animals have been extensively studied, but the data on phages living in invertebrates remain scarce. In fact, they have never been reported for most animal phyla. Our ultrastructural study showed for the first time a variety of virus-like particles (VLPs) and supposed virus-related structures inside symbiotic bacteria in two marine species from the phylum Bryozoa, the cheilostomes Bugula neritina and Paralicornia sinuosa. We also documented the effect of VLPs on bacterial hosts: we explain different bacterial 'ultrastructural types' detected in bryozoan tissues as stages in the gradual destruction of prokaryotic cells caused by viral multiplication during the lytic cycle. We speculate that viruses destroying bacteria regulate symbiont numbers in the bryozoan hosts, a phenomenon known in some insects. We develop two hypotheses explaining exo- and endogenous circulation of the viruses during the life-cycle of B. neritina. Finally, we compare unusual 'sea-urchin'-like structures found in the collapsed bacteria in P. sinuosa with so-called metamorphosis associated contractile structures (MACs) formed in the cells of the marine bacterium Pseudoalteromonas luteoviolacea which are known to trigger larval metamorphosis in a polychaete worm.

[Osteogenic and angiogenic properties of heparin as a system of biomolecule delivery for bone bioengineering: a brief critical review]. / Osteogennye i angiogennye svoistva geparina kak sistemy dostavki biomolekul pri bioinzhenerii kosti: kratkii kriticheskii obzor.

The review discusses the complex, ambiguous and individual effects of heparin and its derivatives on the bone and circulatory systems, in dependence of the dosage, the state of the cells and tissues of recipients. General data on the anticoagulant activity of heparin and its derivatives are presented; aspects of the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis are considered in details. Particular attention is paid to the ability of heparin to bind osteogenic and angiogenic biomolecules: thus us especially important for the development of systems for their delivery and sustained controlled release. A schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering is proposed.

[The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses].

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization.

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense "grains" on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzyme-linked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

[Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating]. / Sekretsiia signal'nykh molekul krovetvornykh nish v usloviiakh osteogennoi differentsirovki mul'tipotentnykh mezenkhimnykh stromal'nykh kletok, indutsirovannoi rel'efnym kal'tsii-fosfatnym pokrytiem.

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

Haloperidol Reduces the Activity of Complement and Induces the Anti-Inflammatory Transformation of Peritoneal Macrophages in Rats.

It is known that psychotropic substances affect the immune system. Unfortunately, chronic antipsychotic administration causes side toxicological effects, associated with oxidative stress. The mechanisms of these effects are still unclear. We investigated the impact of sub-chronic administration of haloperidol (Hal) on parameters of innate immunity and related systems in healthy rats and compared them with Hal content. Hal administration (0.5 mg/kg, 3 weeks) resulted in two-fold decrease of the activity of the complement system and hemostasis. Hal content correlated with the activity of the complement (r = -0.71), phagocytic activity of peritoneal macrophages (r = 0.78), leukocyte elastase (r = -0.71) and glutathione-S-transferase activity (r = -0.67). Hal fully blocked in vitro PMA-induced iNOS expression in macrophages and changed their morphology to "anti-inflammatory" phenotype. The comparison of in vivo and in vitro data showed that Hal has a direct effect on phagocytic component of innate immunity and an indirect effect on leukocyte elastase and antioxidant enzymes. The results obtained in the present study indicated that Hal significantly affects homeostasis and causes a number of complex biological transformations. Graphical Abstract.

[Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus].

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4-5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70-5.80 to 5.25-5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

Emergency Soft Tissue Reconstruction Algorithm in Patients With Open Tibia Fractures.

INTRODUCTION: Tactic of emergency closing of soft tissue defect allows to significantly improve the treatment results concerning patients with severe open fractures. However, a number of certain factors make the implementation of this tactic rather difficult. Injured people's unstable conditions are mong these crucial factors which include, polytrauma in lots of cases, absence of exact recommendations for recovery terms, choice of definite tissue flaps and a type of circulation. THE AIM OF STUDY: is to develop exact, usable and in practice algorithm of emergency reconstruction of leg soft tissues in patients with severe open tibia fractures, based on the usage of the most foolproof and simple methods. DATA PATIENTS AND METHODS: 85 patients with open tibia fractures complicated by soft tissue defects were included in our study. Patients were divided into two groups. The control group consisted of 56 patients. Soft tissue reconstruction in this group was provided without an exact algorithm, after continuous attempts on local healing. After analyzing the treatment process and the treatment results we have developed the algorithm of emergency soft tissue reconstruction. It was used in 29 patients (the study group). This algorithm allows choosing optimal timing for tissue reconstruction and appropriate method to be applied, depending on the patient's condition, the mechanism of soft tissue defect formation, and its square and localization. RESULTS: We observed a statistical decrease in deep wound infection frequency, partial tibia necrosis frequency, chronic osteomyelitis frequency, duration of hospitalization in patients with severe open tibia fractures because of using our algorithm.

Complement-Mediated Death of Ciliate Tetrahymena pyriformis Caused by Human Blood Serum.

Toxicity of human blood serum for ciliate Tetrahymena pyriformis is determined by the complement system. When ciliate are dying after being exposed to blood serum, cell membrane permeability for low-molecular-weight compounds significantly increases, probably due to pore formation. Serine protease inhibitors or exposure to physical factors inducing complement inactivation (e.g., heating up to 56°C) completely prevented ciliate death under the effect of human serum. Activation of serum complement upon interaction with Tetrahymena cells occurred by the classical or lectin pathway, while the contribution of the alternative activation pathway was negligible.

[Surgery of pancreatic injuries]. / Khirurgiya povrezhdenii podzheludochnoi zhelezy.

AIM: To estimate different approaches to treatment of victims with pancreatic trauma with pancreatic trauma. MATERIAL AND METHODS: It was analyzedthe results of treatment of 342 victims with pancreatic trauma in N.V. Sklifosovskiy Research Institute of Emergency Care for the period 1991-2012. RESULTS: It was concluded that for the las decade curative and diagnostic tactics for pancreatic injury in victims with combined abdominal trauma has been changed; current diagnostic markers of pancreatic lesion and adequate intraoperative diagnosis are used. All of this together with timely specific therapy and adherence to guidelines of surgical treatment decreased mortality rate from 17.0 to 11.1% and suppurative complications incidence from 43.8 to 19.9%.

[Diagnosis and treatment of associated injuries of the pelvis].

The aim of investigation is improvement of treatment results of unstable injuries in the pelvic ring in patients with severe associated injuries. An experience of treatment of 533 patients with unstable pelvic injuries is presented. Diagnostic measures take into account the severity of the condition of the victim. Stage treatment with consecutive performing of temporary external immobilization of the pelvis and following final osteosynthesis was optimal. Replacement of external fixation for submersible osteosynthesis held in stable or terminal condition of the victim. Developed treatment of patients with pelvic injuries decreased mortality to 8.4%, number of general complications to 11.6% and number of local complications to 8.7%. Also it decreased length of hospital stay to 35.5 days and provided excellent and good functional results of treatment in 78.9% of cases.

[Mini-invasive technologies in treatment of acute cholecystitis in patients with high operational and anesthetic risk].

The treatment results of 769 patients with acute calculous cholecystitis and high operational and anesthetic risk at admission are presented in the retrospective study. High risk was determined by expressed comorbidities, diseases' terms, the complications of acute cholecystitis, age, which was more than 60 years in most cases. The patients were divided into 2 groups depending on the severity of comorbidity and the possible effects of its correction. The first group included 617 perspective patients for cholecystectomy. And the second group included 152 patients unpromising for this. Concept of stage treatment was used in the first group including primary decompression of the gallbladder by using of percutaneous transhepatic micro-cholecystostomy under ultrasound guidance. Cholecystectomy was performed after correction of comorbidities, complications of acute cholecystitis, and readjustment of extrahepatic bile ducts by endoscopy if necessary. Laparoscopic cholecystectomy was successfully performed in 587 patients. There was open cholecystectomy in 11 cases. Cholecystectomy was done in 19 patients as a result of conversion. Cholecystostomy from minimal access with extraction of stones under local anesthesia was performed in the second group for decompression and as definitive treatment. There was not observed deaths in patients with high operational and anesthetic risk as a result of such tactics. Postoperatively 1.7% of patients had complications that were successfully resolved.

[Tactics of treatment of the acute cholecystitis complicated by choledocholithiasis].

In this article is present the comparative analysis of the results of different surgical approaches of 201 patients with acute cholecystitis complicated by choledocholithiasis. In the 1st group of 70 (34.8%) patients in the first stage of treatment applied percutaneous cholecystostomies (PC). Retrograde endobiliary interventions endoscopic sphincterotomy (ES) were performed in the second stage before LC. In the 2nd group 131 (65.2%) patients underwent for treatment include preoperative endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy (ERCP/ES) followed by laparoscopic cholecystectomy (LC). The complications of retrograde interventions was higher by 3.5 times in the group of patients without prior decompression of the gallbladder. The surgical treatment was performed by 70 patients of the first group and by 79 (60.3%) patients of the second group. LC was performed by 66 (94.2%) patients with percutaneous cholecystostomies. Proved highly effective percutaneous cholecystostomy and low risk of complications stage treatment.

[Efficacy of combined surgical and endoscopic means of diffuse peritonitis treatment].

An analysis of complex examination and treatment of 123 patients with diffuse purulent peritonitis was made. The patients were divided into two groups. The first group consists of 68 patients, whom conservative surgical intervention was performed at the first stage and videoimage endoscopic sanation of the abdominal cavity with the administration of sodium hypochlorite water solution was made at the second stage. The main group consists of 55 patients, who had the same treatment except that the abdominal cavity was processed with pulsating stream of antiseptic with immobilized forms of sodium hypochlorite at the second stage. It is established, that the application of developed technology allows decreasing of the frequency of postoperative complications on 8.3% and postoperative lethality - 4.8%.

[Virus-like particles--a new strategy for production of vaccines against influenza virus].

Numerous studies demonstrated that simultaneous expression of some viral proteins in the cell with the aid of a process of self-assembly might lead to the formation of the virus-like particles (VLP) even in the absence of the viral genome. The morphological and antigenic similarity between VLP and native virions represents a promising approach to the new type of vaccines. In the last decade, the threat of the influenza strains with pandemic potential becomes more important. Therefore, the technology for obtaining a new generation of safe and effective non-embryo culture vaccines was developed on the basis of the influenza VLP produced in various expression systems. This provides great advantages in comparison with existing methods of vaccine production. Such vaccines induced full humoral and cellular immune response in animals and humans. This review is focused on the literature concerning the influenza VLPs obtained in various expression systems including insect, mammalian and plant cells.