Development, production and characterization of SARS-CoV-2 virus-like particles (Coronaviridae: Orthocoronavirinae: Betacoronavirus: Sarbecovirus).

INTRODUCTION: The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. MATERIALS AND METHODS: Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. RESULTS: VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. CONCLUSION: The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.

[Study of the safety and immunogenicity of VLP-based vaccine for the prevention of rotavirus infection in neonatal minipig model].

INTRODUCTION: In Russia, almost half of the cases of acute intestinal infections of established etiology in 2022 are due to rotavirus infection (RVI). There is no specific treatment for rotavirus gastroenteritis. There is a need to develop modern, effective and safe vaccines to combat rotavirus infection that are not capable of multiplying (replicating) in the body of the vaccinated person. A promising approach is to create vaccines based on virus-like particles (VLPs). OBJECTIVE: Study of the safety and immunogenicity of a vaccine against rotavirus infection based on virus-like particles of human rotavirus A in newborn minipigs with multiple intramuscular administration. MATERIALS AND METHODS: Newborn minipigs were used as an animal model in this study. The safety of the tested vaccine was assessed based on thermometry data, clinical examination, body weight gain, clinical and biochemical blood parameters, as well as necropsy and histological examination. When studying the immunogenic properties of the Gam-VLP-rota vaccine in doses of 30 and 120 µg, the cellular, humoral and secretory immune response was studied. RESULTS: The results of assessing the general condition of animals during the immunization period, data from clinical, laboratory and pathomorphological studies indicate the safety of the vaccine against human rotavirus infection based on VLP (Gam-VLP-rota) when administered three times intramuscularly. Good local tolerance of the tested vaccine was demonstrated. The results of the assessment of humoral immunity indicate the formation of a stable immune response after three-time immunization with Gam-VLP-rota, stimulation of the production of antigen-specific IgG antibodies and their functional activity to neutralize human rotavirus A. It was shown that following the triple immunization with the minimum tested concentration of 30 µg/dose, animals developed a cell-mediated immune response. The results of the IgA titer in blood serum and intestinal lavages indicate the formation of both a systemic immunological response and the formation of specific secretory immunity to human rotavirus A. CONCLUSION: Thus, three-time intramuscular immunization of minipigs with the Gam-VLP-rota vaccine forms stable protective humoral and cellular immunity in experimental animals. Evaluated vaccine is safe and has good local tolerability.

[Virus-like particles based on rotavarus A recombinant VP2/VP6 proteins for assessment the antibody immune response by ELISA].

INTRODUCTION: Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. OBJECTIVE: Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. MATERIALS AND METHODS: VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. RESULTS: The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. CONCLUSION: The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.

[Molecular epidemiological analysis of SARS-CoV-2 genovariants in Moscow and Moscow region].

INTRODUCTION: SARS-CoV-2, a severe acute respiratory illness virus that emerged in China in late 2019, continues to spread rapidly around the world, accumulating mutations and thus causing serious concern. Five virus variants of concern are currently known: Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1), Delta (lineage B.1.617.2), and Omicron (lineage B.1.1.529). In this study, we conducted a molecular epidemiological analysis of the most prevalent genovariants in Moscow and the region. The aim of the study is to estimate the distribution of various variants of SARS-CoV-2 in Moscow city and the Moscow Region. MATERIALS AND METHODS: 227 SARS-CoV-2 sequences were used for analysis. Isolation of the SARS-CoV-2 virus was performed on Vero E6 cell culture. Sequencing was performed by the Sanger method. Bioinformatic analysis was carried out using software packages: MAFFT, IQ-TREE v1.6.12, jModelTest 2.1.7, Nextstrain, Auspice v2.34. RESULTS: As a result of phylogenetic analysis, we have identified the main variants of the virus circulating in Russia that have been of concern throughout the existence of the pandemic, namely: variant B.1.1.7, which accounted for 30% (9/30), AY.122, which accounted for 16.7% (5/30), BA.1.1 with 20% (6/30) and B.1.1 with 33.3% (10/30). When examining Moscow samples for the presence of mutations in SARS-CoV-2 structural proteins of different genovariants, a significant percentage of the most common substitutions was recorded: S protein D614G (86.7%), P681H/R (63.3%), E protein T9I (20.0%); M protein I82T (30.0%), D3G (20.0%), Q19E (20.0%) and finally N protein R203K/M (90.0%), G204R/P (73.3 %). CONCLUSION: The study of the frequency and impact of mutations, as well as the analysis of the predominant variants of the virus are important for the development and improvement of vaccines for the prevention of COVID-19. Therefore, ongoing molecular epidemiological studies are needed, as these data provide important information about changes in the genome of circulating SARS-CoV-2 variants.

[Antiviral properties of synthetic histidine derivatives containing membranotropic volumetrical carbocycles in their molecule against SARS-CoV-2 virus in vitro].

INTRODUCTION: Currently, low molecular-weight compounds are being developed as potential inhibitors of CoVs replication, targeting various stages of the replication cycle, such as major protease inhibitors and nucleoside analogs. Viroporins can be alternative protein targets. The aim of this study is to identify antiviral properties of histidine derivatives with cage substituents in relation to pandemic strain SARS-CoV-2 in vitro. MATERIALS AND METHODS: Combination of histidine with aminoadamantane and boron cluster anion [B10H10]2 (compounds IIV) was carried out by classical peptide synthesis. Compound were identified by modern physicochemical methods. Antiviral properties were studied in vitro on a monolayer of Vero E6 cells infected with SARS-CoV-2 (alpha strain) with simultaneous administration of compounds and virus. RESULTS: Derivatives of amino acid histidine with carbocycles and boron cluster were synthesized and their antiviral activity against SARS-CoV-2 was studied in vitro. Histidine derivatives with carbocycles and [B10H10]2 have the ability to suppress virus replication. The solubility of substances in aqueous media can be increased due to formation of hydrochloride or sodium salt. DISCUSSION: 2HCl*H-His-Rim (I) showed some effect of suppressing replication of SARS-CoV-2 at a viral load of 100 doses and concentration 31.2 g/ml. This is explained by the weakly basic properties of compound I. CONCLUSION: The presented synthetic compounds showed moderate antiviral activity against SARS-CoV-2. The obtained compounds can be used as model structures for creating new direct-acting drugs against modern strains of coronaviruses.

In Vitro Antiviral Activity of a New Indol-3-carboxylic Acid Derivative Against SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012-2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 µM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 106 TCID50/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility - high activity (IC50 = 1.06 µg/mL) and high selectivity index (SI = 78.6) - this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

[In situ gels as a modern method of intranasal vaccine delivery].

The continuous emergence of new pathogens and the evolution of microbial drug resistance make it absolutely necessary to develop innovative, effective vaccination strategies. Use of nasal vaccination can increase convenience, safety, cause both local and systemic immune reactions. Intranasal administration nevertheless has a number of shortcomings that can be overcome by using the latest achievements of pharmaceutical science. One of the aspects of such solution may be the use of systems for the production of intranasal vaccines in situ polymer compositions that provide a directed sol-gel transition controlled by the physiological conditions of the nasal cavity. At the same time, the gelation of the administered dose in contact with the nasal mucosa involves prolonged exposure of the drug at the injection site, greater mucoadhesion, counteraction to mucociliary clearance, modified and more complete release. A number of both foreign and domestic manufacturers produces polymers such as chitosan, gums, polyoxyethylene and polyoxypropylene block copolymers (poloxamers, proxanols), carbomers. For effective pharmaceutical development of new intranasal IBD delivery systems corresponding to the QbD concept, not only the knowledge of the range of excipients is necessary, but also simple, accessible, and reproducible methods for determining indicators that define the critical parameters of such delivery systems. In accordance with the conducted scientific search, the main indicators of standardization of in situ intranasal systems were identified: temperature and time of gel formation, gel strength, rheological characteristics, mucoadhesion, release, nasal mucociliary clearance time.

Radiation Inactivation of Coronavirus Infection Pathogen by the Example of Transmissible Gastroenteritis Virus.

In recent years, members of the Coronaviridae family have caused outbreaks of respiratory diseases (MERS, SARS, and COVID-19). At the same time, the potential of radiation-induced inactivation of this group of viruses have been little studied, although radiation technologies can be widely used both in the processing of personal protective equipment and in the sterilization of vaccines. In the present work, the effect of 10 MeV electron beams and 7.6 MeV bremsstrahlung on the coronavirus infection pathogen (transmissible gastroenteritis virus) has been studied in vitro. In the given experimental conditions, irradiation with photons turned out to be more effective. The virus-containing suspension frozen at -86°C was the most resistant to radiation: the dose required for complete inactivation of the virus in this case was from 15 kGy, while for the liquid suspension and lyophilized form the sterilizing dose was from 10 kGy. At lower radiation doses for all samples during passaging in cell culture, residual infectious activity of the virus was observed. These differences in the efficiency of inactivation of liquid and frozen virus-containing samples indicate a significant contribution of the direct effect of radiation.

[Monoclonal antibodies to hemagglutinin of influenza A/H7N3 virus (Orthomyxoviridae: Alphainfluenzavirus: Influenza A virus)].

INTRODUCTION: Variants of influenza virus A/H7 have the same high pandemic potential as A/H5. However, the information about the antigenic structure of H7 hemagglutinin (НА) is considerably inferior in quantitative terms to similar data for H5 НА.The aims of the study were development and characterization of the monoclonal antibodies (MAbs) panel for HA subtype H7 of the influenza A virus. MATERIAL AND METHODS: Viruses were accumulated in 10-day-old chicken embryos. Purification and concentration of the virus, determination of protein concentration, preparation of MAbs and ascitic fluids, hemagglutination and hemagglutination inhibition (HI) tests, assessment of antibodies' activity in indirect enzyme-linked immunosorbent assay (ELISA), as well as determination of MAbs isotypes and neutralization reaction (NR) were carried out by standard methods. RESULTS: The obtained MAbs to А/mallard/Netherlands/12/2000 (H7N3) strain were studied in HI test with a set of strains of different years of isolation belonging to different evolutionary groups. MAbs had a reduced reactivity compared to the immunogen-virus for all the studied strains. Cross-interaction of MAbs 9E11 and 9G12 in HI test with influenza A/H15 virus has been observed. DISCUSSION: Influenza A agent with H7 HA variant could serve as a potential cause of a future pandemic. Development of the MAbs panel for subtype H7 HA is an urgent task for both veterinary medicine and public health. CONCLUSION: The obtained MAbs can be used not only for epitope mapping of the H7 HA molecule (currently insufficiently studied) and as reagents for diagnostic assays, but also for determining common («universal¼) epitopes in HA of different strains of this subtype.

[Synthesis and characterization of human rotavirus A (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) virus-like particles].

INTRODUCTION: Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics. MATERIAL AND METHODS: The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. RESULTS: VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis. CONCLUSION: The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.

[Problems of ante mortem diagnostics of prion diseases].

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.

[Prion diseases and the biosecurity problems.]

The review presents the current state of the problem of prions and prion diseases with an emphasis on theepidemiological and epizootological risks of pathogens that cause fatal neurodegenerative diseases in humans and animals. The results of molecular genetic studies of the conversion of normal PrPc prion protein molecules to infectious forms of PrPd, resistance to physical disinfection methods, in particular exceptional thermal stability, and their ability to overcome interspecific barriers, while increasing virulence, are described. The possibility of infection not only by nutrition, when eating even heat-treated meat of sick animals, but also due to surgical interventions, especially neurosurgical and ophthalmic, as well as the use of immunobiological preparations, are emphasized. Since there are currently no means for the effective treatment of prion diseases in the world, attention is drawn to the high degree of relevance for the biosafety of the country to develop domestic highly sensitive test systems that can effectively detect prion infectious protein in vivo at the preclinical stage of the disease. The latest methods of automatic protein amplification and identification of prion proteins are briefly described as the most promising areas of research in the field of diagnosis of prion diseases.

[Adamantan derivatives capable of inhibiting the reproduction of a Rimantadine resistant strain of influenza A(H1N1)pdm09 virus (Influenza A virus, Alphainfluenzavirus, Orthomyxoviridae).]

INTRODUCTION: Adamantanthane-type drugs such as rimantadine and amantadine have long been used to treat diseases caused by influenza A virus. However, as a result of the mutations, influenza viruses have become resistant to aminoadamantans. The target for these drugs was the protein channel M2. Influenza A virus M2 viroporin in the protein shell forms fairly specific ion channels with a diameter of about 11 Å, specializing in transporting protons inside the viral particle (virion). Restoration of the antiviral properties of adamantanthane-type drugs consists in the selection of advanced functional groups bound by the carbocycle to find new sites of binding to the protein target M2. The purpose of the study is to identify the antiviral properties of new adamantanum derivatives to the pandemic strain of influenza A virus in vitro. MATERIAL AND METHODS: Compounds of aminoadamantans with amino acids and other organic molecules were obtained by classical peptide synthesis methods. The structure of the compound was tested by means of physical and chemical methods. Antiviral properties of synthetic compounds were studied in vitro on monolayer MDCK cells infected with pandemic strain of influenza A/California/07/2009 virus in two schemes of administration of investigated compounds and virus. RESULTS: The reference strain of the influenza virus A/California/07/2009(H1N1) was sensitive to the compounds under test in varying degrees. The antiviral activity of the compounds was expressed in a 50% inhibitory concentration (IС50) ranging from 0.5 to 2.5 мкM, which is generally a good indicator for the Rimantadine/Amantadine resistant strain. DISCUSSION: The values of the IС50 for compounds introduced two hours before contact with the virus were slightly higher than those for single-moment introduction of the substance and virus. The effect of increasing the inhibitory concentration in the prophylactic scheme of compounds was valid for all compounds of the experiment. CONCLUSION: The presented synthetic compounds are active against the variant of influenza A virus resistant to Rimantadine and Amantadine preparations. The obtained compounds can be used as model structures for creation of a new drug of direct action against advanced strains of influenza A virus.

[Comparative molecular and genetic characterization of rabies viruses (Rabies lyssavirus, Lyssavirus, Rhabdoviridae) circulated in the Russian Federation in 1985-2016.]

INTRODUCTION: Rabies caused by the neurotropic virus of the genus Lyssavirus, Rhabdoviridae family, which infects all warm-blooded vertebrates including human beings. The homology level of the amino acid sequences for Lyssaviruses nucleoprotein reaches 78-93%. Aim - study the genetic diversity and molecular epidemiology of Lyssaviruses circulated in the Russian Federation in 1985-2016. MATERIAL AND METHODS: 54 isolates of rabies virus isolated from animals, and 2 isolates from humans, 4 vaccine strains of rabies virus: RV-97, ERA, Shchelkovo 51, ERAG333 used in phylogenetic study. Phylogenetic analysis was performed using Genbank data on genome fragments of 73 rabies virus isolates and 9 EBLV-1 isolates. DNASTAR V.3.12, Bio Edit 7.0.4.1 and MEGA v.10.0.5, Primer Premier 5 programs have been used. RESULTS: Comparative molecular genetic analysis of genomes fragments of 130 Lissaviruses, isolated on the territory of the RF, Ukraine in 1985-2016, vaccine strains of rabies virus, showed their distribution by geographical feature. Comparison of the nucleoprotein fragments of the rabies virus isolates with vaccine strains revealed 4 marker mutations: V56I (Eurasian group), L/V95W (Central group), D101N/S/T, and N/G106D. Phylogenetic analysis of the isolate «Juli¼, isolated from a human bitten by a bat proved his belonging to the European Bat lyssavirus-1a. DISCUSSION: Study of the molecular epidemiology of rabies within the Russian Federation allows for the genotyping of the viruses and helps to study the hidden mechanisms of rabies infection in animal and human populations, and to characterize vaccine strains, including during oral vaccination. CONCLUSION: Further study of the molecular epidemiology of rabies within the Russian Federation and the countries bordering it is important.

Genetic diversity of group A rotaviruses in Moscow in 2018-2019.

Here, we present the results of a study in which 639 samples obtained between October 2018 and April 2019 from patients with symptoms of acute gastroenteritis were tested for the presence of a rotavirus infection. The antigen of group A rotavirus was detected in 160 samples (25% of those tested). To study the genetic diversity of group A rotavirus, RNA was isolated from the samples, and polymerase chain reaction combined with reverse transcription (RT-PCR) with primers specific for the VP4, VP6, and VP7 genes of group A rotaviruses was performed. At least one fragment of the group A rotavirus genome was found in 101 samples (15.8%). These fragments were sequenced, and their G and P genotypes-as well as their combinations-were determined. The predominant G genotypes were G9 (35.8% of all genotyped samples) and G4 (28.4%), but the rare G12 genotype was also found (3.0%). The dominant P genotype was P[8]. The spectrum of certain G/P combinations of genotypes included seven variants. The most common variants were G9P[8] (37.2%) and G4P[8] (30.2%).

[Change of phenotypic properties of escape mutants and readaptants of influenza virus A (H1N1)pdm09 under the influence of selected mutations in the molecule of hemagglutinin.]

INTRODUCTION: After the emergence and spread of pandemic H1N1 viruses in 2009, antigenic epitopes recognized by neutralizing antibodies against the hemagglutinin of influenza A/Moscow/01/09(H1N1)pdm09 viruses were studied. PURPOSE: The purpose of the study was to obtain readapted variants of the virus from a low-virulent escapemutant that has an increased affinity of the avian and the human types cellular receptors compared to the wild type and the comparative study of their antigenic and receptor specificity. MATERIAL AND METHODS: Viruses were accumulated in 10-day-old chicken embryos. The MAB panel against HA of influenza virus strain A/IIV-Moscow/01/09(H1N1)sw1 was used in the form of ascites fluids from mice. Immunization of mice, HI testing, elution of viruses from chicken erythrocytes, PCR and sequencing of readapted variants were performed by standard methods. RESULTS: The amino acid substitution A198E acquired in the process of readaptation leads to changes in the antigenic specificity. A correlation was found between a decrease in virulence of a low-virulent escape mutant associated with the substitution D190N in the hemagglutinin molecule and an increase in the hemagglutinating titer to inhibitors in normal mouse serum. Viruses with low affinity of cellular receptor analogs and carrying amino acid substitutions have an increased ability to elute from chicken erythrocytes. DISCUSSION: The results discuss the effect of mutations in the HA molecule of the influenza A(H1N1) pdm09 virus to the change in antigen specificity; virulence for mice, adsorption-elution at cellular receptors. CONCLUSION: A comparative study of the antigenic specificity and receptor-binding activity of the escape mutants was conducted for the hemagglutinin of the influenza virus A/Moscow/01/2009 (H1N1)swl, and the readapted variants obtained for one of the escape mutants with reduced virulence for mouse. Monitoring the pleiotropic effect of mutations in the hemagglutinin H1 molecule is necessary to predict variants of the virus with pandemic potential.

[Evaluation of the molecularbiological properties of human rotavirus A strain WA.]

BACKGROUND: Rоtaviruses are amоng the leading causes of severe diarrhea in children all over the Wоrld. Vaccination is considered to be the mоst effective way to cоntrоl the disease. Currently available vaccines for prevention of rоtavirus infection are based on live attenuated rotavirus strains human оr animal origin. OBJECTIVES: The aim of this investigation was to study the biological and genetic properties of an actual epidemic human rotavirus A (RVA) strain Wa G1P[8] genotype. METHODS: RVA Wa reproduction in a monolayer continuous cell lines, purification and concentration of RVA antigen, PAAG electrophoresis and Western-Blot, electrophoresis of viral genomic RNA segments, sequencing. RESULTS: Human RVA G1P[8] Wa strain biological and molecular genetic properties were assessed in the process of the adaptation to MARC145 continuous cell line. Cell cultured RVA antigen was purified, concentrated and then characterized by the method of PAAG electrophoresis and immunoblot. To verify RVA Wa genome identity, electrophoresis of viral genomic RNA segments was performed. The lack of accumulation of changes in the RVA Wa genome during adaptation to various cell cultures and during serial passages was demonstrated by sequencing fragments of the viral genome. CONCLUSIONS: RVA Wa strain is stable, it possesses high biological activity: it has been successfully adapted to the MARC145 cell line and RVA Wa virus titer after the adaptation reached 7,5-7,7 lg TCID50/ml. The identity of the cultivated RVA to the original strain Wa G1P[8] was confirmed.

[Assessment of immunogenic activity of the cloned human rotavirus A WA strain.]

INTRODUCTION: Rotovirus infection (RVI) caused by the dsRNA-containing virus from genus Rotavirus, Reoviridae family, belonging to group A (RVA), is the cause of severe diarrhea in human and other mammalian species. Vaccination is the most effective way to reduce the incidence of RVI. At present, the effectiveness of using gnotobiotic piglets as a universal model for reproducing human rotavirus infection and assessing the quality of RVI vaccine preparations has been experimentally proven. OBJECTIVES: Evaluation of immunogenic activity of the cloned RVA Wa strain in the new-born Vietnamese potbellied piglets trial. MATERIAL AND METHODS: Development of viral preparations of the cloned human Wa strain PBA, development of human RVA rVP6, ELISA, polymerase chain reaction with reverse transcription, immunization and experimental infection of newborn piglets. RESULTS: The article presents the results of the experiment on double immunization of newborn piglets with native virus preparations with the infection activity 5.5 lg TCID50/ml, 3 cm3 per dose, HRV with adjuvant 500 µg per dose and mock preparation (control group) followed with experimental inoculation of all animals with virulent virus strain Wa G1P[8] human RVA with infectious activity of 5.5 lg TCID50/ml in 5 cm3 dose. Development of clinical signs of disease and animal death were observed only in control group. RT-PCR system to detect RVA RNA in rectal swabs, samples of small intestine and peripheral lymph nodes was developed. ELISA based on obtained human RVA rVP6 was developed and results on RVA-specific IgG-antibodies in serum samples of experimental piglets are presented. CONCLUSION: In the course of the research, a high immunogenic activity of the native and purified virus of the cloned Wa RVA strain Wa was established and the possibility of its use as the main component of the RVI vaccine was confirmed. The possibility of using conventional newborn pigs instead of gnotobiotic piglets as an experimental model was demonstrated.

[Molecular and biological characteristics of vaccinary ERA-CB 20M of rabies virus.]

The molecular and biological characteristics of the vaccine against rabies virus strain ERA-CB 20M obtained by the Russian rabiologist, doctor of medical sciences S.V. Gribencha by adapting and cloning the strain ERA and SAD in a transplantable BHK-21 C13 cell culture are presented. The spectrum of the most sensitive strain of rabies ERA-CB 20M cell lines was determined and the level of glycoprotein was quantitatively determined. Primary nucleotide sequences of fragments of the genome of the strain ERA-CB 20M (genes N and G) were obtained and phylogenetic analysis was carried out. Molecular analysis showed that this strain belongs to the group of vaccine strains SAD1. When compared with the reference strain SAD1, 10% of the nucleotide differences were revealed in the gene fragment N; 15%, in the gene fragment G.

The DNA of Bacteria of the World Ocean and the Earth in Cosmic Dust at the International Space Station.

Cosmic dust samples from the surface of the illuminator of the International Space Station (ISS) were collected by a crew member during his spacewalk. The sampler with tampon in a vacuum container was delivered to the Earth. Washouts from the tampon's material and the tampon itself were analyzed for the presence of bacterial DNA by the method of nested PCR with primers specific to DNA of the genus Mycobacteria, DNA of the strains of capsular bacteria Bacillus, and DNA encoding 16S ribosomal RNA. The results of amplification followed by sequencing and phylogenetic analysis indicated the presence of the bacteria of the genus Mycobacteria and the extreme bacterium of the genus Delftia in the samples of cosmic dust. It was shown that the DNA sequence of one of the bacteria of the genus Mycobacteria was genetically similar to that previously observed in superficial micro layer at the Barents and Kara seas' coastal zones. The presence of the wild land and marine bacteria DNA on the ISS suggests their possible transfer from the stratosphere into the ionosphere with the ascending branch of the global electric circuit. Alternatively, the wild land and marine bacteria as well as the ISS bacteria may all have an ultimate space origin.