[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.

[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.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

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.

[Evolution of pandemic influenza virus A(H1N1)pdm09 in 2009-2016: dynamics of receptor specificity of the first hemagglutinin subunit (HA1).]

INTRODUCTION: The new reassortant of the swine flu virus A(H1N1)pdm09, which emerged in 2009, overcame the species barrier and caused the 2009-2010 pandemic. One of the key points required for the influenza virus to overcome the species barrier and adapt it to humans is its specific binding to the receptors on the epithelium of the human respiratory tract. PURPOSE: Studying the dynamics of changes in receptor specificity (RS) of the HA1 subunit of the hemagglutinin of the influenza A(H1N1)pdm09 virus strains isolated during the period 2009-2016 on the territory of the Russian Federation, and an analysis of the possible impact of these changes on the incidence rates of the population of the Russian Federation of pandemic influenza in certain epidemic seasons. MATERIAL AND METHODS: Standard methods of collecting clinical materials, isolation of influenza viruses, their typing and genome sequencing were used. For the study of RS of influenza A virus (H1N1)pdm09, the method of solid phase sialosidenzyme analysis was used. RESULTS: It is shown that the change in the parameter W3/6 , which characterizes the degree of a2-3 receptor specificity (a2-3-RS) of the influenza virus A(H1N1) pdm09 over a2-6-RS, coincides with the change in the incidence rates of the Russian Federation's pandemic flu in separate epidemic seasons. There is a tendency to increase the affinity of the virus A(H1N1)pdm09 to α2-3 analogs of the sialyl-glycan receptors of the human respiratory tract epithelium - α2-3-sialoglycopolymers (α2-3-SGP), and falls to α2-6-SGP, with the virus showing the greatest affinity for sulfated sialoglycopolymers. DISCUSSION: Screening for RS strains of influenza A (H1N1)pdm09 virus isolated on the territory of the Russian Federation in 2009-2016 revealed a decrease in the affinity of viruses for a2-6-sialosides, especially for 6'SL-SGP, which is probably due to the presence of amino acid substitutions in the 222 and 223 positions of RBS HA1 viruses. Previous studies have shown that the presence of such substitutions correlates with an increase in the virulence of the influenza A virus (H1N1)pdm09 [16, 23]. Probably, the pandemic virus has evolved towards the selection of more virulent pneumotropic variants. CONCLUSION: Monitoring of the receptor specificity of a pandemic influenza virus makes it possible to identify strains with altered RS to the epithelium of the human respiratory tract and an increased ability to transfer from person to person. Change in the period 2009-2016 the W3/6 parameter characterizing the degree of α2-3-RS excess of the influenza A(H1N1)pdm09 virus over α2-6-RS, coincides with the change in the incidence rates of the pandemic influenza population of the Russian Federation in certain epidemic seasons.

[Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus].

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.

DRIFT OF INFLUENA A(H3N2) VIRUS: BIOLOGICAL, ANTIGENIC AND GENETIC PROPERTIES IN EPIDEMIC SEASON 2016-2017 IN RUSSIA AND COUNTRIES OF THE NOTHERN HEMYSPHERE.

The article presents the features of the influenza virus circulation for the period from October 2016 to May 2017 in some territories of Russia collaborating with the D.I. Ivanovsky Institute of Virology, Federal State Budgetary Institution "N.F. Gamaleya Federal Research Centre for Epidemiology and Microbiology", Ministry of Health of the Russian Federation. One of the 2016-2017 season's peculiarities in Russia and countries of the Northern hemisphere was the earlier start of an increase in ARD morbidity with peak indexes reached towards the end of December 2016 - January 2017. First, influenza A(H3N2) virus was predominant; then, it was followed by influenza B virus activity observed until the end of the season. The indexes of morbidity were higher than in the previous season, while the rates of hospitalization and mortality were lower, lethal cases being detected in persons 65 years old and older. Epidemic strains of influenza A(H3N2) virus belonged to 3c.2a genetic group, reference strain A/Hong Hong/4408/2014, and its subgroup 3c.2a1, reference A/Bolzano/7/2016, that are antigenically similar. Strains of influenza B virus were antigenically similar to the B/Brisbane/60/2008 vaccine virus. Strains were sensitive to oseltamivir and zanamivir. The share participation of non-influenza ARI viruses was similar to preliminary epidemic seasons. WHO has issued recommendations for influenza virus vaccines composition for 2017-2018 for the Northern hemisphere.

[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.

[Next-Generation Techniques for Discovering Human Monoclonal Antibodies].

Monoclonal antibodies have found wide applications in the treatment of cancer, as well as of autoimmune, infectious, and other diseases. Several dozen new antibodies are currently undergoing different stages of clinical trials, and some of them will soon be added to the list of immunotherapeutic drugs. Most of these antibodies have been generated using hybridoma technology or a phage display. In recent years, new methods of obtaining human monoclonal antibodies have been actively developing. These methods rely on sequencing immunoglobulin genes from B lymphocytes, as well as on the creation of antibody-secreting stable B-cell lines. The term next-generation antibody-discovery platforms has already been established in the literature to refer to these approaches. Our review focuses on describing the results obtained by these methods.

[Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86­99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

Next-Generation Techniques for Discovering Human Monoclonal Antibodies.

Monoclonal antibodies have found wide applications in the treatment of cancer, as well as of autoimmune, infectious, and other diseases. Several dozen new antibodies are currently undergoing different stages of clinical trials, and some of them will soon be added to the list of immunotherapeutic drugs. Most of these antibodies have been generated using hybridoma technology or a phage display. In recent years, new methods of obtaining human monoclonal antibodies have been actively developing. These methods rely on sequencing immunoglobulin genes from B lymphocytes, as well as on the creation of antibody-secreting stable B-cell lines. The term next-generation antibody-discovery platforms has already been established in the literature to refer to these approaches. Our review focuses on describing the results obtained by these methods.

[Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin].

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.

[The 2015-2016 epidemic season in Russia and the world: Circulation of influenza viruses, trends in incidence, clinical aspects, and treatment algorithm]. / Épidemicheskii sezon 2015-2016 gg. v Rossii i mire: osobennosti tsirkuliatsii virusov grippa, dinamika zabolevaemosti, klinicheskie aspekty i algoritm lecheniia.

In the 2015-2016 epidemic season, there were dominant influenza A(H1N1)pdm09 strains (over 90%) among the circulating influenza viruses in most countries of the Northern Hemisphere and in Russia. A study of the antigenic properties of influenza A(H1N1)pdm09 strains revealed no differences in those of vaccine virus. Sequencing showed that there were amino acid substitutions in hemagglutinin (receptor binding and Sa sites) and in the genes encoding internal proteins (PA, NP, M1, and NS1). The rise in the incidence in the Russian Federation, which was etiologically associated with influenza viruses, was registered in January-February 2016 with its maximum being observed at 4-5 weeks of 2016. Within the framework of the epidemiological surveillance of circulating influenza viruses in the Russian Federation, which was conducted by the WHO European Office, the D.I. Ivanovsky Institute of Virology, Honorary Academician N.F. Gamaleya Federal Research Centre for Epidemiology and Microbiology, Ministry of Health of Russia, and the Research Institute of Influenza, Ministry of Health of Russia, monitored at the Infectious Diseases Hospital One (IDH-1), Moscow Healthcare Department. Among 1491 examinees, influenza was verified in 104 (21.3%) adults, 208 (42.5%) pregnant women, and 177 (36.2%) children. Influenza A(H1N1)pdm09 was more often diagnosed in the age group of 15-40 years (63.7%); the proportion of influenza patients aged over 50 years increased (22.1%). Most adult patients had moderate influenza; pneumonia complicated the disease in 27.4%. Influenza in the pregnant women was complicated by pneumonia in 4.8% of cases. Influenza was more frequently diagnosed in infants and preschool children aged 0 to 3 years (42.9%), 4 to 6 years (41.2%), and older (15.9%), namely: 7-9 years (10%) and 10-12 years (5.9%). Influenza in the children was complicated by acute tonsillitis (19.4%) and varying degrees of laryngeal stenosis (12.4%). Bronchial obstructive syndrome developed in 2.5%, the rate of pneumonia was 6.2%. Antiviral therapy (AVT) in the early stages of the disease reduces the risk of its severity, the frequency of secondary complications, and the duration and degree of clinical symptoms of influenza. AVT with oseltamivir, zanamivir, imidazolyl ethanamide pentandioic acid (ingavirin), and interferon-a2b (viferon) has been performed in the patients hospitalized at Moscow IDH-1 in the 2015-2016 epidemic season.

Virological, epidemiological, clinic, and molecular genetic features of the influenza epidemic in 2015-2016: prevailing of the influenza A(H1N1)09 pdm virus in Russia and countries of the Northern hemisphere.

This work describes the specific features of the influenza virus circulating in the period from October 2015 to March 2016 in 10 cities of Russia, the basic laboratories of CEEI at the D.I. Ivanovsky Institute of Virology "Federal Research Centre of Epidemilogy and Microbiology named after the honorary academician N.F. Gamaleya" of the Ministry of Health of the Russian Federation. The increase in the morbidity caused by influenza viruses was detected in January-February 2016. The duration of the morbidity peak was 4-5 weeks. The most vulnerable group included children at the age from 3 to 6; a high rate of hospitalization was also detected among people at the age of 15-64 (65%). In clinic symptoms there were middle and severe forms with high frequency of hospitalization as compared with the season of 2009-2010, but much higher in comparison with the season of 2014-2015. Some of the hospitalized patients had virus pneumonias, half of which were bilateral. Among these patients, 10% were children; 30%, adults. The mortality in the intensive care unit of the hospital was 46%. Almost all lethal cases were among unvaccinated patients in the case of late hospitalization and without early antiviral therapy. The predominance of the influenza A(H1N1)09pdm virus both in the Russian Federation and the major part of the countries in the Northern hemisphere was noted. The results of the study of the antigenic properties of influenza strains of A(H1N1)pdm09 virus did not reveal any differences with respect to the vaccine virus. The sequencing data showed the amino acid substitutions in hemagglutinin (receptor binding and Sa sites) and in genes encoding internal proteins (PA, NP, M1, NS1). Strains were sensitive to oseltamivir and zanamivir and maintained resistance to rimantadine. The participation of non-influenza ARI viruses was comparable to that in preliminary epidemic seasons.

[Insertional mutation in the AZOBR_p60120 gene is accompanied by defects in the synthesis of lipopolysaccharide and calcofluor-binding polysaccharides in the bacterium Azospirillum brasilense Sp245].

In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR_p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR_p60120 gene in the A. brasilense Sp245 LPSI- Cal- KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.

[The 2013-2014 epidemic season. Hospital monitoring and antiviral therapy for influenza ].

AIM: To characterize the 2013-2014 epidemic season from the results of detection of influenza infection in patients; to provide the molecular genetic characteristics of the strains isolated from deceased patients. SUBJECTS AND METHODS: The investigators examined 1203 patients (387 children, 509 people older than 16 years of age, 307 pregnant women) admitted to Moscow Clinical Infectious Diseases Hospital One with the clinical signs of acute respiratory viral diseases. Nasal lavage and autopsy specimens were used to isolate viral strains, then to sequence genomic fragments, and to determine receptor specificity. RESULTS: Out of the 1203 examinees, 284 (23.6%) were influenza-positive: 221 (77.8%), 24 (8.5%), and 39 (13.7%) patients had influenza A(H3N2), influenza A(H1N1)pdm09, and influenza B, respectively. Influenza was notified in 42,7% of the pregnant women. There was a preponderance of its moderate form; its severe form developed in single cases having comorbidities. One fatal outcome was registered. The intake of antiviral medications in the first 48 hours of the disease could prevent complications. The investigators revealed mutations in the strain isolated from the bronchoalveolar lavage fluid of a patient with severe pneumonia complicated by acute respiratory distress syndrome. CONCLUSION: There is evidence that there are mutant A(H1N1)pdm09 viruses that have high pneumotropicity. The high risk of their circulation in the population and the risk of severe influenza forms involving the lower respiratory tract remain. Early antiviral therapy in the first 36-48 hours diminishes the clinical manifestations of influenza and reduces the risk of developing complications.

[The peculiarities of the influenza epidemics in some areas of Russia during 2012-2013 season. The influenza A (H1N1) pdm09 virus domination in European countries].

The peculiarities of the influenza viruses circulation in 2012-2013 are discussed. The results were obtained in 10 cities of Russia, where basic laboratories of the Influenza Ecology and Epidemics Center of on the basis of Ivanovsky Institute of Virology, Ministry of Health of the Russian Federation, are situated. The increasing rate of the ARD morbidity caused by influenza viruses was observed in January-March 2013. The highest indices of the morbidity were detected during 6-7 weeks with the following decreasing rate till threshold levels to week 14. The influenza A (H1N1) pdm09, A (H3N2), and B viruses were the cause of the epidemic, but their activity differed over areas of Russia. The results of study of the antigenic and genetic properties of the influenza strains demonstrated closed relatives with respect to vaccine strains. In addition, some heterogeneity of the circulating strains and their drift variants were found as well. All tested strains were sensitive to oseltamivir (excluding one A (H1N1) pdm09 strain), zanamivir, arbidol, and remained resistant to rimantadine. The ratio of the ARD viruses was comparable with the last epidemic seasons.

[Etiology of fatal pneumonia caused by influenza A(H1N1)pdm2009 virus during the pandemic in Russia].

The results of the study of the autopsy materials from 61 patients with the diagnosis of pneumonia received by virological and genetic methods are reviewed. The materials were studied at the Influenza Etiology and Epidemiology Center of the Ivanovsky Institute of Virology, Ministry of Health and Social Development of the Russian Federation, during epidemic seasons 2009-2010 and 2010-2011. The data were analyzed with respect to age, sex, comorbidity diseases and identified on the groups of the risk of severe forms of the disease. The presence of the pandemic influenza virus strain RNA was confirmed in 70.5% of materials; RNA of influenza B was detected in 1.2% cases. The co-infections caused by the bocavirus, adenovirus, parainfluenza virus type 2 and 4, rhinovirus, and streptococcus were detected only in 19.7%. In most cases, the influenza virus was the etiologic agent of lethal pneumonia, which justifies the necessity of the early etiological diagnosis and treatment with antiviral drugs.

[Efficacy of anti-neuraminidase drugs application during and after an influenza pandemic].

The emergent 2009 A(H1N1) pandemic brought into acute focus the problem of choosing the most effective anti-influenza drugs for successive influenza infection spreading control. Oseltamivir and zanamivir, influenza virus neuraminidase inhibitors (NAIs), were recommended by the WHO experts for the treatment and prevention of influenza, including that caused by pandemic strains. A major concern regarding the use of specific antiviral compounds is the emergence of the drug-resistant strains. Oseltamivir carboxylate and zanamivir IC50 values were equal to 0.3-5.2 microM for the most of A(H1N1)pdm09 pandemic strains and 1.6-8.6 microM for the strains of influenza B virus in cell-based ELISA assay (2009-2010 season). All the studied strains of influenza A(H1N1 ) pdm09 (151) and B (22) viruses were sensitive to NAIs (2009-2011 seasons). For the first time in Russia oseltamivir-resistant A(H1N1) pdm09 influenza virus was isolated from the patient on the 5th day of a treatment course of this drug.

[Development of the influenza epidemic in season 2011-2012 in some areas of Russia: results of activity of the Influenza Etiology and Epidemiology Center of the Ivanovsky Institute of Virology].

The results of analysis of the peculiarities of the epidemic 2011-2012 development in the areas of 10 cities of Russia obtained by basic laboratories of IEES on the base of D.I. Ivanovsky Research Institute of Virology, Ministry of Public Health and Social Development of Russia, are presented. The increasing ARD morbidity caused by the influenza viruses was detected rather late--in February-March 2012. The highest indices of the morbidity were detected during weeks 10-13 followed by decreasing to threshold levels by week 27. Children 0-2 and 3-6 years old were involved the most, meantime the high rate of hospitalization was found for 15-64 years old aged group (25%). Influenza A(H3N2) and B viruses were the cause of the epidemic. The results of studies of the antigenic and genetic properties of the influenza strains showed most of them to be close relatives to the vaccine strains. Some heterogeneity of circulating strains and their drift variants were found as well. All tested strains were sensitive to arbidol, oseltamivir and zanamivir, and saved resistance to rimantadine. The ratio of ARD viruses was comparable with the last epidemic seasons.