Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

[Etiological structure of influenza and other ARVI in St. Petersburg during epidemic seasons 2012-2016.]

The etiological structure of influenza and other acute respiratory viral infections including their rate of incidence in St. Petersburg and Leningrad region during 4 epidemic seasons has been studied. Seasonality of some respiratory viruses was shown and peaks of circulation of RSV, adenovirus, parainfluenza viruses, rhinovirus, bocavirus, metapneumovirus and coronavirus were marked. The interference of influenza A viruses and RSV, RSV and rhinoviruses was highlighted. A high incidence of adenovirus infection in organized communities and RSV infection in children was revealed.

Modern Approaches for Identification of Modified Nucleotides in RNA.

This review considers approaches for detection of modified monomers in the RNA structure of living organisms. Recently, some data on dynamic alterations in the pool of modifications of the key RNA species that depend on external factors affecting the cells and physiological conditions of the whole organism have been accumulated. The recent studies have presented experimental data on relationship between the mechanisms of formation of modified/minor nucleotides of RNA in mammalian cells and the development of various pathologies. The development of novel methods for detection of chemical modifications of RNA nucleotides in the cells of living organisms and accumulation of knowledge on the contribution of modified monomers to metabolism and functioning of individual RNA species establish the basis for creation of novel diagnostic and therapeutic approaches. This review includes a short description of routine methods for determination of modified nucleotides in RNA and considers in detail modern approaches that enable not only detection but also quantitative assessment of the modification level of various nucleotides in individual RNA species.

[Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures].

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.

SAFETY AND IMMUNOGENICITY OF COLD-ADAPTED RECOMBINANT INFLUENZA VECTOR EXPRESSING ESAT-6 AND AG85А ANTIGENS OF M. TUBERCULOSIS.

Recombinant viral vectors represent one of the most promising platforms for creating a new generation of vaccines against tuberculosis. We constructed a vaccine candidate based on a cold-adapted influenza vector with a truncated NS1 protein containing an insert of tuberculosis ESAT-6 and Ag85A antigens. The recombinant virus possessed a cold-adapted and temperature-sensitive phenotype and was attenuated for mice when administered intranasally. Immunofluorescent staining and Western blot showed the expression of ESAT-6 protein in MDCK cells infected by recombinant virus. After intranasal administration to mice, the recombinant virus stimulated a specific anti-tuberculosis CD4 + Th1-type response with the formation of polyfunctional antigen-specific T cells.

GENETIC AND ANTIGENIC CHARACTERISTICS OF RESPIRATORY SYNCYTIAL VIRUS STRAINS ISOLATED IN ST. PETERSBURG IN 2013-2016.

Antigenic and genetic characteristics of Russian RSV isolates are presented for the first time. Of the 69 strains isolated in St. Petersburg, 93% belonged to the RSV-A antigenic group. The antigenic variations in the F-protein RSV were analyzed using a panel from 6 monoclonal antibodies by the method of micro-cultural ELISA. Depending on the decrease in the effectiveness of interaction with monoclonal antibodies (relative to the reference strain Long), RSV-A isolates were divided into 4 antigenic subgroups. The results of 24 isolates sequencing showed that more than 60% of them had substitutions in significant F-protein sites compared to the ON67-1210A reference strain of the current RSV genotype ON1/GA2. The most variable were the signal peptide and antigenic site II. When comparing the results of ELISA and sequencing, it was not possible to identify any specific key substitutions in the amino acid sequence of the F-protein that affect the interaction of the virus with antibodies. The nucleotide sequence of the F-gene from 19 of the 24 characterized isolates was close to that of ON67-1210A reference virus and was significantly different from RSV-A Long and A2 viruses. A separate group consisted of 5 strains, in which the F-protein structure was approximated to RSV Long.

[Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents].

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents.

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

[Characterization of cold-adapted influenza strain A/HongKong/1/68/162/35 as a potential donor of attenuation and high reproduction].

Live and inactivated vaccines are currently produced using virus reassortants originating from various gene donors of internal proteins. Based on the pandemic virus A/Hong Kong/1/68 (H3N2), a cold-adapted thermo-sensitive strain A/Hong Kong/1/68/162/35 was generated. It is distinguished for its high reproductive capacity (9-9.5 lg EID50), and hemagglutinating activity (1:1024-1:2048). The strain has ts and ca phenotype: reproductive capacity at t = 39 degrees C is 1.0 lg EID50; at t = 26 degrees C, 8.5 lg EID50. A total of 16 mutations have emerged from comprehensive sequencing of the virus genome. Among them 10 mutations were located in the genes of polymerase complex and NP, with respective amino-acid substitutions. The stability of strain characteristics, such as attenuation to humans and high reproductive capacity, were confirmed by repeated sequencing of the genome after tenfold passing of the virus in chicken embryos. Reassortants of the strain A/Hong Kong/1/68/162/35 with the wild-type viruses have inherited useful features of donor virus.

[Evolutionary variability of influenza B viruses in Russian Federation in 2005-2012].

Specific traits of influenza B viruses circulation in Russia and worldwide in 2005-2012 were studied and the amount of influenza B viruses in the whole population of influenza viruses isolated in Russia was estimated. The trend toward antigenic drift for both Victoria and Yamagata lineages was characterized. The genetic analysis revealed amino acid changes that influenced the antigenic properties of the viruses. The match of the epidemic isolates and vaccine strains was corroborated.

[Genetic diversity and molecular evolution of the influenza A viruses in Russia during 2006-2012].

The results of molecular genetic analysis of more than 280 strains of influenza A virus subtypes H1N1 and H3N2 circulating in Russia in 2006-2012 are presented. The genetic changes underlying the evolution of the virus strains and sensitivity to antiviral drugs were analyzed. Significant changes in the genetic structure of influenza A viruses circulating in the Russian Federation and their phylogenetic affiliation are shown to occur within the studied period. The studies identifying codons under the positive selection in silico in the genes encoding surface proteins of the influenza virus were demonstrated to be efficient for the analysis of the antigenic drift and direction of evolutionary variability of the influenza viruses.

[Characterization of influenza virus reassortants based on new donor strain A/HK/1/68/162/35(H3N2)].

Influenza reassortant viruses A/SPb/HK/09(H1N1), A/Astana/HK/2009 (H5N1), A/Otar/HK/2010(H3N8), and A/Perth/ HK/2011(H3N2), carrying surface antigens of different subtypes, were constructed on the basis of new potential unified donor strain A/HK/1/68/162/35(H3N2). The virulence and reproduction activity of the obtained reassortants were tested. The safety of the candidate live and inactivated influenza vaccines produced from the reassortant viruses was demonstrated. The study demonstrates that A/HK/1/68/162/35 can be used as a unified donor for attenuated and high-yield vaccine reassortants.

[H1N1V influenza epidemic of 2009 in Russia].

The paper describes dynamics, distribution and morbidity rate during the 2009 A(H1N1)v influenza epidemic in Russia. The epidemic appears to have been especially severe in the cities of the Far-East and Siberian Federal Districts where the average morbidity rate ranged from 6.4% to 19.2% (mean 10.3%) and the epidemic duration from 7.8 to 8 weeks. In less affected Southern and Central Federal Districts A(H1N1)v influenza occurred in 5.7% of the population. Schoolchildren aged 7-4 years showed the highest morbidity rate of 28.8%. The age group of 18-53 years accounted for 79.4% of the total lethality. Viral isolates were genetically stable and exhibited 98.9% hemagglutnin (HA) homology with reference viruses. None of the strains had an amino acid substitution at position 275 of neuraminidase (NA) responsible for resistance to oseltamivir. Towards the end of the epidemic, the viral population displayed a significant rise in the number of strains containing mutations in 4 genes (4 HA, 2 NA, 2 PB2 and 1 PA mutations respectively). 26.7% of the viral isolates obtained in the end of the epidemic had D222G substitution responsible for tropism of viruses to lung tissues. Epidemiologically, the 2009 A(H1NI)v influenza epidemic is described as moderate based on the absence of pathogenicity determinants typical of both A(H1N1) influenza virus of 1918 and A(H5N1) virus. The paper compares the 2009 epidemic with those caused by A/Honkong/68 and A/USSR/ 90/77 viruses. The necessity of classification for the discrimination between A(H1N1) subtype viruses is emphasized.

[Analysis of pandemic influenza in Russia as a part of the global process based on data of an influenza monitoring reference center].

AIM: Characterization of features of influenza pandemic development in Russia in relation to global process. MATERIALS AND METHODS: Pandemic monitoring was performed by using results of integrative analysis of laboratory diagnostic and population morbidity data from 49 supporting bases of Federal center of influenza from various cities in Russian Federation. Isolation of influenza virus was carried out in MDCK cells and chicken embryos under BSL-3 conditions. Reference virus A/California/07/09 obtained from CDC (Atlanta, USA) and antisera against this strain contained in WHO kit were used for antigenic analysis; rat antisera, new monoclonal antibodies against pandemic influenza virus developed by Research institute of influenza were also used. RESULTS: Based on PCR monitoring during epidemic peak, rate of pandemic influenza identification reached 45-49% of examined patients. About 53% of lethal cases of respiratory infections were caused by pandemic influenza virus, while predominately young people died from pneumonia and acute respiratory distress syndrome. Russian isolates generally were antigenically and genetically similar to the parent pandemic strain--influenza virusA/California/07/09, but contained S203T substitution in hemagglutinin. A number of strains contained D222G mutation that is responsible for the expansion of substrate specificity, as well as strain specific substitutions in hemagglutinin and neuraminidase molecules. The investigated isolates were resistant to remantadin, but sensitive to oseltamivir. CONCLUSION: Due to the formation of population immunity after the end of the first pandemic wave new drift variants of the virus capable of overcoming this formed immunity should be expected that apparently will require the correction of vaccine composition for the 2011 - 2012 season.

[The 2009 pandemic influenza in Russia. I. Diagnosis and molecular biological characteristics of the virus].

The analysis of 1558 clinical samples revealed influenza virus A(H1N1v) RNA in 339 patients with influenza and 163 fatal cases,which was made in May to December 2009. Data on the antigenic properties of more than 250 of pandemic virus strains isolated at the Research Institute of Influenza and the molecular genetic characteristics of 31 strains are presented. All the test isolates were found to have the S203 substitution in hemagglutinin, which was characteristic of one of 5 minor genome A(H1N1v) virus variants found in the United States and Mexico in 2009. All the test strains contain the S31N substitution in the M2 protein, which determines viral resistance to adamantine, and have no H275Y substitution in neuraminidase, which determines oseltamivir resistance. The substitution of amino acid residue of Asp to Gly at position 222 of HA was found in 8 (73%) of 11 isolates from postmortem lung and trachea samples and in 2 (10%) of 20 isolates from nasopharyngeal swabs. The determination of the pathogenic role of this substitution calls for further investigations.

[Prevention of breast cancer]. / Profilaktika raka molochnoi zhelezy.

Analysis of 60 Russian and foreign publications demonstrated that the incidence of breast cancer and socio-biological characteristics of this disease necessitate improvement of methods for its prevention and treatment. Factors significantly increasing the probability of breast cancer are analyzed. Risk groups in need of thorough prophylactic examinations and treatment can be formed on the basis of these data. Some dyshormonal hyperplasias are considered as precancer. Active prophylactic measures, including surgery, are recommended for patients with such forms of mastopathy. There is a group of patients with 100% risk of breast cancer. Cancer can be prevented in them by prophylactic interventions, from resection of the mammary gland to subcutaneous mastectomy with plasty. Such strategy will have a positive impact on the incidence of breast cancer and the results of its treatment.

[Criteria of selection of patients with breast cancer for organ-sparing operations]. / Kriterii otbora bol'nykh rakom molochnoi zhelezy dlia organosokhraniaiushchikh operatsii.

The analysis is given to a number of criteria guiding the selection of breast cancer patients for preserving surgery. The tumor size is one of the most essential criteria. Breast-sparing surgical treatment within radical resection of the breast according to the above selection proved not inferior by survival characteristics to radical mastectomy surpassing it by social and occupational results.

[Criteria for selecting patients with breast cancer for organ-saving operations]. / Kriterii otbora bol'nykh rakom molochnoi zhelezy dlia organosokhraniaiushchikh operatsii.

The study discusses the results of treatment of 597 cases of stage I-IIa breast cancer who underwent either mastectomy after Halsted, Patey or Pirogov (434 patients), or organ-saving radical resection of the breast (163). The analysis of the data identified biologic, morphologic, psychologic and anthropometric criteria as well as laboratory tests which may serve as criteria for selecting candidates for organ-saving surgery and adjuvant radiotherapy. In a group of patients who met the criteria, sparing surgery yielded good results in terms of five-year survival rate (88.9%) and degree of socio-occupational rehabilitation.