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Introduction. The World Health Organization considers the values of antibody titers in the hemagglutination inhibition assay as one of the most important criteria for assessing successful vaccination. Mathematical modeling of cross-immunity allows for identification on a real-time basis of new antigenic variants, which is of paramount importance for human health. Materials and methods. This study uses statistical methods and machine learning techniques from simple to complex: logistic regression model, random forest method, and gradient boosting. The calculations used the AAindex matrices in parallel to the Hamming distance. The calculations were carried out with different types and values of antigenic escape thresholds, on four data sets. The results were compared using common binary classification metrics. Results. Significant differentiation is shown depending on the data sets used. The best results were demonstrated by all three models for the forecast autumn season of 2022, which were preliminary trained on the February season of the same year (Auroc 0.934; 0.958; 0.956, respectively). The lowest results were obtained for the entire forecast year 2023, they were set up on data from two seasons of 2022 (Aucroc 0.614; 0.658; 0.775). The dependence of the results on the types of thresholds used and their values turned out to be insignificant. The additional use of AAindex matrices did not significantly improve the results of the models without introducing significant deterioration. Conclusion. More complex models show better results. When developing cross-immunity models, testing on a variety of data sets is important to make strong claims about their prognostic robustness.

Introduction. Influenza is a disease caused by a widespread virus with pandemic potential. Frequently, individuals vaccinated against seasonal influenza virus are still susceptible to the disease, indicating the need to improve the immunogenic potential of existing vaccines. To assess the efficacy of influenza virus vaccines, immune response only to a single viral antigen — hemagglutinin molecule, is taken into consideration. However, according to preclinical and clinical studies, neuraminidase (NA) stimulates cross-protective immunity, which is effective against not only homologous but also drifted variants of influenza A virus. Materials and methods. In the present study, we investigated the ability of previously selected conserved linear B-cell NA epitopes (SGYSGK, SWPDGK, EECSCYPK, VELIRGRK) to enhance the immunogenicity of an inactivated whole-virion influenza vaccine based on the model strain PR8 (iPR8). BALB/c mice were injected with iPR8 in combination with one of the peptides intramuscularly three times at two-week intervals. Blood samples were collected 14 days after the last immunization, after which the mice were challenged with heterosubtypic influenza viruses H1N1pdm09 and H3N2. Results. All immunized mice showed induction of H1N1 (PR8)-specific IgG antibodies two weeks after the third immunization. The group of mice immunized with the iPR8 vaccine preparation in combination with VELIRGRK peptide showed the most pronounced induction of IgG antibodies to the H6N1 reassortant strain, the NA of which corresponds to the iPR8 virus, indicating the ability of the NA peptide to stimulate the production of NA-specific antibodies. However, the antibodies produced after immunization were not capable to inhibit the NA enzymatic activity. Despite this, mice immunized with iPR8 in combination with anti-NA peptides showed a higher survival rate after infection with heterologous virulent influenza viruses: A/California/07/09 (H1N1pdm09) and A/Philippines/2/82 (H3N2) compared to the PBS and iPR8 groups. Conclusion. Thus, the study demonstrated the immune-potentiating effect of individual peptides corresponding to conservative linear epitopes of the NA molecule in combination with a standard inactivated influenza vaccine, which made it possible to improve the protective effect of the vaccine against heterosubtypic influenza viruses.

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.

Introduction. Influenza is one of the most pressing global health problems. Despite the wide range of available anti-influenza drugs, the viral drug resistance is an increasing concern and requires the search for new approaches to overcome it. A promising solution is the development of drugs with action that is based on the inhibition of the activity of cellular genes through RNA interference. Aim. Evaluation in vivo of the preventive potential of miRNAs directed to the cellular genes FLT4, Nup98 and Nup205 against influenza infection. Materials and methods. The A/California/7/09 strain of influenza virus (H1N1) and BALB/c mice were used in the study. The administration of siRNA and experimental infection of animals were performed intranasally. The results of the experiment were analyzed using molecular genetic and virological methods. Results. The use of siRNA complexes Nup98.1 and Nup205.1 led to a significant decrease in viral reproduction and concentration of viral RNA on the 3rd day after infection. When two siRNA complexes (Nup98.1 and Nup205.1) were administered simultaneously, a significant decrease in viral titer and concentration of viral RNA was also noted compared with the control groups. Conclusions. The use of siRNAs in vivo can lead to an antiviral effect when the activity of single or several cellular genes is suppressed. The results indicate that the use of siRNAs targeting the cellular genes whose expression products are involved in viral reproduction is one of the promising methods for the prevention and treatment of not only influenza, but also other respiratory infections.

Introduction. Influenza A virus is the cause of epidemics and pandemics that severely affect the health and socioeconomic status of the world's population. The need to develop new methods of etiotropic therapy, and the increasing ability of viruses to counteract the antiviral drugs makes extremely relevant the search for new pharmacologically active substances and the subsequent study of their medicinal properties. The aim of the study is to conduct research into the antiviral properties of melanin obtained from the pharmaceutical chaga mushroom in relation to different subtypes of the influenza A virus. Materials and methods. A sample of water-soluble melanin from Inonotus obliquus obtained by alkaline hydrolysis and dried at 40C was tested for toxicity and antiviral activity. The commercial anti-influenza drug Tamiflu was used as a reference drug. Statistical processing of the obtained data was carried out according to the Spearman-Kerber method. Results. Inonotus obliquus melanin (sample 20-24) toxicity markers, such as a maximum tolerable concentration (MTC) of 237.0 g/mL, and a 50% cytotoxic concentration (CC50) of 153,45 g/mL were established for MDCK cell culture. The assessment of antiviral activity of test sample against three subtypes of the influenza A virus (H5N1, H3N2 and H1N1pdm09) demonstrated a decrease in the infectivity of the influenza virus by 2.53.5 lg with 50% virus-inhibiting concentrations (IC50) of 1.559.52 g/mL. Based on the obtained values of CC50 and IC50, the selectivity indices (SI) of the sample were calculated, characterizing its prospects for further research. Conclusions. Melanin obtained from the pharmaceutical chaga mushroom showed the highest activity against the strain of the human pandemic influenza virus A/California/04/2009 (H1N1)pdm09, caused a decrease in its infectivity by 3.5 lg and had an IC50 of 1.6 g/ml. The obtained results indicate the prospects for creating an antiviral drug based on Inonotus obliquus melanins against the influenza virus.

Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.

Objectives. Evaluation of changes in the viral activity of influenza A/WSN/33 after complex knockdown of combinations of cellular genes FLT4, Nup98 and Nup205 in human lung cell culture A549. Methods. The work was carried out using the equipment of the Center for Collective Use of the I. Mechnikov Research Institute of Vaccines and Sera, Russia. The authors performed transfection of combinations of small interfering ribonucleic acid (siRNA) complexes that cause simultaneous disruption of the expression of cellular genes FLT4, Nup98, and Nup205. Within three days from the moment of transfection and infection, the supernatant fluid and cell lysate were taken for subsequent viral reproduction intensity determination using the titration method for cytopathic action. The dynamics of changes in the concentration of viral ribonucleic acid (vRNA) was determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The nonparametric Mann–Whitney test was used to calculate statistically significant differences between groups.Results. Using all of the combinations of siRNA complexes, cell viability did not decrease below the threshold level of 70%. In cells treated with complex FLT4.2 + Nup98.1 + Nup205 at the multiplicity of infection (MOI) equal to 0.1, a significant decrease in viral reproduction by 1.5 lg was noted on the first day in relation to nonspecific and viral controls. The use of siRNA complexes at MOI 0.01 resulted in a more pronounced antiviral effect. The viral titer in cells treated with siRNA complexes FLT4.2 + Nup98.1 and Nup98.1 + Nup205 decreased by 1.5 lg on the first day. In cells treated with complexes FLT4.2 + Nup205 and FLT4.2 + Nup98.1 + Nup205, it decreased by 1.8 and 2.0 lg on the first day and by 1.8 and 2.5 lg on the second day, respectively, in relation to nonspecific and viral controls. When conducting real-time RT-PCR, a significant decrease in the concentration of vRNA was noted. At MOI 0.1, a 295, 55, and 63-fold decrease in the viral load was observed with the use of siRNA complexes FLT4.2 + Nup98.1, Nup98.1 + Nup205, and FLT4.2 + Nup98.1 + Nup205, respectively. On the second day, a decrease in vRNA was also observed in cells treated with complex A. A 415-fold decrease in vRNA on the third day was noted in cells treated with complex FLT4.2 + Nup205. At MOI 0.01, the concentration of vRNA decreased 9.5 times when using complex B relative to nonspecific and viral control.Conclusions. The study showed a pronounced antiviral effect of siRNA combinations while simultaneously suppressing the activity of cellular genes (FLT4, Nup98, and Nup205), whose expression products are playing important role in the viral reproduction process, and obtained original designs of siRNA complexes. The results obtained are of great importance for the creation of emergence prophylactic and therapeutic drugs, whose action is based on the mechanism of RNA interference.

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

Currently, the assessment of the immunogenic properties of influenza viruses as a part of influenza vaccines is carried out by using seroprotection, seroconversion as well as the rate of increases in post-vaccination antibodies. At the same time, significant differences in the immunogenicity of vaccines related to dynamic formation of high antibody titers responsible for long-term protection of the vaccinated, are neglected. Influenza viruses such as A(H1N1)pdm09 that caused 2009–2010 pandemic continue to circulate in the population, therefore, the assessment of the immunogenic activity of vaccine viruses prepared during the pandemic period is interesting in for the methodology to prepare pandemic vaccines to be used in various groups (adults, children, elderly people). Analyzing immunogenicity of influenza vaccines used during the 2009–2010 swine influenza pandemic and the post-pandemic period up to the year 2014 was carried out by applying the graphical method for assessing immunogenicity (immunographs) measured as follows: for each group of vaccinated subjects (depending on the vaccine used), an increased rate in antibody level was calculated and the graphs of immunogenicity were plotted. An increased rate of serum antibodies magnitude from vaccinated subjects and the number of sera (in %) with a given fold increase rate in antibody level from 1 to the maximum magnitude were plotted on the x- and y-axis, respectively. The proposed method for assessing immunogenicity allows to plot immunogenicity graphs regardless of the serum antibodies level found in volunteers. The assessment described above revealed a several features for developing immune response to the pandemic virus A(H1N1)pdm09 such as the lack of immune response in a substantial number of adult volunteers (25–27%) and young children (60–70%) after monovaccine administration. The reason for such immune response can be both an insufficient dose of vaccine-containing viral antigen and suppressed immune response caused by the influenza A(H1N1)pdm09. A study on the immunogenic properties for seasonal influenza vaccines containing the influenza A(H1N1)pdm09 virus antigen in the years 2010–2014 revealed a variety in emerging humoral immunity ranging from a short-term, low-frequency increase in antibodies from vaccinated children to the formation of high antibody titers in elderly. Practically, immunographic analysis of influenza vaccines particularly those derived from the influenza A(H1N1)pdm09 virus, may result in proposing recommendations to increase an antigenic load at the beginning of a pandemic cycle and/or block the suppressive properties of vaccine-contained viruses in pediatric vaccines, because escalating virus dose in the vaccine may not always be achievable in this case.

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 («universal») epitopes in HA of different strains of this subtype.

Introduction. Influenza is a socially significant infection that causes profound damage to populational health and national economy annually. Preventive vaccination is the most effective means to manage influenza and its complications. Diverse influenza vaccines exist, but their common drawback is the narrow specificity, the need for annual renewal of the strain composition, not always satisfactory immunogenicity and effectiveness. In this regard, close attention is paid to developing universal influenza vaccines aimed to induce cross-reactive immune-related cues against most conserved parts of viral proteins. Antibodies against neuraminidase (NA) are able to provide heterosubtypic protection, which is important due to potential threat posed by influenza viruses differed in hemagglutinin and neuraminidase sequence in comparison to currently circulating viruses. The present study is aimed to search for new and analysis of earlier predicted NA linear B-cell epitopes conserved among all influenza A virus subtypes. Results. Eight conserved linear B-cell epitopes were identified around the active site of neuraminidase, three of which (MNPNQKIITIGS, ILRTQESEC, and DNWKGSNRP) were synthesized de novo, conjugated with bovine serum albumin to be next used for mouse immunization. IgG antibodies were detected by ELISA in the sera of immunized mice. Antibodies were found to specifically bind to various influenza A viruses containing NA subtypes N1, N2, N3, and N9. Immunization with NA peptides did not protect mice from substantial body weight loss after infection with lethal H1N1 influenza virus. However, all immunized mice survived during the observation period, whereas in the control group the survival rate was as low as 28.6%. Analyzing lung viral load in the mice infected with the H1N1 virus revealed no differences in virus titers on either day 4 or 8 post-infection. Nevertheless, the protective effect lacked after the mice were challenged with lethal H7N9 influenza virus: Mortality rate, body weight loss, and lung virus titers were comparable in immunized and control mice. Conclusions. The data obtained evidenced about serum cross-reactive anti-NA antibodies induced by immunization with NA peptides, as well as protective efficacy against infection caused by H1N1 virus, but not H7N9 virus. Such results hold promise and indicate that NA linear B-cell epitopes can be used for designing epitope-directed influenza vaccines, but a deeper and more comprehensive study on the specificity of conserved NA epitopes, as well as optimization of immunization schemes for achieving higher protective efficacy are required.

Aim. The aim is to explore the molecular and biological characteristics of influenza virus subtype H6N8.Material and Methods. Regular collecting of biological samples from wild birds was carried out in 2016 in the south of Western Siberia. Isolation of avian influenza virus strains from birds’ cloacal swabs was performed using three passages in embryonated chicken eggs. Hemagglutination assay was used to detect viruses in the allantoic fluid. Type and subtype of influenza viruses were identified using reverse transcription PCR (RT‐PCR). Results. This is the first time the avian influenza virus H6N8 subtype has been isolated in Russia. Phylogenetic analysis revealed that hemagglutinin (HA) and neuraminidase (NA) segments of A/gadwall/Chany/97/2016(H6N8) strain genome belonged to the Eurasian lineages of avian influenza viruses. Phylogenetic analysis showed that the HA sequence of the strain was closely related to the strain isolated from a duck in Bangladesh in 2015 (A/duck/Bangladesh/25767/2015(H6N1).Conclusion. Avian influenza viruses subtype H6N8 have the ability to circulate among wild waterfowl and poultry, increasing the chance of reassortment and transmission of H6 virus strains among birds. For this reason, it is of crucial importance to strengthen surveillance for avian influenza H6 subtype among wild birds in the south of Western Siberia.

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 рurpose 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.

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Copyright of News of Kazakhstan Science / Novosti nauki Kazahstana is the property of NCSTE (JSC National Center for State Scientific and Technical Evaluations) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This paper describes results of comparison of genetic sequences of inf luenza viruses obtained from clinical samples (nasopharyngeal swabs) and viruses isolated on MDCK cells and in developing chick embryos. The obtained data shows that 1–2x fold passaging of inf luenza viruses in MDCK cells does not lead to additional mutations in the genome of the inf luenza virus, and the genetic material of this virus is identical to the genetic material from the clinical sample. When passaging viruses in chick embryos within 1–2 passages, the genetic material of the virus differs from the genetic material from the clinical sample, because of mutations in the receptor region of hemagglutinin, leading to aminoacid substitution Q223R. The mutation Q223R changes the receptor specificity of hemagglutinin from human-type receptors (α-2.6-sialic acids) to avian receptors (α-2.3-sialic acids). The data obtained coincide with the data of Japanese researchers who showed that a similar substitution in the hemagglutinin gene was detected in Japanese strains of inf luenza virus subtype A (H1N1) pdm09, passaged in chick embryos for 1–2 passages. With their further passaging in chick embryos, the number of acquired mutations in the surface proteins of these viruses increases. Thus, we have shown that for the genetic analysis of epidemic-relevant strains of inf luenza viruses for routine epidemiological surveillance tasks, the use of direct sequencing of clinical samples containing the genetic material of viruses makes it possible to obtain the most reliable data, to increase their efficiency, since it saves time normally spent on virus isolation procedure. If preliminary isolation of viruses is necessary, for example, if it is not possible to carry out full genome amplification from a clinical sample (low content of the material, presence of polymerase chain reaction inhibitors), virus isolation on MDCK cells with a minimal amount of passage is optimal, which is used in practice of epidemiological surveillance.

The aim is to explore the ecological diversity of wild birds in Siberia, which are carriers of the Avian Influenza Virus (AIV).Methods. Biological material in the form of cloacal swabs and intestinal fragments from wild migratory birds were collected in the period 2007-2014 years. The virus has been gained in the allantoic cavity of developing chicken embryos. The presence of virus was determined in hemagglutination and primary identification and subtyping of influenza virus was confirmed by RT-PCR (reverse transcription polymerase chain reaction).Results. It was collected and investigated 2300 samples obtained from wild migratory birds 8 Orders. The influenza virus was detected in 185 birds of the three groups. The main role in the circulation of the AIV in the south of Western Siberia, playing members of the family Anatidae Order Anseriformes, namely species - Teal (Anas crecca), garganey (Anas querquedula), and shoveler (Anas clypeata). In the period from 2007 to 2014, the percentage of virus infection in waterfowl ranged from 5.6 to 20%. Order Charadriiformes was characterized by a lower percentage of virus isolation, of not more than 1.4%.Conclusion. Wild migratory waterfowl orders Anseriformes and Charadriiformes are the main reservoir of AIV in the south of Western Siberia. The area south of Western Siberia plays a key role in the persistence of avian influenza viruses, their evolution and geographical distribution.

The aim is to analyze the long-term results of the avian influenza virus surveillance monitoring of influenza virus in birds of one of the key Northern Eurasia points Lake. Uvs Nuur, the Republic of Tyva. Methods. The analysis of the available sources and our own research results is conducted. We used MEGA 5.2 software to construct a phylogenetic dendrogram. Tree topology is constructed by the method of maximum likelihood. Genetic distance matrix is calculated using the Kimura two-parameter metric method. Results. We conducted a biogeographical analysis of the Great Lakes basin, and an overview of the literature and the original results of highly pathogenic H5N1 influenza circulation and molecular epidemiology at Uvs Nuur Lake. Conclusion. Long-term observations at Lake Uvs Nuur revealed the important role of the biogeocoenose for the preservation and evolution of influenza A virus in wild bird populations. Planned ecological and virological monitoring is the basis for correct conclusions about the dynamics of epizootic process, infection control, as well as for the evaluation of the epidemic and pandemic potential of novel viral strains.

Aim. Study of ts, ca, att phenotype, immunogenicity and protective effectiveness of reas-sortants, obtained by a way of recombination of a new influenza cold-adapted (ca) strain donor of attenuation A/Krasnodar/101/35/59 (H2N2) and virulent strain of influenza virus. Materials and methods. Viruses were used: ca strain A/Krasnodar/101/35/59 (H2N2), virulent strains: A/ Kumamoto/102/02 (H3N2) and A/Bern/07/95. For determination of ts and ca phenotype, titration of viruses in chicken embryos was carried out simultaneously at optimal, decreased and increased temperature. Protective effect of immunization was evaluated during intranasal infection of mice with a virulent strain of influenza virus. Results. All the obtained reassortants possessed 6 internal genes from strain-donor of attenuation and 2 genes, coding HA and NA-proteins from virulent strains. Ca reassortants were characterized by ts and ca phenotype, had antigenic specificity and good immunogenicity, had high protective effectiveness. Conclusion. The data obtained indicate on the perspectiveness of ca strain A/Krasnodar/101/35/59 (H2N2)as a donor of attenuation for live influenza vaccines.

Influenza A virus (IAV) is a worldwide pathogen of humans and animals. In nature IAV is associated with aquatic birds. The regular survey of natural populations of aquatic birds is important to describe the diversity of IAV, evaluate pathogenic potential of circulating strains for human and domestic animals and reveal possible routes of virus spread during seasonal migrations of wild birds. In this paper the results of annual ecological and viroiogicai survey among wild birds of Eastern Siberia performed between 2005 and 2012 are presented. The cloacal swabs and tissue samples from 901 wild birds were analyzed for presence of IAV using virus isolation in chicken embryos and/or PCR assay. The subtype of IAV was established using haemaggiutintion inhibition assay and genome sequencing. Seventeen subtypes of IAV were discovered including H1N1, H5N1, HswNl, H0N1, H1N4, HlNlsw, H2N2, H3N2, H3N5, H3N6, H3N8, H4N6, H7N1, H7N8, H10N7, H13N6, H13N8. Highest prevalence of infection was detected among wild ducks Anas crecca (18.3 %), A. platyrhynchos (13.8 %) and herring gull Larus argentatus (6.5 %). The most diverse virus population was revealed in Irkutsk Region among L. argentatus that hosted subtypes H3, H6N1 and H9. In the Republic of Tuva and Krasnoyarsk territory H3N8, H5N1 and H7 subtypes were detected. The wild birds from Republic of Khakassia were shown to hostH4 subtype, whereas in Republic of Buryatia subtypes H3 and H4 were detected in wild ducks and herring gulls. Possible routes of IAV spread and epidemiological implications are discussed.

Modern concepts of the influence of the proteins from viruses-etiological agents of flu – Influenzavirus A, B and C (Orthomyxoviridae) – on the development of different elements of the main disease pathogenesis are analyzed in the review. In particular, the short description of life cycle of Influenza viruses is alleguered with special attention to those its stages which are capable to modulate pathogenetic mechanisms. The interrelation between the structure of hemagglutinin receptor-binding site and virus tropism as well as the influence of the receptor-destroying virus proteins on this phenomenon is described. The mechanism of suppression of interferon production in the infected cell by virus NS1 protein is presented. The induction of apoptosis by nonstructural PB1-F2 protein of Influenza A virusis described.

Under the influence of polarized light, the reproduction of the influenza A virus in the mice has been slowed. The survival rate of mice infected with a lethal dose of influenza A virus was 50%, compared to the control group.

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.

In this paper the data for mortality from experimental infections and natural contamination in different species of poultry is presented. Mortality of natural contamination was identified during the epizootic of influenza in the Novosibirsk area in July 2005. We infected domestic geese, ducks and two breeds of chickens with highly pathogenic influenza virus A/goose/Krasnoozerskoye/627/05 (H5N1) for the experiments constructed.

The California encephalitis serogroup virus, Batai virus and Geta virus were isolated as result of investigation of distribution of arboviruses in Pribaikalye. The results of the study of influenza A virus in birds in Pribaikalye are presented as well.

Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called "pre-fusion") in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

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.

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H^(+) proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.

Introduction: The emergence of influenza virus strains with drug resistance to antiviral drugs requires finding new compounds, potential direct-acting inhibitors. Аdamantane compounds drugs used since the 1960s have lost their activity the resulting due to resistance. Only neuraminidase inhibitors such as zanamivir and oseltamivir have been approved by WHO for influenza treatment. The Russian pharmaceutical drug Arbidol (Umifenovirum) is actively used in Russia. This drug is used to treat influenza in Russia, China and most post-Soviet republics. This work presents a new derivative of aminoadamantane - dichlorohydrate L-histidyl-1-adamantayl ethylamine (2HCl*H-His-Rim), which showed a high level of inhibition of strains of influenza virus A in vitro., Objectives: Comparison of antiviral properties of the new synthetic low-molecular inhibitor of influenza A virus replication and Arbidol drug pharmacy., Methods: The compound 2HCl*H-His-Rim was obtained by classical peptide synthesis methods. It was identified by methods of mass spectrometry, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Its antiviral properties have been studied in vitro for monolayer of cells Vero-E6 infected with a high-virulent strain of A/duck/Novosibirsk/56/06 (H5N1) influenza virus at various injection schemes of the investigated compounds., The Results: The antiviral activity of the 2HCl*H-His-Rim compound against the highly pathogenic strain of the influenza A/H5N1 virus was slightly higher than for the known pharmacy drug arbidol., Discussion: The difference in antiviral activity of these two compounds is explained by different mechanisms of action on the viral particle., Conclusion: The 2HCl*H-His-Rim compound can be recommended as a candidate for preclinical and clinical trials in order to obtain an etiotropic antiviral drug based on it, due to its high efficacy and economic and synthetic availability. The synthetic compound 2HCl*H-His-Rim acts on influenza A virus variants resistant to Rimantadine and Amantadine., Competing Interests: The authors declare no conflict of interest.

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.

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.

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.

Five influenza pandemics emerged in the 20th and 21st centuries. Data regarding possibility of infection of swine with human influenza viruses-and persistent circulation of these strains among swine with subsequent infection of humans with these viruses were obtained in the recent years. A possibility of prediction of influenza pandemics by constant observation and study of influenza viruses circulating among swine is discussed in the paper.

Malignant glioma is the most frequently occurring primary brain tumor. Despite significant progress in the diagnostics and treatment of neoplastic diseases the prognosis for patients with III-IV grade gliomas, remains extremely unfavorable. Rapidly developing area in oncology is the employment of therapeutic viruses with natural or genetically engineered oncolytic activity. In the present study we demonstrated the oncolytic potential of a recombinant influenza A virus vector with impaired interferon antagonism function of NS1 protein in treatment of malignant glioma. Recombinant influenza A virus (HA-DS-GFP) expressing green fluorescent protein from the NS1 open reading frame was used as a model vector. HA-DS-GFP virus has shown infectivity towards glioma cells both in vitro, and in vivo (experimental glioma model in rats). Intratumoral inoculation of HA-DS-GFP resulted in a substantial inhibition or complete regression of tumor growth. Our data demonstrate that recombinant influenza vectors have promising potential in therapy of malignant gliomas.