[The development of immune enzyme test-systems for detecting potentially pandemic influenza viruses subtype A(H2), A(H5), A(H7) and A(H9).]

PURPOSE OF STUDY: To develop diagnostic test-systems constructed according principle of "sandwich" enzyme-linked immunosorbent assay to detect antigens of influenza viruses with pandemic potential: A(H2), A(H5), A(H7), A(H9).The panels of subtype-specific monoclonal antibodies interacting with molecule of hemagglutinin of potentially pandemic viruses of influenza are obtained and characterized. The viruses of every sub-type were supplied with selected pairs monoclonal antibodies/monoclonal antibodies' conjugate with peroxidase of horse-radish, interacting most effectively with virus-immunogen and lacking non-specific activity related to influenza viruses of geterologic types. Considering established threshold values, the sensitivity of test-systems varied depending on sub-type and strain of viruses within limits of 4 - 30 ng/ml at evaluation of viral purified concentrates and 0.3 - 8 hemagglutinin units at analysis of virus-containing allantoic fluid. The specificity of immune-enzyme test-system manifested in conditions of absence of non-specific interactions with both seasonal and potentially pandemic geterologic viruses. The technique can be applied for fast identification of viruses of influenza non-typeable in conditions of routine functioning of practical laboratories.

[Comparison of the influenza epidemics in Russia caused by the pandemic virus A(H1N1)pdm09 within the period from 2009 to 2013].

Comparative analysis of the three past epidemics with the participation of the pandemic influenza A(H1N1)pdm09 was conducted according to the results of the epidemiological trials of two WHO National influenza centers for the morbidity, hospitalization, and mortality of the influenza in 59 cities of Russia for the period from 2009 to 2013. The first wave of the pandemic of 2009 was the most severe. Compared with this wave, during the next epidemics of 2011 and 2013, the involvement of urban population in the epidemic was reduced, as well as the morbidity in the people 15-64 years old and schoolchildren 7-14 years old. The duration of the epidemic among the adult population, the mortality rate of the total population, and the mortality rates in all age groups were also decreased. Vice versa, the incidence in the children of preschool age and the elderly people and the duration of the epidemic among children (especially preschool children) were increased. The share of patients 65 years and older, children 0-2 years old, and patients with pathology of the cardiovascular systems among the deceased patients increased to 33.6%.

[THE COMPARATIVE ANALYSIS OF EFFECTIVENESS OF QUICK TESTS IN DIAGNOSTIC OF INFLUENZA AND RESPIRATORY SYNCYTIAL VIRAL INFECTION IN CHILDREN].

The analysis was implemented concerning diagnostic parameters of commercial quick tests (immune chromatographic tests BinaxNOW Influenza A&B and BinaxNow RSV Alere, Scarborough Inc., USA) under detection of antigens of influenza virus A and respiratory syncytial virus in clinical materials. The polymerase chain reaction in real-time and isolation ofviruses in cell cultures. The analysis of naso-pharyngeal smears from 116 children demonstrated that sensitivity and specifcity of detection of influenza virus A using device mariPOC in comparison with polymerase chain reaction made up to 93.8% and 99.0% correspondingly at total concordance of results of both techniques as 98.3%. At diagnosing of respiratory syncytial virus using device mariPOC parameters made up to 77.3%, 98.9% and 862% as compared with polymerase chain reaction. The sensitivity, specificity and total concordance of results of immune chromatographic tests BinaxNOW in comparison ofpolymerase chain reaction made up to 86.7%, 100% and 96.2% correspondingly at detection of influenza virus A and 80.9%, 97.4% and 91.6% correspondingly at detection of respiratory syncytial virus. In comparison with isolation technique in cell cultures sensitivity of system mariPOC and immune chromatographic tests proved to be in 1.3-1.4 times higher at detection of influenza virus A and in 1.7-2 times higher in case of isolation of respiratory syncytial virus. There is no statistically significant differences between diagnostic parameters received for mariPOC and immune chromatographic tests at diagnosing influenza virus A and respiratory syncytial viral infection.

[New monoclonal kits for the diagnosis of the adenoviral infection].

Diagnostic properties of new monoclonal antibodies (MAbs) to hexon adenovirus antigen (AB) monoclonal ELISA kit for early diagnosis of adenoviral infection were tested. Developed ELISA kit and FITC-conjugate of new monocional antibodies for immunofluorescent analysis were used for detection of different types of adenoviruses in clinical materials. The availability of their use in clinical and epidemiological practice was validated.

[Epitope analysis of the hemagglutinin molecule of the Victoria lineage influenza B viruses].

A panel of five monoclonal antibodies (MAbs) to the HA1 molecule of the influenza B virus of the Victorian lineage with high virus-neutralizing activity was developed. For identification of the virus neutralizing epitopes in HA1 escape mutants (EM) of the influenza BIShandong/07/97 and B/Malaysia/2506/04 virus were selected using virus- neutralizing antibodies (MAbs). Three EMs had single, two--double and one--triple amino acid substitutions (AAS) in HA1 (H122N, A202E, K203T, K2031, K203N or A317V). In addition, AAS N197S was detected in three EMs. A correlation of AAS identified with peculiarities of interaction of EMs with Mabs was discussed.

[Development of influenza surveillance in Russia in the system of the WHO national influenza center].

Analysis of development influenza activity season 2010-2011 is presented. Significant participation of influenza A(H1N1)pdm09 virus and influenza B of Victoria lineage virus in the epidemic morbidity structure with minor participation ofA(H3N2) virus was revealed. The influenza viruses isolated in Russia according to antigenic properties were similar to the strains included in the vaccine composition. Drift variants of influenza A(H1N1)pdm09 viruses isolated in Astrakhan and St.-Petersburg were recognized using WHO CC in London as representatives of three new genetic groups.

[The application of radial hemolysis technique in detection of antibodies to avian influenza virus A (H5N1) and pandemic virus A (H1N1) pdm09].

The article discusses the results of study that demonstrated the possibility of successful application of radial hemolysis reaction in analyzing the human inoculation immunity to new strains of influenza virus serotype A - A (H5N1) and A (H5N2). The radial hemolysis reaction provides accurate results on introduction of erythrocytes of horse or sheep into hemolytic system instead of erythrocytes of hens applied previously. The technique combines high sensitivity (in comparison with reactions of hemagglutination-inhibition and micro-neutralization, correlation coefficient 0.84-0.85) and total absence of inhibitors impact on the reaction results. During the investigation of immune response of patients who had pandemic virus A (H1N1) pdm09, radial hemolysis reaction demonstrated not only primary detection of antibodies to virus-agent (67%) during pandemic, but also elective heightened sensibility in the zone of low titer serums (1:20) in hemagglutination-inhibition reaction. These characteristics are very important in analysis of antibodies levels at early stages of disease. The radial hemolysis reaction continues to be a reliable instrument in evaluating qualitative and quantitative indicators of humoral immunity in ill patients and persons inoculated with new strains of human influenza virus.

[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.

[Determination of the diagnostic parameters of domestic immunoenzyme test systems versus foreign analogues in the detection of serum rubella virus antibodies].

Russian enzyme immunoassay (EIA) test systems (EATS) and foreign analogues (DSL and BCM-Diagnostics, USA) were compared in the detection of human serum rubella virus (RV) antibodies. Analysis of RV IgM levels ascertained a greater agreement of quality indices for the EATS "EKOlab-Krasnukha-IgM" than for the system "VectoRubella-IgM". As compared with the USA reference systems, the sensitivity, specificity, and total agreement of the data obtained by the EATS "EKOlab" were 100, 97.5, and 97.7%, respectively; those were 88, 84.4%, and 85.4% for "VectoRubella-IgM". In healthy individuals, strictly seropositive cases of IgM detection, revealed by the EATS "VectoRubella-IgM" and unconfirmed by the results obtained by the EATS "BCM-Diagnostics-IgM" (8%) are most likely to be regarded as false-positive due to the presence of serum rheumatoid factor (RF). The principle of indirect EIA used in the system "VectoRubella-IgM" makes it impossible to rule out the impact of RF on test results. The EATS "EKOlab-Krasnukha-IgM" and the systems made in the USA apply the principle of EIA with IgM involvement that, unlike indirect EIA, minimize to have nonspecific results. All three analyzed Russian EATS "EKOlab-Krasnukha-IgG", "Krasnukha-screening", and "VectoRubella-IgG" in the detection of RV IgG show rather high diagnostic parameters as compared with the systems made in the USA. The important additional advantage of the EATS "EKOlab-Krasnukha-IgG" over other Russian systems is that the kit has reference serum with the known content of RV IgG. This allows one to give results in absolute units and to standardize the calculations of some independent experiments. The performed study suggests that out of the Russian test systems, EATS "EKOlab-Krasnukha-IgM" and "EKOlab-Krasnukha-IgG" should be preferred.

[Development of competitive enzyme immunoassay for the detection of influenza A(H5) virus antibodies].

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.

FEATURES OF EPIDEMIC PROCESS OF INFLUENZA A(H1N1)PDM09 AND A(H3N2) IN RUSSIA FROM 2009 TO 2017.

The goal of this work is to compare the key parameters of influenza epidemics of different etiology. Four epidemics of influenza with predominance of influenza A(H1N1)pdm09 and 4 epidemics of influenza A(H3N2) were analyzed using the database of the Federal State Research Institute of Influenza on morbidity, hospitalization, deaths from influenza in 59 cities in the period from 2009 to 2017. The intensity of epidemics involving the influenza A(H1N1)pdm09 decreased from high to medium, while the intensity of epidemics of influenza A(H3N2) increased from low to medium. In the epidemic of influenza A(H1N1)pdm09 in the total population, the incidence of influenza and ARI decreased by a factor of 1.6, while the mortality among patients decreased by a factor of 1.7 in all age groups, except for those over 65 years, for whom the incidence and mortality increased by factors of 1.4 of 2.3, respectively. In the epidemic of A(H3N2), a trend for increasing morbidity and mortality was more pronounced among individuals older than 65 years. Pandemic influenza remains the leading cause of deaths. Among the dead in the epidemic of influenza A(H1N1)pdm09, the proportion of young individuals decreased (including a fourfold decrease of lethality in pregnant women), while the proportion of persons over 65 years increased 7.8 times; in the epidemic of influenza A(H3N2) only 2.5-fold increase was observed. In the epidemic of influenza A(H1N1)pdm09, the proportion of lethality increased among people with cardiovascular diseases and diseases of the internal organs; in the epidemic of influenza A(H3N2), the proportion of lethality increased among people with immunodeficiency, diseases of the internal organs and respiratory system.

[siRNA-mediated inhibition of respiratory syncytial virus replication].

The effect of siRNA against respiratory syncytial virus (RSV) was investigated in the cultured cells. MA104 cells were inoculated by RSV and transfected by different variants of siRNA against RSV phosphoprotein (P) mRNA or non-specific siRNA as a control. The inhibition of RSV was assessed by microscopically studying the cells, titrating the virus, and estimating viral RNA quantity in the culture supernatants by real-time polymerase chain reaction (PCR). The most potent variants of siRNA caused an up to 8-day delay of microscopically detectable syncytium generation to 8 days, an up to 280-fold decrease in viral titers, and an up to 40-fold reduction in viral RNA quantity in the supernatants, as compared to the controls (p < 0.001). RSV mRNA is a suitable target for siRNA-mediated RSV replication inhibition, promising an advance in the treatment of RSV infection.

The efficacy of cell therapy in the treatment of patients with trophic venous ulcers of the lower limbs.

This paper analyzes the results of cell therapy carried out during multimodality treatment of 23 patients with trophic ulcers of the lower limbs because of varicosity and postthrombotic disease. Twenty-five patients were entered into the control group provided the modern wound dressings Suprasorb. It has been demonstrated that cell therapy applying fibroblasts, strain 11 00/14, noticeably upgrades the treatment efficacy. Ulcer healing was attained in all the patients of the basic group. The times of epithelialization averaged 1.5 week for varicose and 3.2 weeks for postthrombotic ulcers. In the control group, the healing of varicose trophic ulcers was recorded in 86% and of postthrombotic ulcers in 78% of patients. The times of healing averaged 3.6 and 3.9 months respectively, Cell therapy as part of multimodality treatment of trophic venous ulcers excels the modern wound dressings as regards the efficacy.

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.

[Cell therapy in treatment of trophic ulcers of lower extremities].

The aim of the work was to study the effectiveness of using human embryo fibroblast culture in complex treatment of trophic ulcers of venous etiology in 23 patients with trophic ulcers of lower extremities. The cause of the appearance of ulcers was postthrombophlebitic disease in 16 patients and varicose disease in 7 patients. A control group consisted of 25 patients (postthrombophlebitic disease in 18 patients and varicose disease in 7 patients). The human embryo cell culture grown on the wound cover "Foliderm" was used at the stage of epithelization in the main group, while in the control group the modern alginate, collagen, hydrogel, polyurethane and hydrocolloid covers Suprosorb--Suprosorb A, Suprosorb C, Suprosorb G, Suprosorb F and Suprosorb H were used. Healing of the varicose trophic ulcers in the control group was achieved in 86% of patients, of post-thrombophlebitic--in 78% of patients. The average period of healing was 3.6 and 3.9 months respectively. Healing of trophic ulcers in the main group took place in 100% of patients. The average period of healing was 1.5 week for varicose and 3.2 weeks for postthrombophlebitic ulcers. The cell therapy was shown to be a highly effective method in treatment of venous trophic ulcers.

[Ultrastructural changes in the cells of human embryo lung fibroblasts in the reproduction of the coronavirus HCoV/SPb/01/03].

The reproduction of the new coronavirus HCoV/SPb/01/03 in the cultured human embryo lung fibroblasts (HELFBs) was electron microscopically studied. The virus was shown to replicate in the cultured HELFBs, by using for this a cell membrane system and causing profound changes in its morphology. After 24 hours of cell infection, there were mature and defective HCoVISPb/01/03 virions were detected in the vacuoles near the peripheral cisterns of the Golgi apparatus. Some of the vacuoles contained folded membranous structures along with virions.

Improved methods of influenza virus propagation. I. Enhancement of virus reproduction in cell cultures.

Comparative studies on the reproduction of a set of influenza A and B virus strains in different cell cultures were carried out. A cultivation system yielding influenza virus with high infectivity, based on the use of roller cultures of bovine embryo kidney cells, of virus strains adapted to this culture, and of a maintenance medium of improved composition, was developed.

Improved methods of influenza virus propagation. II. Characteristics of cell culture and allantoic virus preparations.

Regular reproduction of influenza A virus with infectious titres of 7.4--8.5 log EID50/0.2 ml was observed in roller bovine embryo kidney cell cultures. The haemagglutinating activity of the cell culture preparations was 8-33-fold lower than that of the allantoic preparations, the infectivity being similar. Cell culture preparations of influenza A/Victoria/35/72 and A /Leningrad/538/74 viruses were markedly immunogenic in laboratory animals and antigenically active in complement fixation (CF) test. The CF activity of liquid and lyophilized preparations remained unchanged for 12--13 months (the period of observation). The stability of infectivity of lyophilized cell culture virus preparations was similar to that of allantoic virus preparations.

Comparative studies on influenza B virus. II. The effect of host cells on biological properties and polypeptide composition.

The reproduction patterns of various influenza B virus strains isolated in 1970-1976 using roller cultures of MDCK cells and chick embryos (CE) were compared. The cultural and allantoic virus populations did not differ in their sensitivity to non-specific inhibitors from mammalian sera and in their reactivity with specific haemagglutinins (HA). The content of infectious virus and HA in the harvested allantoic fluids as compared to medium fluids were 94- and 8-fold higher, respectively, even though the fluids did not differ in the titres of complement-fixing (CF) antigen. The mol. weight (MW) of HA1 polypeptide of the B/Len/75 virus prepared in culture was higher than that Of the allantoic virus (57.5 K and 55.5 K, respectively). Under reducing conditions, the HA of the virus from culture was represented mostly by the uncleaved HA0 polypeptide, while that of the allantoic virus by the HA1 and HA2 subunits. Under non-reducing conditions, the virus from medium fluid was found to contain glycopeptide D with MW of 90 K.

Clinico-immunologic and allergologic studies with the inactivated influenza virus vaccine purified and concentrated by gradient centrifugation.

Vaccination activity and safety of inactivated influenza centrifugal divaccine have been studied in groups of subjects aged 18 to 22, 15 to 16, and 9 to 14 years. The vaccine tested contained either a standard dose (6-8 micrograms) or double a dose (12-16 micrograms) of the haemagglutinin (HA) of influenza viruses A(H1N1) and A(H3N2). The double antigenic load of the vaccine did not enhance its reactogenicity for adults or adolescents aged 15 to 16 years. It enhanced, however, the production of antihaemagglutinine antibodies to the vaccine strains. The range of the antibodies formed and their persistence were independent on the virus dose. The increased dose (12-16 micrograms) of HA decreased the humoral immune response in school children aged from 11 to 14 years. Neither standard nor double dose of the vaccine caused any changes in the biochemical or haematological parameters of blood and urine. No allergic response was registered by the indirect mast cell degranulation (IMCD) test in rats.