[ANALYSIS OF POPULATION IMMUNITYAGAINST INFLUENZA PRIOR TO 2014 AND 2015 EPIDEMIC SEASONS].

AIM: Control for the population herd immunity against seasonal influenza viruses as well as for emergence of antibodies against influenza with pandemic potential in human blood sera. MATERIALS AND METHODS: HAI reaction against vaccine and epidemic influenza viruses as well as HPAI viruses A/rook/Chany/32/2015 (H5N1) (clade 2.3.2. lc.) andA/Anhui/01/2013 (H7N9). RESULTS: Among all the sera samples collected in the autumn of 2014 and 2015, none had reacted in HAI against A(H5N 1) and A(H7N9) antigens even at 1:10 dilution. Among samples collected in autumn 2014, 41% were positive to A/California/07/09(H1Nlpdm9) virus, 36% - A/Texas/50/2012 (H3N2), 40% - B/Brisbane/60/2008 (Vict.lin.) and 47% reacted in HAI against the B/Massachusetts/2/2012 (Yam.lin.) strain. 22% of all the samples had a titer of at least 40 against all the antigens and only 10% in HAI had a titer of 40 or more against all the vaccine strains. Among the samples collected in autumn 2015, the number of seropositive against A/California/07/09(HlNlpdmO9) varied from 31% in the Urals FD to 46% in the Southern FD. The amount of seropositive against A/Switzerland/9715293/13 (H3N2) strain was at the level of 4 - 13% in all the FDs except Urals, where this parameter was slightly above 30%. The amount of seropositive against vaccine influenza B viruses varied from 23 to 76%. Only 2% of sera had titers in HAI of 40 or above against all the vaccine strains, 29% of all the samples were seronegative. CONCLUSION: Population immunity in Russia against influenza A(H3N2) is at a very low level, thus socially significant consequences of influenza epidemics in many aspects will depend on the vaccination campaign of autumn 2016.

Highly pathogenic influenza H5N1 virus of clade 2.3.2.1c in Western Siberia.

In the spring of 2015, avian influenza virus surveillance in Western Siberia resulted in isolation of several influenza H5N1 virus strains. The strains were isolated from several wild bird species. Investigation of biological features of those strains demonstrated their high pathogenicity for mammals. Phylogenetic analysis of the HA gene showed that the strains belong to clade 2.3.2.1c.

[Analysis of population immunity of residents of Russian Federation regions against seasonal and highly pathogenic strains of influenza virus].

AIM: Determine the level of antibodies against socially significant types/serotypes of influenza virus in sera of individuals residing in various regions of Russia. MATERIALS AND METHODS: 1525 samples of blood sera collected in August-December 2013 in 8 regions of Russian Federation were studied in hemagglutination inhibition reaction (HAI) with antigens obtained from A/California/07/09, (H1N1)pdm09, A/Victoria/361/2011(H3N2), B/Brisbane/60/2008 (Victoria line), B/Massachusetts/2/2012 (Yamagata line), A/Commongull/Chany/2006 (H5N1), A/Anhui/01/2013 (H7N9) influenza virus strains. RESULTS: None of the blood sera samples had significant HAI titers against A/H5 and A/H7 antigens. Of all the 1525 samples, 788 (52%) were positive with A(H1N1)pdm09 antigen; 734 (48%) reacted with A(H3N2) antigen; 1010 (66%) samples were positive with B/Victoria antigen and 602 (39%) samples were positive with B/Yamagata antigen. CONCLUSION: Healthcare institutions should pay attention to the correction of population immunity profile in regions for the reduction of social-economic losses from seasonal influenza epidemics.

[Neutralizing single-chain antibodies against the tick-borne encephalitis virus].

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA 13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the sc13D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified sc13D6 was (3.0 +/- 0.2) x 10(7) M(-1) for the equilibrium state and (2.8 +/- 0.3) x 10(7) M(-1) in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC50 value for sc13D6 was 16.7 microg/ml.

[Comparison of in vitro and in vivo methods of evaluation of the efficacy of influenza virus hemagglutinin inhibitors].

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.

A prototype prophylactic anti-influenza preparation in aerosol form on the basis of Abies sibirica polyprenols.

This research investigates a promising antiviral compound based on polyprenols from Siberian silver fir (Abies sibirica). The physico-chemical characteristics of a preparation developed in aerosol form and an estimation of its protective efficacy against aerosol challenge of laboratory animals are presented. It is shown that (1) by using a simple ultrasonic disperser one can obtain aerosol of three formulations studied with about 70% of its mass accumulated in the size range below 1.8 microm; (2) 40-100% of aerosol particles contain preparation for different formulations; (3) after delivering under specified schedules, the preparations as developed can protect up to 100% of mice against 5 LD(50) of influenza A/Aichi/2/68 (H3N2) virus aerosol infection. Animals inhaled twice the preparation doses (which were 100 times lower than injection ones of the same efficacy) and did not exceed 10 microg/mouse. It was shown that the mode of action of this immunomodulating preparation was nonspecific stimulation of immune cells' various activities.

[An intensification of antiviral and interferon effects of ridostin (an interferon inducer) by cationic liposomes in vitro and in intranasal administration]. / Usilenie kationnymi liposomami protivovirusnogo i interferonogennogo deistviia induktora interferona ridostina in vitro i pri intranazal'nom primenenii.

Combined application of ridostine with catonic liposomes was shown to essentially enhance the interferon-inducing and antiviral activity of the former in experiments with cell cultures L-929, which is apparently related with an improved efficiency of intracellular delivery of dsRNA. A comparative study demonstrated that ridostine, when combined with liposomes, is needed by 10(3)-10(4) times less as when it is used alone. A pretreatment of the cellular monolayer by cationic liposomes contributes also to enhancing the activity of ridostine, which can be explained by an enhanced permeability of cells for dsRNA holding on-for as long as 30 minutes after the removal of liposomes from the liquid culture. A separate successive administration of, first, liposomes and, then, of ridostine in BALB/c mice (20 mg/kg) leads to a more intensified induction of interferon in the upper respiratory tract tissues as compared with the administration of ridostine alone.

[Experimental molecular design of recombinant vaccines]. / Eksperimental'noe modelirovanie molekuliarnykh konstruktsii kombinirovannykh vaktsin.

A method was elaborated to construct combined artificial immunogens mimicking virus particles. The gist was exposing protein antigenic determinants of one virus on the particle surface and delivering plasmids with genes for antigenic proteins of another virus to specialized immune cells. Such immunogens were constructed and shown to induce biosynthesis of specific antibodies against HIV-1 and the tick-borne encephalitis virus. The level and duration of the humoral and cell responses were assayed.

[Mechanisms of mice resistance to influenza virus A/AICHI/2/68 after preventive injection with polyprenols ]. / Mekhanizmy rezistentnosti myshei k virusu grippa A/AIChI/2/68 pri profilakticheskom vvedenii poliprenolov.

Preventive effect in influenza can be attained by intramuscular injections of fir (Abies) polyprenols. One of 5 tested polyprenol preparations (No. 1), injected 2 days before aerogenic infection with influenza virus, reliably protected mice from disease. Mice pretreated with polyprenol preparations or Hanks' solution did not differ by accumulation of interferon in the lungs One day after aerogenic infection. Three days after injection of polyprenol preparation No. 1 the weights of the spleen and thymus significantly decreased. One day after injection cell count in the bronchoalveolar tract of mice was almost 2-fold higher than in the control at the expense of lymphocytes and macrophages. After 3 days the relative and absolute counts of macrophages decreased and those of lymphocytes decreased significantly. Three days after injection macrophages were 2-fold more active in absorption of zymosan granules. Preparation No. 1 affected the production of superoxide anion radicals, whose production by all macrophages in the bronchoalveolar tract of mice was significantly higher on day 1 postinjection than on day 3 and higher than on days 1 and 3 after injection of preparation No. 2.

Effect of intramuscularly injected polyprenols on influenza virus infection in mice.

This study demonstrates the possibility of achieving a prophylactic effect by intramuscular injection of Abies sibirica polyprenols for the control of influenza virus infection in mice. One of the five polyprenol preparations tested, preparation N1, which had the lowest hydrophilic-lipophilic balance (8.6), produced a significant protective effect when injected in a dose of 2000 microg/mouse 2 days before aerosol infection of mice with influenza virus. A moderate protective effect was also observed using a second preparation, designated N2. One day after aerosol infection, animals pre-treated with 2000 microg doses of the polyprenol preparations or Hanks' solution showed no difference in the level of interferon accumulation in the lungs. Three days after injection of preparation N2 and N1, a significant decrease in spleen and thymus weights was, observed in the mice. One day after injection of these preparations, the number of lymphocytes in the bronchoalveolar tract of the mice exceeded almost twice that seen in mice treated with placebo. After 3 days, relative and absolute numbers of macrophages decreased, whereas those of lymphocytes increased significantly. Three days after the administration of preparations N1 and N2, macrophages became approximately twice as active in absorbing zymozan granules. Preparation N1 affected the system of superoxide radical anion production to a greater extent than preparation N2. The production of radical anions by the macrophages of the bronchoalveolar tract in the mice, 1 day after intramuscular injection of preparation N1, was significantly higher than that seen on day 3 and that induced by preparation N2 1 and 3 days after injection. These data indicate that emulsions of polyprenols that have relatively low hydrophilic-lipophilic balance, inhibit influenza virus infection in mice through a modulation of the host immune response.

[Immunogenicity of a recombinant strain of vaccinia virus, expressing a Venezuelan equine encephalomyelitis virus structural protein gene in peroral immunization]. / Immunogennost' rekombinantnogo shtamma virusa ospovaktsiny, ékspressiruiushchego geny strukturnykh belkov virusa venesuél'skogo éntsefalomielita loshadei pri peroral'noi immunizatsii.

Immunogenicity of recombinant vaccinia virus strain (VR26) expressing Venezuelan equine encephalomyelitis (VEE) virus structural protein genes was studied by oral immunization. Sera of animals immunized with VR26 contained antibodies specific to VEE virus, among which antibodies with virus-neutralizing activity were present. Evaluation of the protective efficiency of oral immunization with VR26 demonstrated a high level of animal protection from lethal doses of VEE virus. Rabbits immunized orally were highly resistant (protection index 142.9) to intranasal infection, which is of priority importance for antiVEE vaccine. Comparative analysis of the results of scarification and oral immunization with VR26 indicates that the type of immune response depends on the method of immunization. These results demonstrate good prospects of oral vaccination with recombinant VR26 strain for immunoprophylaxis of VEE.

[Comparative study of aerosol and oral methods of infection of Lympantria dispar L. caterpillars with nuclear polyhedrosis virus]. / Sravnitel'noe izuchenie aérozol'nogo i oral'nogo metodov infitsirovaniia gusenits neparnogo shelkopriada (Lymantria dispar L.) virusom iadernogo poliédroza.

The aerosol and oral routes of infection of Gypsy moth larvae with nuclear polyhedrosis virus are compared. The virus in aerosol retains its biological activity. The virus output/expenditure ratio is virtually the same in the studied routes of infection. Aerosol method of inoculation saves 30% components of media and is 6-8 times less labor consuming. This method permits complete automation of infection of larvae, thus essentially improving the efficacy of baculovirus production.

[Sensitivity of white mice to influenza virus (A/Aichi/2/68) in aerogenic infection]. / Chuvstvitel;nost' belykh myshei k virusu grippa (A/Aichi/2/68) pri aérogennom zarazhenii.

Polydispersed aerosols from allantoic fluid of chick embryos induced with influenza virus with different median weight aerodynamic diameters of corpuscles (0.5, 0.8, 1.1, 2.2, and 6.0 mu are effectively deposited in respiratory organs of mice weighing 18-19 g. The sensitivity of mice of different weight to aerogenic infection with influenza virus (strain A/Aichi/2/68) was virtually the same. The efficacies of aerogenic 50% infective and lethal doses (1.8-2.5 lg) for mice of the same weight were different. The sensitivity of mice to aerogenic infection and of developing chicken embryos to the virus (ID50 = EID50) is the same. Mice weighing 10-19 g can be infected via airways with adapted influenza virus in studies of therapeutic and prophylactic effects of drugs.

[Dynamics of interferon induction in albino mice by interferon inducer ridostin administered by various routes]. / Izuchenie dinamiki interferonoobrazovaniia v organizme belykh myshei pri razlichnykh putiakh vvedeniia induktora interferona ridostina.

The time dependence of interferon production in blood, tissues of the respiratory tract, brain and olfactory tract of mice BALB/c was investigated after administration of the interferon inductor ridostin by various routes. Intraperitoneal injection of ridostin in a dose of 5 mg/kg induced intensive accumulation of interferon in the blood serum with the peak in 8 hours (2560 U/0.2 ml) while no interferon was detected in the tissues of the respiratory tract and brain of the animals. Intracerebral injection of ridostin in the same dose induced accumulation of interferon in both the tissues of the brain (maximum 160 U/0.2 ml in 24 hours) and the blood serum (maximum 1280 U/0.2 ml in 8 hours). After respiratory administration of ridostin interferon was detected only in the site of the administration in the tissues of the upper respiratory tract and lungs of the mice.

[Studying the possibility of respiratory immunization against tick-borne encephalitis]. / Izuchenie vozmozhnosti respiratornoi immunizatsii protiv kleshchevogo éntsefalita.

There are known 3 likely mechanisms of virus conveyance into the central nervous system (CNS). These include hematogenic penetration, spread along the peripheral nerves, and the olfactory pathway which begins from the infected olfactory neuroepithelial cells. The possibility of viral spread into CNS via the olfactory pathway was shown for the representatives of togaviruses, herpesviruses, coronaviruses, rhabdoviruses, and for some others. This study suggests that the olfactory pathway of viral conveyance into CNS may be blocked by specific mucosal antibodies in the nasal mucosa. The recombinant TK- variant of WR vaccinia strain with inserted genes coding structural and nonstructural proteins of TBE virus is accumulated in the branches of the respiratory tract only while the parenteral vaccinia strain is detected in the brain regions, spleen, respiratory tract, and in blood. The protective activity of recombinant strain and inactivated TBE vaccine after mice immunization by escarification or intranasally, or subcutaneously was comparatively studied. The findings indicate that intranasal immunization by recombinant strain is the most protective against intraperitoneal challenge by TBE virus. The mucosal and humoral immune response that was induced by intranasal immunization seems to provide the highest levels of protection, which was experimentally observed.

[Some aspects of interferon formation in white mice]. / Nekotorye aspekty interferonoobrazovaniia v organizme belykh myshei.

White mice weighing 14-16 g were intranasally infected with LD50 of influenza virus (A/Aichi/2/68 strain). High levels both of virus and interferon were detected in the lung. Sufficient virus accumulation in the nasal cavity occurred with low interferon induction. At the same time high blood interferon levels corresponded to sporadic low viremia. Intraperitoneal injection of the interferon inducer ridostin (a pharmacological formulation of dsRNA) to BALB/c mice (18-20 g) in a dose of 5 mg/kg induced intensive blood accumulation of interferon with its peak at 8 hours postadministration (2560 U/0.2 ml), but interferon was not detected in the respiratory tract and brain of these mice. Intranasal (15 mg/kg) and aerogenic (0.4-0.6 mg/kg) administration of ridostin induced interferon mainly in the upper respiratory tract and lung. The regularities found are in agreement with the data on interferon induction by other dsRNA preparations, which makes it necessary to design dosage forms of interferon inducers for respiratory application in influenza.

[Expression of the gene for vaccinia virus 36K nonstructural protein in Escherichia coli and study of the protective properties of expression products]. / Ekspressiia gena nestrukturnogo belka 36K virusa ospovaktsiny v Escherichia coli i izuchenie protektivnykh svoistv produktov ékspressii.

The nonstructural 36K protein of vaccinia virus (VV) mapped in HindIII-P and HindIII-J fragments of VV strain L-IVP has been expressed in E. coli as a fusion protein. The products of 36K gene preserved the antigen homology with the native protein 36K and the capacity to bind specific immunoglobulins of rabbit antiVV serum. The protective properties of 36K gene products and their joint effect with the immunodominant protein 35K were investigated. The non-structural 36K gene products showed no protective activity, but increased the production of specific antibodies in mice immunized with a mixture of both protein preparations, this increase being compatible with that observed after immunization with the inactivated virus preparations.

[Some pathogenetic characteristics of influenza infection in white mice]. / Nekotorye patogeneticheskie kharakteristiki grippoznoi infektsii u belykh myshei.

Influenza virus strain A/Aichi/2/68 replicated only in the lungs, nasal cavity, and trachea after intranasal challenge of white mice weighing 14-16 g. Rapid accumulation of the virus was associated with a somewhat delayed accumulation of endogenous interferon in the lungs, the concentration of interferon gradually decreasing by the end of the disease. An appreciable accumulation of the virus in the nasal cavity was coupled with weak interferon induction in this organ. On the other hand, a high level of interferon in the blood was concomitant with the development of slight sporadic viraemia.

[Study of the treatment-prophylactic effect of immunomodulators in experimental infections, caused by Marburg, Ebola, and Venezuelan equine encephalitis viruses]. / Izuchenie lechebno-profilakticheskogo deistviia immunomoduliatorov pri éksperimental'nykh infektsiiakh, vyzvannykh virusami Marburg, Ebola i Venesuél'skogo éntsefalomielita loshadei]

Therapeutic and prophylactic effects of immunomodifiers ridostin, reaferon, and polyribonate used alone and in various combinations were assessed in experiments on guinea pigs infected with Venezuelan equine encephalomyelitis (VEE) (strain Trinidad), Marburg (strain Popp), and Ebola (M/C-8 variant of Zaire strain) viruses at doses 5 to 20 respiratory LD50 through the respiratory airways. Urgent prophylactic simultaneous intramuscular and intranasal administration of ridostin protected the animals infected with Marburg virus (p = 0.1) and prolonged their life span by 2.4 days (p = 0.15). In Ebola infection a combination of ridostin and reaferon appreciably prolonged the mean life span: by 2.9 days (p = 0.04). In VEE ridostin alone or in combination with reaferone appreciably increased the share of survivors; ridostin with reaferon and polyribonate notably prolonged the mean life span of infected animals. None of these drugs or combinations produced an appreciable therapeutic effect in any of the studied infections.