Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012.

In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

[Study of the effect of antiviral drugs on the reproduction of the respiratory syncytial virus by enzyme immunoassay]. / Ispol'zovanie immunofermentnogo analiza dlia izucheniia deistviia protivovirusnykh preparatov na reproduktsiiu respiratorno-sintsitial'nogo virusa.

A test system based on EIA was developed for evaluating the efficiency of drugs active towards the respiratory syncytial virus (RSV) in cell culture. Virasole and its structural analog ribamedii active towards RSV infection and arbidol whose activity in RSV infection is unknown were tested. Like virasole and ribamedil, arbidol inhibited the expression of RSV antigens, the inhibitory effect increasing with the drug concentration and decreased with increase of the multiplicity of virus infection. MIC50 for arbidol, virasole, and ribamedil were 10, 5, and 6 micrograms/ml, respectively. These data prompt clinical trials of arbidol in RSV infection.

Alteration in the antigenic structure of M1 protein of influenza A virus mutant resistant to a new antiviral compound mopyridone.

Using 14 monoclonal antibodies (MoAbs) in solid-phase ELISA it was found that influenza virus A/Hong Kong/1/68 (H3N2) mutants resistant to the antiviral compound mopyridone as compared to the mopyridone-sensitive mutant manifested significant changes in the antigenic structure (sites 1A, 2 and 3) of M1 protein. No differences in M1 were found between rimantadine-resistant and rimantadine-sensitive mutants of influenza virus A(H3N2).

Analysis of internal proteins of influenza A (H2N2) viruses isolated from birds in East Germany in 1983.

Proteins and RNAs of influenza A (H2N2) viruses isolated from birds in 1983 in East Germany were compared antigenically with those of H2N2 human strains. The electrophoretic mobility of the viral proteins and of the S1-treated double-stranded RNAs from two human and six avian strains, as well as the results of EIA-tests using monoclonal antibodies to their matrix protein and nucleoproteins indicate an antigenic relationship between the avian isolates and human strains of H2N2 subtype. One of the avian strains had a reduced amount of matrix protein.

[The detection of antibodies to HIV protein p24 in human blood sera by the method of lanthanide immunofluorescent analysis]. / Vyiavlenie antitel k belku p24 VICh v syvorotkakh krovi cheloveka metodom lantanidnogo immunofliuorestsentnogo analiza.

A competitive time-resolved fluoroimmunoassay (TR-FIA) system for the detection of antibodies to protein p24 of HIV was developed on the basis of monoclonal antibodies. The advantages of this test system over analogous enzyme immunoassay system and commercial test system "Antigen" (USSR) were demonstrated. The newly developed test system of TR-FIA was used for examination of sera from HIV-infected persons.

[The strain-specific diagnosis of influenza by using lanthanide immunofluorescence analysis based on monoclonal antibodies to the hemagglutinin of the influenza A virus]. / Shtammospetsificheskaia diagnostika grippa s ispol'zovaniem lantanidnogo immunofliuorestsentnogo analiza na osnove monoklonal'nykh antitel k gemaggliutininu virusa grippa A.

Nine monoclonal antibodies (MCA) to hemagglutinin of influenza A/Taiwan/1/86 (H1N1) virus and 5 MCA to influenza A/Mississippi/1/85 (H3N2) virus were generated and characterized. The MCA were used for the development of diagnostic test systems on the basis of time-resolved fluoroimmunoassay. The same MCA were used as primary and detecting antibodies in the test system specific for HA of the H1 serosubtype, whereas in the test system specific for influenza A serosubtype H3 virus MCA of different epitope appurtenance were used as primary and secondary antibodies. The sensitivity of the test system for HA of serosubtype H1 was found to be 10 ng/ml and that for serosubtype H3 5 nh/ml. The developed test systems were tried on the clinical material collected during the epidemic periods of 1983-1989.

[The use of sectional polystyrene plates in the set-up of lanthanide immunofluorescence analysis]. / Ispol'zovanie razbornykh polistirolovykh planshetov pri postanovke lantanidnogo immunofliuorstsentnogo analiza.

The authors have examined the possibility of using sectional polystyrene plates, made in this country, in time-resolved fluoroimmunoassay (tr-FIA) with Venezuelan equine encephalomyelitis and tick-borne encephalitis arboviruses, and with influenza A virus. The plates presensitized with specific antibodies were found fit for the detection of the antigens of the above viruses. These plates are not recommended for the detection of influenza A virus-specific proteins adsorbed directly onto the microplate surface.

[Standardization of conditions for detecting the internal proteins of the influenza virus in studying their antigenic properties in solid-phase immunoenzyme analysis]. / Standartizatsiia uslovii vyiavleniia vnutrennikh belkov virusa grippa pri issledovanii ikh antigennykh svoistv v tverdofaznom immunofermentnom analize.

The influence of the conditions of adsorption and virion destruction by freezing-thawing and detergents on the detection of M1 and NP proteins of different influenza virus strains by solid-phase enzyme immunoassay with direct virion adsorption on polystyrene was studied. It was found that for the detection of M1 protein the optimal conditions included virion disruption with detergent and adsorption to polystyrene at 4 degrees C, and for NP protein disruption by freezing-thawing at adsorption to polystyrene at 37 degrees C. In the study of the antigenic properties of protein M1 of different influenza virus strains using monoclonal antibodies it was shown to be necessary, first, to achieve maximum detection of proteins and, second, to standardize the amount of the adsorbed antigen with polyclonal antibodies.

[Stat-diagnosis of influenza using lanthanide immunofluorescence analysis]. / Ekspress-diagnostika grippa s pomoshch'iu lantanidnogo immunofliuorestsentnogo analiza.

A test system for influenza diagnosis has been developed on the basis of monoclonal antibodies to influenza A and B virus proteins using the principles of lanthanide immunofluorescence analysis. The diagnostic test system was shown to be highly specific in detecting influenza A and B virions in model systems. For the one-step variant of a double sandwich used in the study, the total time of diagnostic experiment using clinical materials was shown to be reduced to 15-20 min.

[Indication of the influenza virus in clinical samples using immunoenzyme and lanthanide immunofluorescence analyses]. / Indikatsiia virusa grippa v klinicheskikh obraztsakh s pomoshch'iu immunofermentnogo i lantanidnogo immunofliurestsentnogo analizov.

A comparative study of the potentials of enzyme-immunoassay (EIA) and lanthanide immunofluorescent assay (LIFA) for influenza virus detection in nasopharyngeal aspirates and nasopharyngeal washings was carried out. Monospecific and polyclonal antisera to hemagglutinin polyclonal antisera to matrix protein as well as polyclonal and monoclonal antisera to nucleoprotein were used. The nasopharyngeal aspirates were shown to contain virus-specific antigens in the amounts sufficient for influenza virus detection with polyclonal and monoclonal antibodies. There was a good correlation between chromophore results by EIA and fluorescence level in LIFA. The sensitivity of the test systems used (EIA and LIFA) was shown to be insufficient for the detection of virus-specific material in the nasopharyngeal washings. Besides, there was no correlation between the results obtained by different test systems. It was concluded that nasopharyngeal aspirates should be collected for rapid influenza diagnosis by EIA and LIFA.

[Heterogeneous distribution of influenza A matrix protein in polyacrylamide gel, detected using an immunoblotting method with monoclonal antibodies]. / Geterogennoe raspredelenie v poliakrilamidnom gele matriksnogo belka virusa grippa A, vyiavliaemoe metodom immunoblota pri ispolzovanii monoklonal'nykh antitel.

The immune blotting method using monoclonal antibodies to Ml protein showed protein Ml to migrate in several bands in polyacrylamide gel electrophoresis (PGE) of influenza A virus polypeptides. The heterogeneous distribution of Ml protein in PGE is due to the formation of aggregates: dimers, trimers, and polymers of a higher order. The capacity of Ml protein for aggregation is typical not of all influenza virus strains and most likely is not associated with gel overloading. Since dimers and trimers of Ml protein comigrate in the gel with virus-specific proteins such as NP and proteins of a polymerase complex, this circumstance should be take into consideration in using PGE for isolation of pure influenza virus proteins.

Antigenic reactivity of matrix protein and nucleoprotein of influenza virus as detected by EIA after dissociation with different detergents.

Solid phase enzyme-immunoassay (EIA) was employed to assess the antigenic reactivity of matrix protein (M) and nucleoprotein (NP) of influenza A virus adsorbed to polystyrene in the presence of different detergents such as beta-octaglucoside (OG), Triton X-100, Tween-20, sodium dodecylsulphate (SDS), sodium deoxycholate (Doch-Na), Nonidet P-40 (NP-40), and sarcosyl at concentrations ranging from 0 to 2%. The antigenic reactivity of NP was the highest in the absence of detergents. For M protein, Doch-Na, SDS, NP-40 and sarcosyl of 0.05-0.1% enhanced the chromatophoric response in EIA 1.5-2 times. In contrast, the antigenic reactivity of M protein remained unchanged after OG or Triton X-100 treatments, and it decreased in the presence of Tween-20.

[Isolation and characteristics of monoclonal antibodies to influenza virus types A and B]. / Poluchenie i kharakteristika monoklonal'nykh antitel k virusam grippa tipov A i B.

Monoclonal antibodies (MCA) to influenza type A (10F) and B (5H and 6H) viruses have been prepared. By immunoblotting method, MCA 10F were found to be specific for NP-protein of influenza A virus, and MCA 5H and 6H to be specific for hemagglutinin of influenza B virus. It was established that the 10F clone interacted with all the investigated influenza A virus strains with different antigenic formulae (H1N1, H2N2, H3N2) and could be used for typing of this virus type. Clones 5H and 6H react specifically with hemagglutinins of influenza B viruses isolated in 1940, 1979, and 1983 which makes them useful for diagnosis of influenza B.

Simultaneous determination of the level of antibodies to influenza virus surface and internal proteins by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.

[Immunoenzyme test system based on antibody F(ab)2-fragments for detecting the NP protein of the influenza virus]. / Immunofermentnaia test-sistema na osnove F(ab)2-fragmentov antitel dlia vyiavleniia belka NP virusa grippa.

A test system has been proposed for the detection of influenza A virus NP protein by solid-phase enzyme immunoassay in a modified system of "double" antibodies: the primary antibodies were F(ab)2-fragments of immunoglobulin class G isolated from rabbit antiserum specific for NP protein which was also used as the source of secondary antibodies. The antigen-bound antibodies were detected by protein A (from St. aureus) conjugate with horseradish peroxidase. The high specificity and sensitivity of the test system (0.3 ng/ml NP protein) and a minimal level of nonspecific reactions were demonstrated. The advantages of this method are discussed.

The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties.

ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.