[Improvement of erythropoietin bioassay.]

The relevance of bioassay standardization results from the lack of consistent national regulatory requirements for evaluation of recombinant human erythropoietin quality and the need to harmonize these requirements with international ones. Precision studies were carried out in 6 experiments on Balb/C mice. The factors that can influence the accuracy of the method were altered during the experiments. Each experiment included three levels: 20, 40 and 80 IU/ml, and 8 replicates for the reference and test samples. The trueness was estimated by bias relative to the reference value at 5 levels: 10, 20, 40, 80 and 160 IU/ml, and 4 replicates for the reference and test samples at each level. The test samples were prepared by a series of independent dilutions of the reference standard. Reticulocyte count was performed using a flow cytometer. 5 µmol acridine orange solution was used as a dye. Experimental study of accuracy and optimization of erythropoietin bioassay procedure helped to obtain two validation characteristics (trueness and precision). It was shown that logarithms of erythropoiesis registered values could reasonably be used in statistical calculations of erythropoietin specific activity and evaluation of the method's validation parameters. The theoretically and experimentally justified test procedure includes three levels of doses: 20, 40 and 80 IU/ml, and 8 animals for each level, which is consistent with the international requirements for accuracy. According to the results of experimental studies, the trueness is characterized by a bias of no more than 9 % and does not exceed the range of the calculated activity (80-125 %). Statistical processing of the test results by the parallel-line method makes it possible to check the assumption of equivalence of the test and reference samples and to calculate the test sample activity. The confidence limit of the calculated activity for intra-laboratory precision of 5.6 % is equal to 76-131 % which complies with the proposed range (64-156 %, P=0.95).

[Bolivian hemorrhagic fever].

Analysis of data of the available literature on epidemiology of Bolivian hemorrhagic fever, manifestations of human disease, biological properties of the causative agent and development carried out abroad of means and methods of diagnostics, prophylaxis and therapy of this infection that presents a potential threat for the population and economy of the Russian Federation in case of introduction of the causative agent is presented.

[Experimental assessment of the possible significance of argasid ticks in preserving the natural foci of West Nile virus infection].

Experiments indicated that the argasid ticks Alveonasus lahorensis were highly susceptible to West Nile virus when inoculated in the hemocoel. The virus concentration in the ticks reached high values when very low doses (0.01 PFU) of the pathogen were administered. The ticks kept at 3.0 +/- 1.0 degrees C retained the pathogen up to 116 days (a follow-up period). The infection rate of the ticks depending on the virus dose administered was in the range from 12 to 80%. The contaminated specimens successfully transmitted the virus to rabbits by blood suckling. The findings suggest that the argasid ticks may be involved in the preservation of West Nile virus in the interepidemic period and be responsible for the outbreak of this infection in summer and autumn months.

[Rift valley fever].

In the last quarter of century virus of Rift valley fever (RVF) sharply extended its distribution by moving from Africa to Asia and evolving from low- to high pathogenic for humans causing severe hemorrhagic disease, practically equaling in this respect with some members ofa group of extremely dangerous pathogens. Morbidity and epidemics of RVF are analyzed. Evolution of epidemic development of the infection is examined. Necessity of development of means and methods for diagnostics, prophylaxis and therapy of RVF is underlined.

[Argentine hemorrhagic fever].

Features of the Argentine hemorrhagic fever are described in the review. Epidemiology, etiology, clinical presentation and pathogenesis of the disease are examined. Special consideration is given to the features of the pathological agent of Argentine hemorrhagic fever--the Junin virus. Features of the disease diagnostics are indicated--virological and serological studies, immunochemical and molecular-biological methods of identification of the pathological agent and antibodies against it. Approaches to etiotropic therapy of this disease and vaccination are examined. Based on the foreign experience perspective guidance for the creation of the system of protection of the population of the Russian Federation against Argentine hemorrhagic fever are presented.

[Hantapulmonary syndrome].

Features of hantavirus pulmonary syndrome are considered in the review - zoonosis natural focal polyetiological viral infection, that is characterized by lung injury. Etiology of the disease, main characteristics of the agents, epidemiology, contagiousness, pathogenesis, clinical presentation of this pathology are examined. Laboratory diagnostics, therapy and prophylaxis ofhantavirus pulmonary syndrome are described.

[Monkeypox: features of spread after cancellation of mandatory pox immunization].

Features of spread of monkeypox after eradication of smallpox and cancellation of mandatory pox immunization are examined. In the condition of cancellation of mandatory pox immunization, a decrease of population immunity to pox in the population, a lack of vigilance in most of the medical specialists to diseases caused by other pathogenic for human orthopoxviruses was noted. This situation complicates the prognosis of the development of possible outbreaks of infection of humans by monkeypox. In such situation only constant vigilance with respect to this zooanthroponosis, use of express diagnostics methods, as well as means of effective protection will allow to stop outbreaks of monkeypox at the early stages of the development.

[Evaluation of safety of prophylactic use of immunoglobulins against viral hemorrhagic fevers from horse blood sera].

AIM: Evaluate safety of prophylaxis of viral hemorrhagic fevers by specific heterologous immunoglobulins. MATERIALS AND METHODS: Clinical-laboratory examination of 24 individuals after intramuscular administration of heterologous Ebola immunoglobulin was carried out. Anaphylactogenicity of the immunoglobulins was studied by WD 42-28-8-89 in guinea pigs compared with commercial preparations. RESULTS: Immediate type reactions were not observed. In individuals with normal anamnesis the number of local reactions was 31%, general in the form of lung serum disease - 13%. In individuals with unfavorable anamnesis against the background of desensitization therapy there were almost no reactions; without it local reactions were present in 50%, mild severity serum lung disease - in 17%, medium - in 33%. Immunoglobulins against especially dangerous viral agents by anaphylactogenic properties did not differ from commercial heterologous preparations. CONCLUSION: Application of specific immunoglobulins from horse blood sera (the main means of protection from dangerous and especially dangerous exotic viral infections) with compliance by desensitization principles is relatively safe. Safe level of sensitization properties is characterized by anaphylaxis index up to 3.7 for guinea pigs.

[Cowpox: features of spread after cancellation of mandatory pox immunization].

Features of spread of cowpox in the contemporary conditions are examined. A decrease of population immunity to pox in the population of Russia caused by cancellation of pox immunization, hidden circulation of cowpox virus in various species of rodents, as well as lack of vigilance to pathogenic orthopoxviurses in healthcare workers were noted to create the real preconditions for the emergence of infection of humans caused by cowpox virus. Thereby presence of means of express laboratory diagnostics of cowpox and means of effective medical protection for the prevention of development of this disease in the population of Russia becomes an actual necessity.

[Study of immunogenicity and protective efficacy of a novel inactivated vaccine with chitosan against influenza A/H1N1/2009].

AIM: Study of immunogenicity and protective efficacy of a novel inactivated vaccine with chitosan against influenza A/H1N1/2009. MATERIALS AND METHODS: Influenza virus A/California/7/2009 (H1N1) strain was used in the study. Mice were immunized twice (21 day interval) with experimental samples of inactivated influenza vaccine: No. 1--without the addition of chitosan, No. 2--with addition of chitosan. The blood was obtained 21 days after the first and 10 days after the second immunization with the vaccines and was treated with RDE. Antibody levels were evaluated in HI reaction. RESULTS: HI reaction method showed that antibody titers induced after immunization of vaccine No. 2 were higher than those induced after immunization with vaccine No. 1. Evaluation of protective efficacy of the vaccines against an experimental form of influenza infection in mice showed that after immunization with vaccine that does not contain chitosan the level of virus accumulation does not differ from the control statistically significantly (p < or = 0.05), at the same time the level of virus accumulation in the lungs of infected animals immunized with chitosan containing vaccine significantly (significantly with 95% probability) decreased by an average 3.01g when compared with control. CONCLUSION: Comparative analysis of immunogenicity and protective efficacy of experimental samples of inactivated influenza vaccine against influenza A/H 1N1/2009 showed that the vaccine with the addition of chitosan stimulates the formation of a higher immune response and promotes a more significant suppression of influenza A infectious agent reproduction in the lung target-organ.

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy.

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.

[Experience with empirical treatment of severe acute respiratory syndrome due to coronavirus, genotype IV].

Foreign experience with empirical management of severe acute respiratory syndrome (SARS) due to coronavirus, genotype IV is described. It is indicated that the data on the efficacy of ribavirin with respect to SARS in the patients are contradictory. The efficacy of two chemotherapeutics, i. e. lopinavir and ritonavir used in the SARS foci is confirmed. The drugs are at present applicable all over the world in retrovirus therapy of HIV infected subjects. The search for efficient Russian unspecific medicines for control of SARS is actual.

[Present state of in vitro and in vivo studies on efficacy of medical nonspecific protective agents with respect to genotype IV coronavirus causing severe acute respiratory syndrome].

The results of the in vitro studies on the efficacy of medical nonspecific protective agents from various pharmacological groups showed that some drugs, such as velferon, alferon, betaferon, ribavirin and lopinavir were active against TOPC virus, that permitted to recommend them for estimation of their activity on laboratory animals. The data on the in vivo activity of pharmacological drugs with respect to TOPC virus are rather scanty and it is difficult to predetermine their efficacy. The danger of TOPC virus latent circulation among wild animals in China requires research of new efficient medical agents for protection of the people from the pathogen in the Russian Federation.

[Experience with use of immunomodulators in treatment of tonsillitis developed at the background of preimmunization by TEOVac].

The use of immunomodulators in the treatment of subjects with postvaccinal reactions to TEOVac was investigated. The most effective schemes were shown to be those with the use of viferon or combination of arbidol and licopide. The terms of the response signs cupping off were much shorter vs. the cases treated with polyoxidonium. The immunomodulating factors did not affect the intensity of the immunity to the vaccine virus.

[Experimental study of Grippferon antiviral activity against Mexican pandemic influenza virus A/H1N1/2009 in vitro].

Analysis of the efficacy of Grippferon vs. the reference drugs Realdiron and Reaferon-EC against the influenza virus A(H1N1)/2009 in susceptible static cell cultures showed that in the concentrations tested it was efficient in inhibition of the virus cytopathic activity and generation of specific hemagglutinin.

[Therapeutic efficacy of Triazavirin, a novel Russian chemotherapeutic, against influenza virus A (H5N1)].

Therapeutic activity of Triazavirin against experimental influenza A was studied on albino mice intranazally infected with influenza virus A/Chicken/Kurgan/Russia/02/05 (H5N1) vs. reference drugs (Oseltamivir, Remantadin and Arbidol). The study showed that in a therapeutic dose of 1 mg/kg Triazavirin was efficient in protection of the animals from death. Its protective therapeutic efficacy (36.7+/-1.7%) was close to that of Oseltamivir (50.0+/-0.0%), comparable with that of Remantadin (38.3+/-1.7%) and higher than that of Arbidol (11.7+/-1.7%). During the whole observation period (up to the terminal phase) Triazavirin inhibited the influenza virus A accumulation in the lungs of the infected albino mice by more than 3 lg.

[Stabilization of peroxidase conjugates used in enzyme immunoassay systems to detect Ebola and Marburg virus antigens].

The time course of changes in the activity of solutions of horseradish peroxidase conjugates with immunoglobulins against Ebola and Marburg fevers was studied in the presence of different components. The series of the conjugates of ELISA kits for the detection of Ebola and Marburg virus antigens, which were prepared on the basis of the designed stabilizing solution, preserved at less than 90% of its baseline activity during 10 months at a storage temperature of 2 to 8 degrees C.

[The nosological structure of the mortality of fetuses\infants with extremely low weight (acccording to the 2004-2008 records of the Kursk Regional Morbid Anatomy Bureau)].

The authors present the results of studying the mortality of fetuses/infants with extremely low weight from the 2004-2008 records of the Children Pathology Department, Kursk Regional Morbid Anatomy Bureau. The nosological structure of pathology in late miscarriages points to the potential possibilities of reducing mortality rates, by rendering medical aid to pregnant women. The problems of early detection of fetal malformations and those of postmortem diagnosis of intrauterine infections retain their topicality.

[Estimation of in vitro Monclavit-1 virucide activity against influenza virus A (H5N1)].

The virucide activity of Monclavit-1 against the influenza virus A/Chicken/Kurgan/2/Russia/05 (H5N1) was investigated in the MDCK cell culture. It was shown that at a temperature of 14.0 +/- 1.0 degrees C Monclavit-1 in a 20-fold dilution within an hour inactivated the cytopathic activity of the virus and the specific hemagglutinin generation by 100 and 87.5% respectively. In a 40-fold dilution it inactivated at the average by 50% both the cytopathic activity and the specific hemagglutinin generation. In an 80-fold dilution it inactivated the cytopathic activity at the average by 20%. In a 160-fold dilution it did not inactivate the pathogen. At a temperature of 25.0 +/- 1.0 degrees C Monclavit-1 inactivated the influenza virus A/Chicken/Kurgan/2/Russia/05 (H5N1) by 90.0% only in the highest concentration.

[Interferon inductor cupping of postvaccinal complications after variolation].

Efficacy of arbidol and ridostin in cupping postvaccinal complications due to variolation was studied by the clinico-virological, hematological and biochemical indices and it was shown that arbidol was efficient in cupping development of dermal complications, lowered the severity of the postvaccinal reaction and stimulated the cellular and humoral immune response. Ridostin, a high molecular interferon inductor, was highly efficient in cupping all the forms of the postvaccinal complications, including the neurological and cutaneous ones.