Vol 95, No 3 (2018)

Aim. Determination of potential epidemic hazard degree of migration in the possibility of cholera importation into the RF constituents with (without) the international border crossing points by air, sea, road and rail transport, as a component when determining the epidemic potential of the territory. Materials and methods. The data of the Rospotrebnadzor Directorates for 83 Russian Federation constituents, including 61 subjects with air, sea, road and rail transport border crossing checkpoints, taking into account the links with countries affected by cholera, and in 22 without international checkpoints ( 2011 — 2015) were used. The analyses of demographic indicators for characterizing intensity and determination the potential epidemic hazard degree of migration was carried out. Results. The increase in population due to interstate migration by various transports was determined in 83,6% subjects. The algorithm of epidemiological assessment was developed, with calculating the potential epidemic hazard degree (PEHD) of population migration in the possibility of cholera importation into 83 administrative subjects. The highest PEHD of migration was determined in 17 of 61 subjects with checkpoint sets; in 39 — higher than normal and in 5 — low; in 22 subjects without international checkpoints — in 8, 12 and 2, respectively. Conclusion. The obtained results indicate the presence of epidemiological risks associated with migration.

Aim. Experimental substantiation of possibility to determine the rabies virus-infected Vero cell line portion in culture by flow cytometry (FC) and FITC labeled diagnostic anti-rabies immunoglobulin (DAI), manufactured in Russia. Materials and methods. Fixation and permeabilization of Vero cells, infected by rabies virus strain «Moscow 3253», was carried out by means of Суtofix/Cytoperm reagent (BD Biosciences, USA) according the Vengatesan D. et al. method (2006) and then intracellular rabies antigen was stained by DAI. Percentage of infected cells was determined by FC in 24, 48 and 72 h and as well in 48 h when the cell cultures were infected with tenfold dilutions of virus-containing fluid from 10-1 to 10-8. Results. There was a significant increase in the percentage of infected cells on average from 30 to 70% in time interval from 24 to 48 h. With 1000-fold dilution of viral-containing fluid the FC method detected the 6,9±0,21% of infected cells in Vero cultures (P<0,001, n=3). Conclusion. FC has proved to be a fast, sensitive and reliable method for determining the relative number of virus- infected Vero cells in cultures. The drug DAI had a sufficient activity for its effective use in the automated version of MFA based on the FC method. The use of FC is possible at various stages of anti-rabies drug production and control, and is also promising in terms of further improving of the rabies diagnosis.

Aim. In this study we aimed to develop the methodology to change the antigen specificity of chimeric antibodies by replacing the variable region genes in the previously designed universal plasmid constructions pLK DT-17 and pHG DT-17 encoding the DT-17 antibody against the diphtheria toxin (DT) to the genes of antibody binding to another DT epitope — DT-22. Materials and methods. The genes of the light and heavy chain variable regions of mouse anti-DT antibodies — DT-22 were amplified from the hybridoma producing monoclonal antibodies to DT by reverse transcription and PCR methods. Genetic engineering methods were used to replace the variable regions of DT-17 antibody in the recombinant plasmids pLK DT-17 and pHG DT-17 encoding the light and heavy chains of DT-17 antibody, respectively to the relevant genes of DT-22. Subsequently, a «supervector» pSV DT-22, containing the genes of both chains of the chimeric antibody, was designed. CHO cells were transfected with a «supervector» and a highly productive clone, secreting chimeric antibodies to DT was obtained. Immunochemical and cultural methods were used to evaluate antibody activity. The affinity chromatography was used to purified preparative amounts of antibodies. Results. The yield of purified secreted chimeric DT-22 antibodies was 4 mg from per liter of culture medium. The minimum concentration of chimeric antibodies at which DT was neutralized in the CHO cells was 22 μg/mL of medium. Conclusion. Thus it has been shown how to generate new vector coding synthesis of light and heavy chains of a chimeric DT-22 antibody specific to another DT epitope using previously constructed universal recombinant plasmids pLK DT-17 and pHG DT-17 encoding, light and heavy chains of antibodies against DT DT-17, respectively.

Aim. Analysis of the arp gene internal fragment nucleotide sequences variability in modern russian T. pallidum subsp. pallidum strains. Materials and methods. 57 T. pallidum isolates obtained from specialized dermatovenerologic clinics of the Central (Kaluga), the North Caucasus (Stavropol) and the Siberian (Tyva) regions in 2016 — 2017 were used in the study. The sequensing of the arp gene was performed using capillary electrophoresis technology. Results. A two-round amplification of the arp gene have been proposed, which ensures a correct reading of its internal region. Four variants of 60-nucleotide repeats in the internal arp fragment are described, which differing in 6, 8 and 15 — 17 codons compositions. Various combinations of these repeats, corresponding to the reference Nichols strain, globally distributed Street 14 genogroup, and the firstly described Stavropol regional variant are shown. Conclusion. The prospect of arp gene sequencing as a way to increase T. pallidum molecular typing efficiency is postulated.

Aim. The clinical and epidemiological significance of the Latin American Mediterranean (LAM) genetic family of Mycobacterium tuberculosis determines the importance of the correct detection of LAM strains. In this study, a complex of molecular methods was used to analyze LAM strains in the population of M. tuberculosis in the Omsk region of Western Siberia, which is characterized by a high incidence of drug-resistant tuberculosis. Materials and methods. The collection included 207 strains of M. tuberculosis, isolated in the Omsk region in 2015 — 2016. The strains were subjected to spoligotyping, analysis of LAM-specific SNP Rv0129c 309G>A, and whole genome sequencing followed by bioinformatics analysis. Results. A comparison of the obtained CRISPR-spoligotyping profiles with the international SITVIT_WEB database, assigned 11 strains (5.3%) to the LAM genotype. At the same time, based on analysis of phylogenetic SNP in the gene Rv0129c, 30 isolates (14.5%) were assigned to LAM. Whole genome sequencing was performed for 4 isolates with different spoligotyping profiles. Conclusion. The results of this study show the limited utility of the decision rules implemented in SITVIT_WEB to define LAM family for isolates with long deleted blocks of spacers or abridged spoligoprofiles. The following approach can be recommended for detection of LAM isolates (1) primary spoligotyping, comparison with SITVIT_WEB, and mandatory interpretation in the light of expert knowledge; (2) detection of LAM-specific SNP (e.g., using PCR-RFLP).

Infections caused by Streptococcus pneumoniae are relevant for Russia and the world. One of the key factors in the pathogenicity of pneumococcus is a polysaccharide capsule. The structure of polysaccharide antigens is described more than 90 serotypes of the pathogen. The experience of using polysaccharide and conjugated pneumococcal vaccines shows that these preventive drugs protect against a limited number of serotypes of the pneumococcus. It is of interest to study the protective properties of pneumococcal proteins, as they are conservative and have high homology within the species, potentially expanding serotype non-specific protection level. Thus, the efforts of researchers focus on the development of protein vaccines or conjugated vaccines based on proteins of S. pneumoniae. The review considers the biological properties of the most well-known proteins of pneumococcus and provides data on preclinical studies of the obtained recombinant proteins as experimental vaccine preparations. Immunization with various proteins of S. pneumoniae provides protection of animals from nasopharyngeal colonization, pneumonia and sepsis. Currently, clinical trials (I/II phases) are being tested with several experimental protein vaccines. In the near future it will be possible to assess the real effectiveness of such vaccines.

The review expounds the main content issues of biological safety in the modern period. Theproblems of postgraduate education in the field of biological safety through professional retraining and advanced training programs, as well as training of highly qualified specialists were discussed. The need to form a separate specialty “Biological Safety”for specialists in medical and biological profiles was noted.

Ebola virus disease is dangerous viral infection, occurring in the form of hemorrhagic fever, characterized by acute clinical symptoms and high mortality rate due to multiple organ failure. Ebola virus natural foci are located in forested areas of the central and western parts of Africa. It was believed for many years, the incidence of Ebola virus disease has been sporadic and the burden of it is true only in endemic areas. However, the unprecedented Ebola epidemic caused by Zaire virus in 2013 — 2016, has significantly changed our understanding of this disease and the patterns of its distribution. We have also identified weaknesses in the organization of anti-epidemic measures, the effectiveness of which was not very effective at the onset of the epidemic, in particular due to weak development of in vitro diagnostics (IVD). However, during the elimination of the epidemic in West Africa, anti-epidemic system has been modified substantially, largely due to quickly developed IVD kits. This review is devoted to analysis of trends in IVD for Ebola virus disease based on the experience obtained in the course of the West-African epidemic in 2013 — 2016.