Relevance. Combined foci of zoonotic infections are found in all regions of the Russian Federation. A feature of combined foci is the common biocenosis for populations of pathogens of various etiologies (bacteria, viruses, parasitic infections) and the formation of natural foci by them. Aim. Epizootological study of the territory in the south of the Arkhangelsk region for the presence of combined foci of zoonotic infections (tularemia, leptospirosis and hemorrhagic fever with renal syndrome – HFRS). Materials and methods. 135 small mammals were captured and examined in the Ustyansky district of the Arkhangelsk region. Established the species composition and mixed infection among small mammals pathogens of tularemia, leptospirosis, hemorrhagic fever with renal syndrome. Results. In the south of the Arkhangelsk Region in the Ustyansky District, in 2019, for the first time, a combined natural focus of the forest type was identified and characterized. The bank vole and the common shrew are the main carriers of the causative agents of these infections. Five species of small mammals are mixed-infected with the causative agents of these infections. Infected animals with one, two or three pathogens were simultaneously detected in all studied biotopes. This indicates the presence of a combined natural focus of tularemia, leptospirosis, and HFRS. Conclusion. The existence of a combined natural focus of tularemia, leptospirosis and HFRS in middle taiga ecosystems in the south in the Ustyansky district of the Arkhangelsk region was revealed for the first time. Thus, this area can be considered enzootic for tularemia, leptospirosis and HFRS. This focus belongs to the forest type in terms of landscape and geographical characteristics. Mixed infection of small mammals suggests the possibility of simultaneous infection with pathogens of two or more infections, not only animals, but also people located on the territory of natural foci. and non-simultaneous infection of humans with tularemia, leptospirosis and HFRS in combined foci.

Leptospirosis holds one of the leading places among zoonoses due to the breadth of distribution of natural and economic foci, as well as the severity of the clinical manifestation and mortality rate. A relatively low incidence rate in some regions is usually accounted for by unsatisfactory level of differential diagnostics, including insufficient coverage by diagnostic tests. Leptospira are characterized by marked phenotypic and genotypic polymorphisms, affecting epidemiological features and clinical manifestation of leptospirosis. Therefore, laboratory methods are important tool for epidemiological surveillance and diagnostics of leptospirosis. Serological methods primarily the microscopic agglutination test (MAT) currently remain the leading tool in investigating leptospirosis. To study a near 60 year-long contribution of various leptospira serogroups to etiological pattern of leptospirosis, we have analyzed archived MAT serum data from patients suspected of leptospirosis retrieved from our laboratory journals during from the years 1962 to 2020. It was shown that antibodies to the Grippotyphosa serogroup (25.1%) were found most often in patients, which might be associated with the wide range of reservoir host animals and resistance of this leptospira serogroup to environmental factors. At the same time, it should be noted that at different periods antibodies to various leptospira serogroups prevailed in the sera of patients, and in many cases this data matched with the contribution of these pathogens to the etiological morbidity pattern. In particular, dominance of pathogens of the Sejroe serogroup is currently the global trend in the etiological pattern of leptospirosis that was also observed by us at some years. The data obtained might be useful for epidemiological surveillance of leptospirosis infection, including predicting potential outbreaks, as well as for developing approaches to its diagnostics, treatment and prevention.

Relevance. Currently, the study of the combination of natural foci of zoonotic infections has become one of the urgent problems in the field of human infectious pathology, since there is a real possibility of simultaneous or not simultaneous infection of people with two or more natural focal pathogens of different etiologies and the formation of mixed infections in them. Combined lesions are characterized by territorial and morphological structure of the landscape, the presence of common reservoir hosts and types of circulating pathogens (bakterialno-bacterial, bacterial-viral, bacterial, viral, parasitic, etc.). Aims. Conduct an analysis of epizootic and epidemic activity of natural foci of tularemia, leptospirosis and hemorrhagic fever with renal syndrome (HFRS) and identify the reasons for the occurrence of mixed infections in humans. Materials and methods. To assess the activity of natural foci and to identify epizootics in populations of mouse-like rodents, the materials of long-term epizootological studies in Russia were used. A comprehensive study of rodents captured in the natural foci of the Central Federal District and the Northwestern Federal District for tularemia by serological, molecular genetic and biological methods was carried out. Statistical data on the incidence of tularemia, leptospirosis and HFRS in the territory of the Russian Federation for 2015-2020 are presented. Results. The analysis of our own research and literature data allowed us to characterize the combined foci of tularemia, leptospirosis and HFRS as bacterial-viral, according to the degree of combination in the parasitic system of common reservoir hosts, such as common, red, water voles, forest, field and house mice, insectivores. According to the level of combination of the morphological structure of the landscape, the foci belong to steppe, meadow-field, forest and floodplain-swamp, and by type these foci are characterized as infectious geographically combined. The assessment of the epizootic manifestation of foci is determined by the number of natural carriers of infections, isolation of cultures of pathogens, detection of antigen, antibodies or DNA from environmental objects, as well as the recorded incidence of people. The intensity of the circulation of infectious agents in natural foci varies in time and space depending on biotic (number of carriers, abundance of vectors, etc.) and abiotic (temperature, humidity, etc.) environmental factors. Adverse weather conditions (frosts, thaws, etc.), lack of food supply force wild rodents to migrate to populated areas, creating additional risks of human infection. To assess the epizootic manifestation of foci, the volumes of epizootic studies are of great importance, which have been reduced to a minimum and according to their results it is impossible to predict the epidemic manifestation of a particular focal area. The incidence of tularemia, leptospirosis and HFRS in humans depends on the characteristics of human-animal contacts. The epizootological situation and nosological profile in different regions have significant differences, which determines the difference in the risk of human diseases. In recent years, the number of officially registered patients with tularemia and leptospirosis in the Russian Federation is relatively small and ranges from several tens to hundreds of cases. However, the figures of officially registered patients do not reflect the real picture of the incidence of these infections, and to a greater extent indicates not the wellbeing of these nosoforms, but rather the problems of identifying (diagnosing) sick people of a particular infection. The analysis of the incidence of each case of tularemia over the past ten years has allowed to identify mixed-infected patients with pathogens of zoonotic natural focal infections. These are mainly patients with moderate-severe and severe manifestations of the disease with a primary diagnosis (acute respiratory infections, acute respiratory viral infections, purulent lymphadenitis of various localization, angina, chronic pyelonephritis, pneumonia, hepatitis, etc.). Diagnostics is aimed at determining the nosology, clinical form, severity of the condition, detection of complications and indications for treatment. For natural focal infections, information on epizootic and epidemiological examination of the focus of an infectious disease is necessary. The diagnosis is established on the basis of clinical and epidemiological data: a combination of fever and intoxication, determine the relationship of the development of the disease with work and the circumstances in which the patient was. Currently, as a result of the examination of sick people, mixed infections with natural focal infections are increasingly being registered: HFRS + tularemia; tularemia+ leptospirosis; leptospirosis + HFRS; tularemia+ borrelliosis; rickettsiosis +tularemia, etc., the presence of two pathogens of zoonotic infections was detected in the patient during successive studies. It was found that the infection of people occurred in areas enzootic for these infections. It should be remembered that a high-quality comprehensive epizootological examination of focal areas allows you to make a forecast and determine the factors of real epidemiological risk. Conclusions. In the epidemiology of any natural focal disease, it is necessary to take into account the presence of combined foci in a particular territory. Lack of experience in the diagnosis of natural focal infections of tularemia, leptospirosis and HFRS often leads to diagnostic errors, and the lack of alertness to their appearance makes it difficult to identify sporadic cases of diseases. Most nosoforms of hemorrhagic fevers lack specific prevention (HFRS among them), therefore it is necessary to strengthen measures aimed at improving the focal areas, i.e. to carry out deratization work in the most active foci near settlements and mass recreation areas of the population. Mixed infection can lead to both activation and suppression of the protective mechanisms of the human body. The exchange of information between medical and veterinary services will make it possible to better organize and carry out comprehensive measures for epizootic and epidemiological surveillance on the territory of natural foci of zoonotic infections. The formation of modern tactics of differential laboratory diagnostics will ensure the identification of mixed-infected patients with natural focal infections.

Детская Городская Больница Святой Ольги,

МИКРОБИОМ РЕСПИРАТОРНОГО ТРАКТА ДЕТЕЙ С МУКОВИСЦИДОЗОМ Н. Н. Рыжова1, О. Л. Воронина1, Э. В. Лосева2, е. И. Аксенова1, М. С. Кунда1, Н. е. Шарапова1, В. Д. Шерман3, А. Л. Гинцбург1 1 Национальный исследовательский центр эпидемиологии и микробиологии им. почетного академика Н. Ф. Гамалеи, Москва 123098, Российская Федерация 2 Институт биологии и химии Московского педагогического государственного университета, Москва 129164, Российская Федерация 3 Медико-генетический научный центр, Москва 115478, Российская Федерация

Highly pathogenic avian influenza viruses (HPAIV) A(H5N8) of group B (Gochang1-like) have emerged in the Tyva Republic of eastern Russia in May 2016. Since November 2016, HPAIV A(H5N8) has spread throughout the European part of Russia. Thirty-one outbreaks were reported in domestic, wild and zoo birds in 2017. The present study aimed to perform a comparative analysis of new HPAIV A(H5N8) strains. Phylogenetic analysis revealed four genetically distinct subgroups in HPAIV A(H5N8) from the 2016-2017 season. Russian strains consisted of three subgroups with differences between isolates from Tyva, Siberia (Chany Lake), and the European part of Russia. Strains from the European part of Russia showed the beginnings of divergent evolution. Slight differences of the Voronezh strains were suggested by sensitivity to antiviral compounds. Testing for host-specific mutations in sequenced strains revealed the absence of mutations associated with possible increased tropism/virulence in mammalian species, including humans. Only one residue of polymerase basic-1, 13P, is discussed, because the L13P mutation increased complementary RNA synthesis in mammalian cells. We concluded that the evolution of HPAIV A(H5N8) is continuous. Surveillance in Russia revealed new cases of HPAIV A(H5N8) and led to the elaboration of prevention strategies, which should be implemented.

Введение БЦЖ (Бацилла-Кальмета-Герена) вакцина - единственная разрешенная к применению, позволяющая противостоять Mycobacterium tuberculosis. Разработка международных требований к производству и контролю БЦЖ вакцины находится под контролем экспертного комитета ВОЗ по биологической стандартизации (Expert Committee on Biological Standardization, ECBS). В 2009 г. ECBS учредил референсы ВОЗ для БЦЖ вакцины на основе трех субштаммов: Danish1331, Tokyo 172-1 и Russian BCG-I, а также требования к контролю, включающие генетическую характеристику произведенных и посевных серий вакцинного субштамма. Согласно требованиям ВОЗ и надлежащей производственной практики (GMP) было выполнено секвенирование трех посевных серий BCG Russia на платформе 454 Roche в нашей лаборатории. БЦЖиты (лимфадениты, остеомиелиты - осложнения при вакцинации БЦЖ) составляют 1% у здоровых реципиентов, однако занимают третье место среди поствакцинальных осложнений в Российской Федерации. Цель наших исследований заключалась в выявлении генетических особенностей субштамма BCG Russia для разработки экспресс-типирования при осложнениях. Методы Полногеномное секвенирование на платформе 454 Roche. MIRU-VNTRplus database для анализа отличий в профилях BCG субштаммов. Набор биоинформационных ресурсов для аннотирования и исследования геномов. Результаты Геном BCG Russia 368 был секвенирован и депонирован в GenBank (Accession Number NZ_CP009243.1). Сравнение геномов последней генерации субштамма и двух предшествующих продемонстрировало стабильность субштамма BCG Russia. Отличие от ближайшего раннего субштамма BCG Tokyo 172 включало 2 RD (region of difference): инсерцию 22 bp и делецию 1602 bp, десять ins/del (1-9 bp) и 52 SNP. Профиль выявленных профагов в геномах M. bovis и M. bovis BCG отражал эволюцию вакцинных субштаммов. Геномы M. bovis содержали профаги 7.5 и 20.3 kb, ранние BCG субштаммы имели профаги 7.5 and 11.2 kb. Большинство поздних субштаммов утратило профаг 11.2 kb. Исключение составили BCG Montreal and BCG Tice, в геномах которых выявлено 6 и 15 профагов, соответственно. Анализ 27 MIRU-VNTR локусов в геноме BCG Russia 368, их сравнение с профилями локусов штаммов M. bovis и BCG субштаммов в MIRU-VNTRplus database позволили обосновать специфичность семи локусов для субштамма BCG Russia. Мы выбрали Mtub21 и Mtub04 - локусы без повторов в геноме BCG Russia, Mtub-30, QUB-26, QUB-323222 -наиболее вариабельные среди M. bovis и M. bovis BCG, и VNTR-3820 и VNTR-4120 как гипервариабельные локусы. Фрагмент анализ этих локусов позволит провести экспресс тестирование при БЦЖитах. Заключение Полногеномное секвенирование - успешный подход в сравнении близкородственных субштаммов и в выявлении специфических локусов для диагностической панели.

The genes of ESAT-6, CFP-10 and Ag85A secreted antigens of Mycobacterium tuberculosis were cloned in Escherichia coli. Fusion proteins Esat6-CBD, Ag85A-CBD and CFP10-CBD with thermostable cellulose binding domain (CBD) of Anaerocellum thermophilum providing high affinity to cellulose were constructed. Content of the fused proteins in overproducing strains was 20% of total protein for CFP10-CBD and 15% for both Esat6-CBD and Ag85A-CBD. Immobilization of the fusion proteins on cellulose is a one-stage process that provides simultaneous antigen purification and absorption on cellulose. The proteins retain their antigenic properties and can be used as the components for subunit tuberculosis vaccine production or in diagnostic kits.

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

Immunization of CBA mice with killed group A streptococcus (type 5) vaccine changed the counts of stromal precursor cells (CFC-F) in bone marrow transplants at different donorrecipient combinations (normal, N, or immune, I). CFC-F counts in bone marrow transplants from normal mice transplanted to immunized animals decreased 4-6-fold depending on the transplant age in comparison with similar transplants in normal recipients. The percentage of CFC-F colonies with alkaline phosphatase (osteogenesis marker) activity decreased more than 2-fold. Similarly, the count of CFC-F in the transplants was 2-fold lower during delayed (7 months) period after bone marrow transplantation from immunized donors (8–12 days after the end of immunization) to intact recipients, while 2 months after transplantation it was 3-fold lower. The mean optical density of the bone capsule in preparations stained for glycogen and alkaline phosphatase was 1.5-3 times lower in the N → I and I → N experiments in comparison with the control (N → N). On the other hand, CFC-F count in the femoral bone marrow of immunized animals was significantly (3.5-2.5 times) higher during the period from 8 days to 8 months after the end of immunization compared to CFC-F count in the femoral bone marrow of intact mice. These results attest to a significant prolonged effect of streptococcal antigens on the bone marrow stromal tissue. These data also indicate that not all CFC-F, the counts of which increased in response to antigens, are responsible for transplantability of the stromal tissue in heterotopic transplantation. Immunization by streptococcal antigens seemed to suppress transplantability and osteogenic activity of stromal stem cells. The efficiency of CFC-F cloning in mouse bone marrow cultures increased significantly (2-3-fold) in the presence of sera from immune mice. The levels of TNF-α and IFN-γ were low in this serum (2.7 and 6 times lower, respectively) in comparison with normal serum. Presumably, the effects of streptococcal antigens on stromal tissue were mediated through serum cytokines.

AIM:To clone the DNA fragment encoding conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein.MATERIALS AND METHODS:E. coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes--according to "Qiagen company's protocols. Extraction and purification of proteins were performed using original method.RESULTS:DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E. coli. Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit.CONCLUSION:Recombinant domain of LigA in chimeric protein produced in E. coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

Bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein factor which plays a dominant role in growth and regeneration of bone tissue. In clinical practice, bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials have not been produced yet. The fragment of the bmp-2 gene encoding a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on the basis of E. coli BL21(DE3). The level of rhBMP-2 production was approximately 25% of total cell protein. Biologically active dimeric form of rhBMP-2 was obtained as a result of isolation and purification of protein from inclusion bodies with subsequent refolding. The obtained rhBMP-2 sample contained more than 80% of the dimeric form and was able to interact with specific antibodies to BMP-2. Biological activity of the rhBMP-2 samples was verified in in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On a model of ectopic osteogenesis, it was shown that the obtained rhBMP-2 exhibited biological activity in vivo, causing tissue calcification in the site of injection. The protein activity in vivo depends on the way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The elaborated method of rhBMP-2 isolation and purification results in an increased common protein yield and ensures the maintenance of biologically active dimeric form compared to the analogues described in the literature.

AIM:To assess effect of continued immunization of mice from CBA line with inactivated group A streptococcal vaccine on levels of serum cytokines and number of stromal bone marrow progenitor cells in immunized mice and in heterotopic transplants after different variants of transplantation.MATERIALS AND METHODS:CBA mice were immunized during 3 weeks with heat-killed vaccine prepared from group A streptococci type 5. Levels of pro- and antiinflammatory cytokines were measured with BioPlex device. Number of stromal progenitor cells was determined on quantity of colonies formed by cells explanted to monolayer cultures.RESULTS:Significant 2.3-fold increase of number of stromal progenitor cells in femoral bone marrow of immune mice was demonstrated. Experiments with heterotopic transplants showed that bone marrow in transplants of mice immunized with streptococci--variant Normal-->Immune (N-->1)-- is defective both on efficacy of cloning and number of stromal progenitor cells. Even short-term presence of stromal tissue in immune organism (variant I -->N) significantly changed these parameters, especially at late time after transplantation. In serum of immune mice changes of cytokines levels, especially TNFalpha, were observed. The level of the latter was decreased (mean--2.5-fold) in all immune serum samples compared to normal serum.CONCLUSION:Immunization of mice with group A streptococci leads to changes in stromal tissue and, possibly, to damage of microenvironment functions including hemo- and lymphopoiesis.

Костный морфогенетический белок 2 (BMP-2) – фактор остеоиндукции, играющий основную роль в процессах роста и регенерации костной ткани. Костнопластические материалы на основе препаратов рекомбинантного белка BMP-2 человека (rhBMP-2) широко применяются в клинической практике, однако российские аналоги подобных материалов отсутствуют. Нами клонирован фрагмент гена bmp2, кодирующий зрелый белок. На основе штамма Escherichia coli BL21(DE3) создан эффективный штамм-продуцент, уровень синтеза белка rhBMP-2 в котором составляет примерно 25% суммарного белка клетки. Разработана методика выделения и очистки rhBMP-2 из телец включения с последующим рефолдингом для получения биологически активного димерного белка rhBMP-2. Получен препарат, содержащий более 80% димерной формы rhBMP-2, который взаимодействует со специфическими антителами к BMP-2. Биологическая активность препарата rhBMP-2 показана в экспериментах in vitro по индукции синтеза щелочной фосфатазы в культурах клеток С2С12 и С3Н10Т1/2. На модели эктопического остеогенеза показано, что rhBMP-2 обладает биологической активностью in vivo, вызывая образование кальцификата в области инъекции. При этом активность белка in vivo зависит от способа введения и особенностей препарата: использования раствора rhBMP-2 или препарата белка с носителем, кислого или основного буферного раствора. Предложенный способ синтеза и очистки белка rhBMP-2 позволяет повысить общий выход белка и увеличить содержание в нем биологически активной димерной формы по сравнению с известными аналогами.

DNA fragments that encode two collagen-binding decapeptides from human von Willebrand factors vWF-H1 and vWF-H2 were cloned in Escherichia coli cells. The effective chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD, which produce strains that contain corresponding decapeptide sequences, Gly-Ser spacer, and a cellulose-binding domain (CBD) from Anaerocellum thermophilum were constructed. Highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained using the one-stage purification method on cellulose and their ability to bind collagen was studied. The obtained constructions are planned to for use in the development of recombinant collagen-binding proteins with different biological activity, which will be used for the further development of a new generation of products and materials for medical purposes, e.g., various kids of implants and coatings.

Development of new technology allows different antigens of a necessary degree of cleanliness to be obtained. This development is a major problem of modern medical biotechnology. A promising approach to this problem includes use of the affinity domains (tags) incorporated in structure of a recombinant antigen and capable to bind to corresponding sorbents. The method of preparation of ready-for-use injections containing complexes formed by soluble antigens on insoluble cellulose immunosorbent (not chemical conjugates) in one stage is based on the fusion protein technology. This approach includes preparation of two-component recombinant proteins containing an antigen of interest and the cellulose-binding domain (CBD), which spontaneously binds to cellulose containing sorbents with high binding constant. Research into the immunogenic properties of the CBD in the complex with cellulose and in the preparation of recombinant CBD in a rat model was performed. The titers of specific antibodies in rat serum induced by recombinant CBD and CBD in the complex with cellulose was evaluated. The CBD in the complex with cellulose was more immunogenic in comparison with CBD alone. The spectrum and levels of cytokines in collected rat serum induced by developed preparations was also measured using the microsphere-based Luminex Flowmetrix system (BioPlex). It was found that the amorphous cellulose was not an immunotolerant sorbent, because it induced the expression of the proinfammatory cytokines in vivo.

The efficiency of cloning of stromal precursor cell in mouse bone marrow culture increases significantly (2–3-fold) in the presence of serum from mice immunized with type 5 group A streptococcus antigens (5-20 μl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-α and IFN-γ are significantly reduced (2.7 times and more than 6-fold, respectively) in the sera of immunized mice in comparison with normal serum. Serum levels of IL-2, -4, -5, -10, and -12 were about the same in both groups; no granulocyte-macrophage CSF was detected. These data attest to appreciable effect of immunization with streptococcal antigens on the bone marrow stromal tissue; this effect is presumably mediated through serum cytokines.