Search

Cellular immunity plays an important role in the pathogenesis and formation of protective immune defense against the SARS‑CoV‑2 virus.The aim of the work was to study the cellular immunity of rhesus monkeys applying flow cytometry after experimental infection with the SARS‑CoV‑2 virus.Materials and methods. Male rhesus monkeys were intranasally inoculated with the SARS‑CoV‑2 virus, Isolate B strain and hCoV-19/Russia/SP48-1226/2020 strain (abbreviated name U-2), at a dose of 5.0 lg PFU. Using flow cytometry, the levels of 21 populations/subpopulations of mononuclear cells in the peripheral blood of animals were determined before experimental infection with the pathogen and on day 14 after infection. SARS‑CoV‑2 coronavirus RNA was assessed using real-time polymerase chain reaction. Determination of the titer of virus-neutralizing antibodies to the SARS‑CoV‑2 virus in the blood sera of animals was conducted through neutralization test evaluating the ability to suppress negative colonies.Results and discussion. Infection with Isolate B strain culture has led to an increase in the relative content of total T-lymphocytes (p˂0.2), cytotoxic T-lymphocytes (p˂0.1), as well as monocytes expressing the early activation marker CD25 (p˂0.2). The decrease in levels has been observed for total B-lymphocytes (p˂0.2) and T-helper cells (p˂0.1). Infection with the U-2 strain culture revealed an increase in the relative content of monocytes expressing the early activation marker CD25 (p˂0.2). Thus, for the first time in the Russian Federation, flow cytometry was used to study the cellular immunity of rhesus monkeys before and after experimental infection with the SARS‑CoV‑2 virus. The obtained information can be used for studying the pathogenesis of SARS‑CoV‑2 infection, course, and outcome of the disease, and developing strategies for vaccination and treatment.

Introduction. The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease . The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry). Material and methods. Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry. Results and discussion. The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p 0.05). Conclusion. For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

Ebola outbreak in eastern parts of the Democratic Republic of the Congo in 2018–2020 proved that the virus remains highly hazardous for humans, and the outbreak in West Africa in 2014–2016, which was the largest Ebola outbreak in history, showed that it could be imported to other continents, including Russia. In 1993 the Federal State Budgetary Institution “48th Central Scientific Research Institute” of the Russian Ministry of Defence developed a specific equine immunoglobulin for emergency prophylaxis of Ebola in risk groups. The evaluation and improvement of the product’s properties is an important area in the development of biological defence technologies.The aim of the study was to examine the properties of the equine anti-Ebola immunoglobulin which had been stored for a long time at 2–8 °C.Materials and methods: the authors studied batches of heterologous anti-Ebola immunoglobulin that had been stored for 17–22 years. The properties of the product were evaluated according to the requirements of the State Pharmacopoeia of the Russian Federation, 14th ed. (Ph. Rus. 14 ed.). The specific activity of the product was determined in a plaque reduction neutralisation test using Ebola virus and African green monkey kidney cells (GMK-AH-1(D)). Immunoglobulin molecular parameters were determined by size-exclusion high-performance liquid chromatography using the test methods described in the European Pharmacopoeia 9.6 and Ph. Rus. 14 ed.Results: the storage of anti-Ebola immunoglobulin for 17–22 years at 2–8 °C resulted in a four-fold reduction of the level of virus-neutralising antibodies against Ebola, decrease in the proportion of monomers from 98 to 74–90%, increase in the proportion of dimers and polymers, and formation of immunoglobulin molecules’ fragments. Signs of toxicity for mice were observed in one of the three product batches. Conclusions: the obtained results suggest the need to perform more studies to test the quality of antiEbola immunoglobulin batches that were stored for shorter periods of time in order to assess the stability of their initial characteristics.

The Arenaviridae family consists of a large group of single strand ambisense RNA viruses that are separated phylogenetically, serologically and geographically into Old World and New World viruses. Recent studies indicate that cellular entry of arenaviruses requires a series of cellular protein interaction and molecular mechanisms. The arenaviruses entry into cells is initiated by the interaction of viral glycoprotein with one or more receptors on the surface of host cells. The main host cell factors that are involved in filovirus entry are attachment factors (α-dystroglycan for Old World and human transferrin receptor 1 for New World viruses), endolisosomal host cell factors (cathepsins B and L and Niemann-Pick C1 protein). The review presents the modern knowledge about the role of structural proteins of arenaviruses and some cell factors in pathogenesis of the diseases, caused by arenaviruses.

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401–4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).

Problems of etiology, taxonomy and nomenclature of filoviruses, epidemiology, morbidity with a little-known by Russian medics especially dangerous exotic infectious disease - Ebola fever are examined. Significant distinguishing features of 2013 - 2015 epidemic in West Africa were detected - along with its unprecedented length, a decline did not take place as in previous outbreaks, neither causative agent virulence, nor infectivity of the infection during multiple generations from human to human. Literature data analysis allowed to assume that in the process of epidemic focus formation Ebola virus changes its properties and cyclically passes through several successive interconnected phases: an initial reservation phase in unknown ecosystems - animals, either plant, soil or water; intermediate phase of epidemic spread with primary acquisition of high virulence for humans, and then its decline; final stage of hidden circulation of causative agent that is ap-athogenic for humans. This hypothetical chain of natural phases’ transitions of Ebola virus allows to explain and link together phenomenology of this causative agent - rapid fall of virulence and infectivity for humans in foci in dynamics of epidemic outbreaks, quite a high population immunity in nosoareal of the causative agent in Africa, that contradicts the established understanding of its high lethality for humans.

Ebola virus, representative of the Ebolavirus genus, Filoviridae family, causes severe hemorrhagic fever in humans, with lethality rates amounting up to 90 %. The members of Ebolavirus genus infect a broad range of mammalian cells. Recent studies indicate that entry of Ebola virus into cells requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses, while others are commonly used by all viral glycoproteins. The cellular factors deployed by filoviruses for their entry into permissive cells are defined incompletely. The aim of this review is to analyze peculiarity of the Ebola virus penetration into permissive cells at molecular level. The Ebola virus entry into cells is initiated by the interaction of viral glycoprotein with one or more receptors on the surface of host-cell. The main host-cell factors, involved in filovirus entry, are: attachment factors (cell lectins and human T-cell mucin 1 (TIM-1)), signaling factors (tyrosinkinase receptors and α5β1-integrin), and endolisosomal host-cell factors (cathepsins B and L and Niemann-Pick C1 protein. The study of the complex set of virus entry events provides potential avenues for the development of antiviral therapies against Ebola fever.

Aim. Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. Materials and methods. Junin virus - causative agent ofArgentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Yhro B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. Results. An experimental study of the effect of various factors of impact on the sample under investigation («thawing-freezing», presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. Conclusion. During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.