Preparation of Class Y Immunoglobulins that Neutralize the Marburg Virus

The aim was to study the possibility of inducing Marburg-neutralizing chicken antibodies (MARV) using various immunogens.Materials and methods. Recombinant vaccinia virus expressing the surface glycoprotein (GP) transgene MARV of Musoke strain and pseudovirus particles exhibiting GP of three strains of MARV – Popp, Musoke and DRC2000 based on lentivirus and recombinant strain of vesicular stomatitis virus (VSV) were used as immunogens. Two groups of birds were involved in the study. Chickens were immunized 9 times: first time they were injected with the recombinant vaccinia virus, and then 8 times – with pseudovirus particles (based on lentivirus and a recombinant strain of the vesicular stomatitis virus). The accumulation of specific antibodies was evaluated by enzyme-linked immunosorbent assay (ELISA). We used recombinant VSV exhibiting GP MARV, and natural MARV strain Popp for the analysis of accumulation of neutralizing antibodies.Results and discussion. We have developed an effective immunization schedule for chickens with three recombinant constructs presenting GP MARV, which results in the induction of chicken IgY antibodies against Marburg virus with a titer in ELISA from 1:100 to 1:1 million. The obtained IgY neutralize MARV pseudoviruses (Popp, DRC2000, Musoke) at a dilution of 1/256 to 1/1024 and the natural MARV virus of the Popp strain at a dilution of 1/8. More stable results were demonstrated by immunization using Freund’s incomplete adjuvant

Immunogenic and Protective Features of the Recombinant Vaccinia Virus Strain Expressing Cassette of Genes of Marburg Virus Structural Proteins

The aim of the study was to create a highly immunogenic vaccine construct based on a recombinant variant of a replication-defective MVA strain of vaccinia virus, expressing virus-like particles that mimic natural infection with Marburg virus. Materials and methods. The recombinant virus was obtained through recombination between homologous viral DNA sequences and the insertion plasmid pDel2-GP-VP-Pat which carries transgenes of the structural proteins GP and VP40 of Marburg virus, flanked by fragments of MVA strain genome. Structure of the recombinant virus was confirmed in PCR and using sequencing, transgenes expression was analyzed by Western blotting, viruslike particles formation was recorded using electron microscopy. Evaluation of immunogenicity and protectivity was carried out using a guinea pig model. The antibody titer was determined in enzyme-linked immunosorbent assay. To assess T-cell response, the intracellular staining of cytokines was used, followed by analysis of samples on a flow cytometer. Results and discussion. On the basis of highly attenuated MVA strain of vaccinia virus a recombinant variant MVA-GP-VP40-MARV has been constructed, carrying a cassette of transgenes, GP and VP40, of Marburg virus in the region of deletion II of the genome. The expression of transgenes in MVA-permissive CER cells infected with recombinant MVA-GP-VP40-MARV strain and secretion of GP and VP40 proteins into culture medium have been demonstrated. Electron microscopy analysis has revealed the presence of Marburg virus-like particles in the culture medium of cells 12 hours after infection. Double vaccination of guinea pigs with MVA-GP-VP40-MARV strain at a dose of 108 PFU/animal induced the formation of antibodies to Marburg and vaccinia viruses, as well as 100 % protection against lethal Marburg virus infection (50 LD50). Using original TEpredict software, the structure of T-helper epitopes of GP protein has been predicted. Using the ICS method, the biological activity of these epitopes has been experimentally confirmed and it was shown that they provide the induction of a T-cell immune response as part of the MVA-GP-VP40-MARV vaccine construct

Highly Effective xMAP Multiplex Assay for the Detection and Identification of Hemorrhagic Fever Agents, Including Ebola Virus

Developed has been the oligonucleotide liquid biochip based on xMAP technology, designed for the laboratory detection of particularly dangerous viral pathogens such as Ebola and Marburg filoviruses, and Machupo , Junin, and Lassa arenaviruses. The suggested approach allows for the detection of up to 100 viral genome equivalents in a sample. The sensitivity and specificity of oligonucleotide biochip is 100 % when the laboratory panels of positive and negative samples are used. These results indicate that the xMAP multiplexing for the detection and identification of tropical hemorrhagic fever agents, including Ebola virus, is not inferior to the conventional method such as real-time RT-PCR and can be applied for evaluation of viral load, and further on can easily be expanded for both the analysis of new viral agents and for the detection of critical mutations in viral genomes

Obtainment of Monoclonal Antibodies and Prospects of Their Application as Basis for Immunodiagnostic Aids for Crimean-Congo Hemorrhagic Fever Virus Detection

) as framework for the production of tools for CCHF virus detection and identification in artificially contaminated samples and clinical specimens containing CCHF antigens was proven efficient

Experience of Application of the ELISA Method for Detection of Antibodies to Ebola Virus during the SAET Team Work in the Republic of Guinea

The aim of the work was to develop a kit for detection of IgM and IgG antibodies to Ebola virus in ELISA.Materials and methods. Ebola virus strain Zaire H.sapiens-wt/GIN/2015/Kalidie-Kindia-1022 grown on cell culture was used as antigen.Results and conclusions. The kit was used by the Rospotrebnadzor SAET team in its work in Guinea while investigating cases during Ebola fever epidemics. It was established that specific IgG antibodies persisted in Ebola fever survivors for 2 years. Application of this kit in the laboratory diagnostics permits ELISA to become the main and confirmatory laboratory method of Ebola fever detection

Susceptibility to SARS-CoV-2 Virus Variants of Concern in Mouse Models

The aim of the research was to assess the susceptibility of mice of different lines to newly emerging variants of SARS-CoV-2.Materials and methods. The SARS-CoV-2 virus strains belonging to variants of concern (VOC) circulating in the territory of the Russian Federation were used in the study. Experiments involved three inbred mouse lines (BALB/c, CBA and C57Bl/6z) and CD1 outbred mice taken from the nursery of the SSC VB “Vector” of the Rospotrebnadzor. The infectious titer of coronavirus in tissue samples obtained from the laboratory animals was determined on a Vero E6 cell culture. The (Ct) threshold value in RT-PCR was considered an additional parameter for monitoring the viral load in the samples. The severity of lung tissue damage was assessed using histological preparations.Results and discussion. The susceptibility of various mouse lines to the genetic variant Beta of the SARS-CoV-2 virus has been investigated. During intranasal infection of the inbred and outbred mice with strains of VOC at a dose of 2·103 TCID50, the virus replicated in the lungs with maximum concentrations 72 hours after infection. The pathogenicity of genetic variants of the SARS-CoV-2 virus for BALB/c mice has been assessed, a 50 % infectious dose for intranasal infection (ID50) determined. Histological analysis showed COVID-19-specific lung tissue lesions in infected animals. Our study proves that BALB/c mice can be used as a model animal in screening studies when evaluating the effectiveness of therapeutic, vaccine preparations and studying the pathogenesis caused by VOC of the SARS-CoV-2 virus: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Omicron (B.1.1.529) and the like

Studies of Sensitivity to Avian Flu Virus A/H5N1 in Chickens

) appear to be highly virulent for chickens. The chance of AFV infection of chickens in case of intranasal challenge is 20 times as great as in the case of peroral one, and 300 times as great as in the case of intragastral one, which bears evidence to higher sensitivity to AFV of the tissues of avian respiratory organs, in comparison with the tissues of gastro-intestinal tract. Therewith, primary target organ for virus in intranasal infected birds is their respiratory channel (mucous membrane of the nasal cavity in particular). Registered is the possibility of existence of fecal-nasal AFV transfer mechanism in chickens

Pathogenicity of the SARS-CoV-2 Virus Variants of Concern for the Syrian Golden Hamster

The aim of the work was to study the pathogenicity of newly emerging variants of SARS-CoV-2 on the model of the Syrian golden hamster.Materials and methods. We used the strains of SARS-CoV-2 virus related to the VOC circulating in the territory of the Russian Federation. The experiments were carried out on outbreed Syrian hamsters obtained from the nursery of the SSC VB “Vector”. The infectious titer of coronavirus in tissue samples collected from infected laboratory animals was determined on a Vero E6 cell culture. The Ct in RT-PCR was considered an additional parameter for monitoring the viral load in the samples. The severity of lung tissue damage in Syrian hamsters with COVID-19 was assessed by histological preparations.Results and discussion. 50 % infecting doses in case of the intranasal infection have been determined, histological analysis of lung tissues performed. The pathogenicity of various variants of the SARS-CoV-2 virus for the Syrian hamster has been evaluated, differences in infecting doses and pathological changes in the lungs have been revealed. SARS-CoV-2 viruses belonging to Beta genetic variant have the highest virulence, while Alpha variant has the lowest one when comparing the studied strains by the ID50 value. The Delta and Omicron variants have a matched ability to cause specific damage to the tissues of the respiratory tract, while being inferior only to the Beta variant. It has been demonstrated that Syrian hamsters are an adequate model for assessing the pathogenicity of the SARS-CoV-2 virus variants of concern. Variants of SARS-CoV-2 virus during intranasal infection has shown different degree of pathogenicity in the Syrian hamster model

Mice as Animal Model for Evaluation of Therapeutic Efficacy of Preparations against Monkeypox

of MPV after 7 days postinfection, applying peroral administration once within 24 hours (24 hours before infection and 7 days after infection of mouse with 60 µg/g of a preparation). Displayed is the feasibility of using 8–15-days-old ICR mice (body weight 9–11 g) as an animal model for evaluation of therapeutic efficacy of the preparations under development against Monkeypox and smallpox

Analysis of Ebola virus Zaire 2014 isolates

Analysis of 5 Ebola virus Zaire 2014 isolates passaged in cell cultures or in mice, demonstrated presence of unique mutations in the genome RNA in some cases. All identified nucleotide substitutions are singular, stochastically located, synonymous or fall within non-coding regions. Variability level of nucleotide sequences is equal to 0.005-0.01 %, suggesting extremely high genetic stability of Ebola virus Zaire, the causative agent of the outbreak. Confirmed is suppression of non-synonymous mutations accumulation in ebolavirus variants in the course of time. Detected are alterations in glycosilation sites and mucin-like domain of ebolavirus glycoprotein