The tissue distribution of SARS-CoV-2 in transgenic mice with inducible ubiquitous expression of hACE2

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARSCoV- 2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre

Candidate antirheumatic genotherapeutic plasmid constructions have low immunogenicity

Rheumatoid arthritis (RA) is a serious systemic disease of connective tissue, mainly affecting joints but also with different extra-articular manifestations. In the course of RA the degenerative changes occur in cartilage surfaces of affected joints and also in subchondral bone tissue, joints get deformed and lose their mobility. RA affects about 1 % of the global human population. Biological therapy with recombinant protein inhibitors of inflammatory cytokines is an effective and well-accepted treatment of RA. TNF inhibitors such as recombinant receptors or monoclonal antibodies are the most widely used biotherapeutics in clinical practice. However, this treatment has some serious side effects. The patients treated with TNF inhibitors are more susceptible to infection diseases, they are also at higher risk of developing neoplastic or autoimmune disorders. Biotherapeutics become less effective or even lose their efficiency with evoking specific antidrug antibodies. These drawbacks are in general associated with repeated systemic injections of large amounts of recombinant protein required to achieve the therapeutic efficacy. Genetic therapy might provide a good and effective solution. Viral genes coding for immunomodulatory factors could be used to create new gene therapy products to treat RA and other human disease. Poxviruses, as compared to other viral families, have an unprecedentedly rich set of such immunomodulatory genes. In particular, they have genes encoding TNF-binding proteins. Previously in a variety of laboratory models we have shown that recombinant TNF-binding protein CrmB can effectively block TNF. In this work we demonstrated that candidate antirheumatic genotherapeutic plasmid constructions encoding poxviral TNF-binding proteins have low immunogenicity

Development and laboratory production of virus-like immune-stimulating complexes based on saponins and evaluation of their adjuvant potential using mice immunisation with influenza antigens

The COVID-19 pandemic has exacerbated the public’s need for effective vaccines. Consequently, significant financial support has been provided to developers of a number of innovative vaccines, including the vaccines with saponin-based adjuvants. In 2021, the World Health Organisation recommended Mosquirix, the first malaria vaccine, which contains a saponin adjuvant. An anti-covid vaccine by Novavax is in the approval phase. A promising approach to vaccine development is presented by the use of virus-like immune-stimulating complexes (ISCOMs) containing saponins and by the creation of combinations of ISCOMs with antigens. The aim of the study was to develop, produce and characterise virus-like immune-stimulating complexes based on saponins of Quillaja saponaria, as well as similar saponins of Russian-sourced Polemonium caeruleum. Materials and methods: The ISCOM adjuvants, Matrix-BQ and Matrix-BP, were produced using liquid chromatography and examined using electron microscopy. Balb/c mice were immunised intraperitoneally and intramuscularly with ISCOM-antigen preparations. Afterwards, the immunised animals were challenged with the influenza virus strain, A/California/4/2009(H1N1)pdm09, adapted and lethal to mice. The serum samples were examined using haemagglutination inhibition (HI) tests. Results: The authors produced the ISCOMs containing saponins of Quillaja saponaria and Polemonium caeruleum. After one intramuscular injection of either of the ISCOM-antigen preparations with 1 µg of each of A/Brisbane/02/2018 (H1N1) pdm09, A/Kansas/14/2017 (H3N2), and B/Phuket/3073/2013 haemagglutinin antigens (HAs), HI tests detected serum antibody titres to the corresponding antigens of ≥1:40. Two intramuscular injections of the ISCOM-antigen preparation containing 50 ng of each of the HAs and Matrix-BQ resulted in a protective response. In some animals, two intraperitoneal injections of ISCOM-antigen preparations resulted in the maximum antibody titre to the A/Kansas/14/2017 (H3N2) vaccine strain of 1:20,480. Two intramuscular injections of a test preparation containing 5 µg, 1 µg, 200 ng, or 50 ng of each of the HAs and Matrix-BQ or a control preparation containing 5 µg, 1 µg, or 200 ng of each of the HAs (commercially available vaccines) to the mice that were afterwards infected with the lethal influenza strain protected the experimental animals from death. Conclusions: The ISCOM-based preparations had high immunostimulatory activity in the mouse-model study. The presented results indicate the potential of further studies of ISCOM-based preparations in terms of both vaccine and immunotherapeutic development

An optimized method for counting viral particles using electron microscopy

Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. When studying samples containing viruses, one confronts an unavoidable question of the quantitative determination of viral particles in the sample. One of the simplest and efficient approaches to quantitative determination of viral particles in preparation includes the use of electron microscopy; however, a high detection threshold is a significant limitation of this method (107 particles per ml). Usually, such sensitivity is insufficient and can result in error diagnosis. This study aims to develop a method making it possible to detect the number of viral particles more precisely and work with samples in which the concentration of particles is lower than 107/ml. The method includes a concentration of viral particles on the polyethersulfone membrane applied in centrifugal concentrators and subsequent calculation using an electron microscope. We selected env-pseudoviruses using a lentiviral system making it possible to obtain standardized samples of virus-like particles that are safer than a live virus. Suspension of viral particles (a volume of 20 ml) was placed into the centrifugal concentrator and centrifuged. After that, we took a membrane out of the centrifugal concentrator and evaluated the number of particles on the ultrathin section using an electron microscope. The number of viral particles on the whole surface of the filter (a square of 4 сm2) was 4×107 virions, the initial concentration of pseudoviruses in the sample was 2×106 per 1 ml (4×107 particles per 20 ml). As a result, the developed method enables one to evade the major disadvantage of quantitative determination of viruses using electron microscopy regarding a high detection threshold (concentration of particles 107/ml). Furthermore, the centrifugal concentrator makes it possible to sequentially drift a considerable volume of the suspension through the filter resulting in enhancement of test sensitivity. The developed approach results in increased sensitivity, accuracy, and reproducibility of quantitative analysis of various samples containing animal, plant or human viruses using electron microscopy

Successful surgical treatment of massive thrombosis of right parts of the heart in a woman-patient with oncological anamnesis

Successful surgical treatment of massive thrombosis of right parts of the heart in a woman-patient with oncological anamnesi

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

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

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

Possibility of using a mouse SCID as a model animal to variola virus for evaluating anti-smallpox drug efficacy

At present, there is no animal model for smallpox that reflects the weakened immune system in people and can therefore help assess the prophylactic (highly preventive) efficiency of antiviral drugs. To fill in the gap, we have explored the possibility of using outbred immunodeficient SCID mice as a model animal for smallpox with the aid of virolo­gical, histological and electron microscopic and sta­tistical methods. There was no clinical evidence of disease by intranasal infection of mice at a dose of 5.2 log10 PFU (plaque forming units). At the same time, the 50 % infective dose (ID50) of VARV estimated for animals by registering the presence of the virus in their lungs after 4 days post i.n. infection was 3.5 log10 PFU and was relatively similar to that in humans, theoretically determined by identification of the clinical picture of the disease. Virus replication was detected only in the respiratory organs of mice challenged i.n. with VARV at a dose of 5.2 log10 PFU (50 ID50). The values for its concentrations in the lungs and nose resembled those for affected people and well-known animal models (Macaca cynomolgus and ICR mice), respiratorily infected with VARV at similar doses. The existing model animals were not significantly different from SCID mice in the duration of viral presence in the lungs. Moreover, in SCID mice, as in humans and other animal models, similar pathomor- phological changes of inflammatory necrotic nature in the respiratory organs have been reported. Using SCID mice in assessing the prophylactic efficacy of the antiviral drugs NIOCH-14 and ST-246 demonstrated the adequacy of the results obtained to those described in the literature. This opens up the prospect of using SCID mice as an animal model for smallpox to develop antiviral drugs intended for people with severe immuno­suppressive states

Evaluation of a strategy for tumor-initiating stem cell eradication in primary human glioblastoma cultures as a model

Primary cultures of human glioblastoma were obtained from the surgical material of patients K. (female, 61 years, Ds: relapse of glioblastoma) and Zh. (female, 60 years, Ds: relapse of glioblastoma). The effectiveness of a new therapeutic approach aimed at destroying the cancer cell community was evaluated on the primary cell lines of human glioblastoma culture by employing a new strategy of tumor-initiating stem cell synchronization and a domestic strategy of their eradication "3+1". The key elements of the strategy were the following indicator results: (1) evaluation of the presence of tumor-initiating stem cells in a population of cells from analyzed cultures by their ability to internalize double-stranded labeled DNA (TAMRA+ cells); (2) determination of the reference time points of the repair cycle of DNA interstrand cross-links induced by cross-linking cytostatic mitomycin C; (3) evaluation of cell cycle synchronization; (4) determination of the time (day after therapy initiation) when TAMRA+ cells were synchronously present in phase G1/S of the cell cycle, sensitive to the therapy; and (5) establishment of the TAMRA+ (tumor-initiating stem cells) eradication schedule. The cultures were treated with cross-linking cytostatic mitomycin C and a compositional DNA preparation. After the treatments, cell division slows down, and the cultures degrade. The K cell line completely degraded within 30 days of observation. The cell number of the Zh culture fell to nearly one-third of the starting value by day 15 of observation. On day 15, this indicator constituted 1/7.45 for mitomycin C and 1/10.28 for mitomycin C + DNA with reference to the control. The main target of the mitomycin C + DNA regimen was TAMRA+ tumor-initiating stem cells of the glioblastoma cell populations. The action of mitomycin C alone or in the combination with DNA demonstrated effective elimination of TAMRA+ tumor-initiating stem cells and the whole primary cultures of human glioblastomas