[Comparative study of Wuhan-like and omicron-like variants of SARS-CoV-2 in experimental animal models].

INTRODUCTION: The variability of SARS-CoV-2 appeared to be higher than expected, the emergence of new variants raises concerns. The aim of the work was to compare the pathogenicity of the Wuhan and BA.1.1/Omicron variants in BALB/c mice and Syrian hamsters. MATERIALS AND METHODS: The study used strains of SARS-CoV-2: Dubrovka phylogenetically close to Wuhan-Hu-1, and LIA phylogenetically close to Omicron, BALB/c mice, transgenic mice B6.Cg-Tg(K18-ACE2)2Prlmn/HEMI Hemizygous for Tg(K18-ACE2)2Prlmn, Syrian golden hamsters. Animals were infected intranasally, pathogenicity was estimated by a complex of clinical, pathomorphological and virological methods. RESULTS: Comparative studies of SARS-CoV-2 Dubrovka and LIA strains on animal models demonstrated their heterogeneous pathogenicity. In parallel infection of BALB/c mice with Dubrovka and LIA variants, the infection proceeded without serious clinical signs and lung damage. Infection with the LIA strain resulted to a systemic disease with a high concentration of viral RNA in the lungs and brain tissues of animals. The presence of viral RNA in mice infected with the Dubrovka strain was transient and undetectable in the lungs by day 7 post-infection. Unlike the mouse model, in hamsters, the Dubrovka strain had a greater pathogenicity than the LIA strain. In hamsters infected with the Dubrovka strain lung lesions were more significant, and the virus spread through organs, in particular in brain tissue, was observed. In hamsters infected with the LIA strain virus was not detected in brain tissue. CONCLUSION: The study of various variants of SARS-CoV-2 in species initially unsusceptible to SARS-CoV-2 infection is important for monitoring zoonotic reservoirs that increase the risk of spread of new variants in humans.

Biological characterization of cold-adapted SARS-CoV-2 variants

Introduction. The emergence of new epidemiologically significant variants of SARS-CoV-2 has shifted emphasis to development of a live vaccine, which would be able to provide protection against a wide range of antigenic variants of the virus. The aim of the study was to obtain SARS-CoV-2 variants attenuated through cold adaptation and to provide their biological characterization. Materials and methods. The Dubrovka laboratory strain of SARS-CoV-2 and its variants were cultured on Vero and Calu-3 cells. The virus quantification was performed by virus titration in Vero cells and by real-time reverse transcription-polymerase chain reaction. SARS-CoV-2 virions were analyzed using transmission electron microscopy. Genome sequences of the virus were identified by nanopore sequencing. The attenuation (att) phenotype of SARS-CoV-2 variants was identified using Syrian hamsters as an animal model for COVID-19. Results. Cold-adapted (ca) SARS-CoV-2 variants – Dubrovka-ca-B4 and Dubrovka-ca-D2 were produced by continued passaging of the Dubrovka strain in the Vero cell culture at the temperature being gradually decreased to 23ºC and by subsequent cloning. Up to 20 nucleotide substitutions and 18 amino acid substitutions were detected in genomes of ca-variants. Ca-variants, as distinct from the parent Dubrovka strain, actively replicated at 23ºC, while the Dubrovka-ca-D2 variant had a temperature-sensitive (ts) phenotype (did not replicate at 39ºC). Ca-variants of the virus replicated poorly at 37ºC in the Calu-3 human lung cell culture, which, along with the ts-phenotype, can be a marker of virus attenuation for humans. In the intranasally infected Syrian hamsters, ca-variants of the virus demonstrated an attenuation phenotype: they did not cause loss of appetite, fatigue, drowsiness, did not slow down weight gain, replicating much more slowly in the lungs and brain compared to the virulent Dubrovka strain. Conclusion. The obtained attenuated SARS-CoV-2 ca-variants, Dubrovka-ca-B4 and Dubrovka-ca-D2, should be studied further as candidate vaccine strains for a live attenuated vaccine against COVID-19. © Team of authors, 2022.

[Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2]

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

Immunogenic properties of SARS-CoV-2 inactivated by ultraviolet light.

Vaccination against COVID-19 is the most effective method of controlling the spread of SARS-CoV-2 and reducing mortality from this disease. The development of vaccines with high protective activity against a wide range of SARS-CoV-2 antigenic variants remains relevant. In this regard, evaluation of the effectiveness of physical methods of virus inactivation, such as ultraviolet irradiation (UV) of the virus stock, remains relevant. This study demonstrates that the UV treatment of SARS-CoV-2 completely inactivates its infectivity while preserving its morphology, antigenic properties, and ability to induce the production of virus-neutralizing antibodies in mice through immunization. Thus, the UV inactivation of SARS-CoV-2 makes it possible to obtain viral material similar in its antigenic and immunogenic properties to the native antigen, which can be used both for the development of diagnostic test systems and for the development of an inactivated vaccine against COVID-19.

Possibilities of Suppressing The Cytopathogenic Effect of SARS-CoV-2 Coronavirus According to The Results of the Antiviral Activity of Cytovir®-3 In Vitro Study

Introduction.The COVID-19 pandcmic has stimulated the search for drugs with specific antiviral activity against the new pathogenic strain of the SARS-CoV-2 coronavirus. First of all, scientific search was aimed at studying drugs with already proven efficacy against influenza and ARVI. The aim of this worfc was to study the antiviral activity of Cytovir∗-3 in vitro in relation to the cytopathogenic effect of the SARS-CoV-2 virus. Material and methods. The antiviral activity of the drug Cy- tovir∗-3 against the SABS-CoV-2 virus was studied in experimental models in vitro on Vero CCL81 cell culture (ATCC).The maximum tolerated concentration and the 50% cytotoxic dose were determined using a quantitative microculture tetra- zolium test assay to calculate the working range of the concentrations of the test drug. Results and discussion. As a result of the study, it was shown that the greatest activity of the drug was manifested when it was added to the cells 24 hours before and 1 hour and 24 hours after viral infection, the inhibition level reached 53% (>IC50) at the drug concentrations of 105,55, and 85 fig/ml, respectively. Cytovir∗-3 suppressed the viral activity of SARS-CoV-2 in the dose range from 10 pg/ml to 105 pg/ml under the indicated infection conditions. It was found that the drug did not exhibit cytotoxic effects on the Vero cell culture in the range of antiviral doses. Conclusion. The antiviral activity of Cytovir∗-3 against the SARS-CoV-2 virus has been proven due to the achievement of IC50, which is below the maximum tolerated dose of 149 pg/ml.

Local Antiviral Activity of The Drug Thymogen, Nasal Dosed Spray, Against SARS-CoV-2 Coronavirus In Vitro

On account of the COVID-19 pandemic, the global pharmaceutical industry has achieved impressive results in the development and introduction of various types of vaccines causing the formation of acquired immunityagainst the SARS-CoV-2 coronavirus Into clinical practice. However, none of them currently show the declared one hundred percent guarantee of protection. In the case of the COVID-19 disease, patients with concomitant pathologies are the most vulnerable to the occurrence of severe complications. The aerosol route of transmission of SARS-CoV-2 contributes to the emergence of outbreaks of the new coronavirus infection in crowded places and closed rooms with poor ventilation. In this regard, an urgent problem is the search for drugs with local antiviral activity, which, together with restrictive measures and mask wearing policy, can potentially reduce the likelihood of contracting coronavirus. In this experimental in vitro study on Vero CCL81 cell culture (ATCC), the local anti¬viral activity of the drugThymogen∗ spray against the SARS-CoV-2 virus was studied in comparison with the antiseptic Miramistin® solution. As a result of the experiment, no toxic effects on Vero ceils were detected in the drugs in the studied concentrations. In a series of experiments, Thymogen∗ spray showed local antiviral activity against SARS-CoV-2 when the virus titer was 5,2 lg TCID50. Therefore, the drugThymogen® dosed nasal spray has high potential as a topical drug for prevention and treatment of COVID-19 disease, which requires additional confirmation in relevant clinical studies. © 2021 Media Sphera Publishing Group. All rights reserved.

Genetic Diversity and Evolution of the Biological Features of the Pandemic SARS-CoV-2.

The new coronavirus infection (COVID-19) represents a challenge for global health. Since the outbreak began, the number of confirmed cases has exceeded 117 million, with more than 2.6 million deaths worldwide. With public health measures aimed at containing the spread of the disease, several countries have faced a crisis in the availability of intensive care units. Currently, a large-scale effort is underway to identify the nucleotide sequences of the SARS-CoV-2 coronavirus that is an etiological agent of COVID-19. Global sequencing of thousands of viral genomes has revealed many common genetic variants, which enables the monitoring of the evolution of SARS-CoV-2 and the tracking of its spread over time. Understanding the current evolution of SARS-CoV-2 is necessary not only for a retrospective analysis of the new coronavirus infection spread, but also for the development of approaches to the therapy and prophylaxis of COVID-19. In this review, we have focused on the general characteristics of SARS-CoV-2 and COVID-19. Also, we have analyzed available publications on the genetic diversity of the virus and the relationship between the diversity and the biological properties of SARS-CoV-2, such as virulence and contagiousness.

[Reactivation of Epstein-Barr virus (Herpesviridae: Lymphocryptovirus, HHV-4) infection during COVID-19: epidemiological features].

INTRODUCTION: Immunodeficiency underlying the development of severe forms of new coronavirus infection may be the result of mixed infection with SARS-CoV-2 and other pathogens, including Epstein-Barr virus (EBV).The aim is to study the prevalence and epidemiological features of co-infection with SARS-CoV-2 and EBV. MATERIAL AND METHODS: A cross-sectional randomized study was conducted in Moscow region from March to May 2020. Two groups were examined for EBV-markers: hospital patients (n = 95) treated for SARS-CoV-2 infection and blood donors (n = 92). RESULTS: With equal EBV prevalence the detection of active infection markers in donors (10.9%) was noticeably lower than in SARS-CoV-2 patients (80%). Significant differences in this indicator were also found when patients from subgroups with interstitial pneumonia with the presence (96.6%) and absence (97.2%) of SARS-CoV-2 in the nasopharyngeal smear were compared with the subgroup of patients with mild COVID-19 (43.3%). The average IgG VCA and IgG EBNA positivity coefficients in donor group were higher than in patient group (p < 0.05). Patients with active EBV infection markers were significantly more likely to have pneumonia, exceeding the reference values of ALT and the relative number of monocytes (odds ratio - 23.6; 3.5; 9.7, respectively). DISCUSSION: The present study examined the incidence and analyzed epidemiological features of active EBV infection in patients with COVID-19. CONCLUSION: A significantly higher rate of detection of active EBV infection markers in hospital patients indicates a combined participation SARS-CoV-2 and EBV in the development of interstitial pneumonia. Low levels of specific IgG EBV serve as predictors of EBV reactivation. Exceeding the reference values of ALT and the relative number of monocytes in patients should serve as a reason for examination for active EBV infection markers.