[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.

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.

[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.

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.

Potential of application of the RNA interference phenomenon in the treatment of new coronavirus infection COVID-19.

COVID-19 has killed more than 4 million people to date and is the most significant global health problem. The first recorded case of COVID-19 had been noted in Wuhan, China in December 2019, and already on March 11, 2020, World Health Organization declared a pandemic due to the rapid spread of this infection. In addition to the damage to the respiratory system, SARS-CoV-2 is capable of causing severe complications that can affect almost all organ systems. Due to the insufficient effectiveness of the COVID-19 therapy, there is an urgent need to develop effective specific medicines. Among the known approaches to the creation of antiviral drugs, a very promising direction is the development of drugs whose action is mediated by the mechanism of RNA interference (RNAi). A small interfering RNA (siRNA) molecule suppresses the expression of a target gene in this regulatory pathway. The phenomenon of RNAi makes it possible to quickly create a whole series of highly effective antiviral drugs, if the matrix RNA (mRNA) sequence of the target viral protein is known. This review examines the possibility of clinical application of siRNAs aimed at suppressing reproduction of the SARS-CoV-2, taking into account the experience of similar studies using SARS-CoV and MERS-CoV infection models. It is important to remember that the effectiveness of siRNA molecules targeting viral genes may decrease due to the formation of viral resistance. In this regard, the design of siRNAs targeting the cellular factors necessary for the reproduction of SARS-CoV-2 deserves special attention.

[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.

[Detection methods for results of a loop-mediated isothermal amplification of DNA.]

The loop mediated isothermal amplification (LAMP) was developed by T. Notomi et al. in 2000. It has become one of the most promising methods for point-of-care diagnostics due to its accuracy, sensitivity and ease of execution. In this review, various methods for detecting the results of the LAMP reaction are considered; their advantages and disadvantages are revealed. Methods for detecting LAMP results can be divided into indirect and direct. Indirect methods aimed at detecting changes in the chemical composition of the reaction mixture include real-time turbidimetry, fluorescence detection with calcein, colorimetric detection with hydroxynaphthol blue, and detection using modified gold nanoparticles. Direct methods based on the detection of accumulation amplicons during the reaction include fluorimetric detection with intercalating dyes, resonance fluorescence energy transfer, enzyme immunoassay, immunochromatography, using cationic polymers and gold nanoparticles. The development in the field of point-of-care diagnostics is characterized by a pronounced tendency to miniaturization, the LAMP reaction on microchips and microfluidic devices with an electrochemical or optical detection method. The most promising for the diagnosis of infectious diseases are turbidimetry methods and the use of intercalating dyes. The development of portable domestic instruments for detecting of LAMP results based on real-time fluorescence detection or turbidimetry will contribute to the widespread introduction of the method into clinical laboratory diagnostic practice. A literature research was conducted in the Pubmed ncbi based on keywords.

[The potential of synthetic small interfering RNA-based antiviral drugs for influenza treatment].

Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.

[SOME FEATURES OF MANIFESTATIONS OF EPIDEMIC PROCESS DURING ACUTE INTESTINAL INFECTIONS IN MOSCOW].

AIM: Study manifestations of epidemic process during acute intestinal infections to establish reasons of low effectiveness of the prophylactic measures carried out and evaluation of the role of rotavirus infection in general disease structure of intestinal infections of unknown etiology. MATERIALS AND METHODS: Data on morbidity of acute intestinal infections of population of Moscow were analyzed. Hospitalized patients with acute intestinal infections were examined using real-time PCR method test-systems of laboratory of molecular virology of Mechnikov RIVS with subsequent typing. RESULTS: Evaluation of multi-year manifestations of epidemic process of morbidity of acute intestinal infections in Moscow has shown, that the cumulative morbidity does not have a tendency of reduction, because the proportion of infections with undeciphered etiological factors is almost 80% of the total aggregate morbidity. The proportion of rotavirus infection in total morbidity of AII of established etiology increased from 53.2 in 2004 to 82.6% in 2014. Morbidity in childrenwith rotavirus infection is 6 times higher than morbidity in adults. CONCLUSION: The results obtained give evidence on the necessity of carrying out specific prophylaxis against viral intestine infection, mostly of rotavirus and norovirus infections. The highest effect should have been expected from use of a bi-vaccine, development of which seems quite an actual problem.

[The prevalence of the human rhinoviruses and coronaviruses circulating in the Moscow region during 2007-2012].

The rhinoviruses and coronaviruses are the most common causative agents of the acute upper respiratory tract infection in humans. They include several species that vary in the pathogenicity, some causing severe respiratory tract diseases. In this work, the species prevalence of rhinoviruses and coronaviruses was studied in 92 virus-positive clinical patients that were collected at the area of the Moscow region during the period from 2007 to 2012. Using the real-time PCR the virus circulation has been established for all species common in humans, including three rhinoviruses, HRV A, HRV B, and HRV C, and four coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. For eight patients, the identity of the rhinoviruses, including 4 cases of HRV-C, 3 cases of HRV-A, and a single case of HRV-B, was corroborated using partial sequencing of the 5 non-coding regions and phylogenetic analysis. The viruses of HRV-C, HCoV-NL63, and HCoV-OC43 were prevalent in children with severe respiratory diseases.

[Evaluation of real-time multiplex PCR effectiveness for group a rotavirus genotyping].

AIM: Evaluate resolution and diagnostic significance of real-time multiplex PCR (MP RT-PCR) as a platform for group A rotavirus G/P genotyping test-systems. MATERIALS AND METHODS: Primer and DNA probe construction for an experimental test-system based on MP RT-PCR was carried out by using specialized PC programs and sequence databases GenBank NCBI, EMBL Nucleotide Sequence Database etc. The experimental genotyping test-system was tested using 116 clinical samples with confirmed rotavirus infection and 14 biosamples negative for group A rotavirus RNA. Selective sequencing of VP7, VP6, VP4 gene mark-erloci was carried out as a reference method forverifying determination of rotavirus genotype. RESULTS: Specific interaction between primers and DNA probes with genotype-specific loci of retrovirus genome segments and a lack of false-negative signals, complete match ofgenotyping results obtained by MR RT-PCR and sequenc- ing methods were established. CONCLUSION: The resolution of MP RT-PCR methods allows designing test-systems that can confidently identify rotavirus genotypes with effectiveness of 90% and above.

[Rubella virus genetic determinant of attenuation].

Vaccination is the most effective and available way to prevent Rubella. Presently, 9 vaccine strains were registered. Nevertheless, the molecular mechanisms of the attenuation were poorly elucidated for the rubella virus. However, the study of these mechanisms identifying genotypic and phenotypic markers of attenuation, which together with sequence analysis could be used for the genetic stability control of vaccine strains, is still of current interest. Common trends of genetic changes in the process of adaptation to cold were found due to comparison of nucleic acid and amino acid sequences of the Russian strain C-77 with corresponding positions of the known rubella virus strains and its wild type progenitors, if available.

[Studying of molecular mechanisms of rubella virus attenuation evidence from Russian strain C-77].

Live attenuated rubella vaccine is used for vaccination. Temperature-sensitive (ts) phenotype was proved for almost all rubella vaccine strains, and the acquisition of the ts phenotype during cold adaptation was strongly correlated with the attenuation of the wild-type viruses. Nevertheless, the molecular mechanisms of the attenuation have been insufficiently understood for rubella virus. Study ofthese mechanisms, identifying genotypic markers of attenuation, which together with the sequence analyses could be used for genetic stability control of vaccine strains, is still of current interest. In this work, we determined nearly complete genome sequences of attenuated (ca) and the wildtype progenitor (wt) of the rubella virus strain C-77 isolated in Russia. Possible genetic determinants of attenuation were detected. Thus, 13 nucleotide differences leading to 6 amino acid substitutions were found. Four amino acid substitutions were found to be almost unique. Special consideration should be given to Tyr1042Cys substitution in the protease domain of C-77 strain, because it most probably plays the crucial role in acquisition of ts-phenotype.

[Evaluation of the efficiency of differential diagnosis of influenza by multiplex real-time polymerase chain reaction].

The paper gives the results of a comparative analysis of the detection of influenza viruses in clinical samples, by using multiplex real-time polymerase chain reaction (RT-PCR) and by virus isolation in MDCK cell cultures. The investigation employed 267 nasopharyngeal swab specimens obtained from patients with influenza symptoms during two epidemic seasons (2008-2009 and 2009-2010). Influenza viruses were found in 104 samples (48 with influenza A virus (IAV) and 56 with influenza B virus (IBV)) by multiplex RT-RCR and in 84 samples (35 with IAV and 49 with IBV) by a cultural technique. The results of detection of influenza viruses by the two methods showed 89.4% agreement. The diagnostic sensitivity of multiplex RT-PCR testing a panel of the clinical samples in question was estimated to be 94.3% for IAV and 95.9% for IBV. The diagnostic sensitivity of multiplex RT-PCR in virus detection was demonstrated to be not only highly competitive with virus isolation, but also superior to the latter.

[Quick test for measurement of rubella virus titer in virus-containing fluid using RT-PCR].

AIM: To develop method of rubella virus titer measurement in virus-containing fluid using real-time PCR (RT-PCR) with fluorescent detection. MATERIALS AND METHODS: Measurement of infectious titer of rubella virus (Wistar RA 27/3 strain) cultivated on Vero cells was performed simultaneously by RT-PCR and cytopathic effect assay (CEA) on PK-13 cell culture and then results obtained by each method were compared. RESULTS: Time interval after inoculation, in which difference between virus titer measured by both methods did not exceed 0.3 1gTCD50/ml (value acceptable by WHO), was 2 - 7 days. Pearson correlation coefficient between two values for the mentioned interval was close to 1, which point to good agreement of results. In control sample--international vaccine standard of rubella virus--difference in virus titer determined by RT-PCR and CEA was within 0.2 1gTCD50/ml that lower than value acceptable by WHO. CONCLUSION: Method for measurement of rubella virus titer in virus-containing fluid using RT-PCR was developed, which characterized by high specificity, sensitivity, standard performing, shorter time needed for procedure compared with classic methods and, at the same time, high correlation of its results with results obtained by the latter methods during defined time interval.

[Development and preclinical study of new generation virosomal split influenza vaccine "Grifor"].

New Russian virosomal split vaccine against influenza "Grifor" was developed. The vaccine is represented by mix of highly purified protective external and internal antigens of influenza A (H1N1 and H3N2) and B viruses. Developed technology of manufacture allowed to provide presentation of external antigens of influenza virus in the form of virosomes, and presentation of internal antigens in the form of micelles with maximal preservation of their antigenic activity. Using electron microscopy, electrophoresis in 10% polyacrilamide gel with sodium dodecyl sulfate, and polymerase chain reaction, morphologic and biochemical properties of the vaccine were studied. Preclinical study, including assessment of antigenic characteristics of "Grifor" vaccine compared to vaccine "Vaxigrip" (France), was performed. It was established that administration of the vaccine did not result in death of experimental animals, decrease of body mass, development of pathologic (including inflammatory, dystrophic and necrobiotic) changes in viscera or render adverse effects on blood hematologic and biochemical parameters and on the immune system. The vaccine was not pyrogenic and allergenic, did not have local irritating effects. Obtained results supported the appropriateness of conducting the clinical trials of "Grifor" vaccine on limited number of volunteers.

[Use of multiplex PCR method with real-time detection for differential diagnosis of respiratory viral infections].

Multiplex real-time polymerase chain reaction test-system with fluorescent detection (RT-PCR) for simultaneous identification of main agents of acute respiratory viral infections: influenza A (IAV) and B viruses (IBV), parainfluenza viruses types 1, 2, 3, 4 (PIV 1 - 4), adenoviruses (ADV), respiratory syncitial virus (RSV), rhinoviruses (RV) and enteroviruses (EV), in presence internal positive control (IPC) represented by vaccine strain of rubella virus RA 27/3. Using multiplex RT-PCR method, respiratory viruses were detected in 116 out of 226 clinical samples (nasal swabs) obtained from patients with symptoms of acute respiratory infection: in 68 (58.6%) samples--IBV; in 21 (18.1%)--IAV; in 12 (10.3%) --RV; in 6 (5.2%)--PIV 2; in 4--(3.4%) ADV; in 3 (2.6%)--RSV; in 2 (1.7%)--EV; in 2 (1.7%)--PIV 4; in 1 (0.9%)--PIV 3; in 1 (0.9%)--PIV 1. Mixed infection was observed in 4 (3.4%) patients. PCR assay allowed to reveal various respiratory viruses in 51.3% of samples. At the same time samples were tested for the presence of 12 respiratory viruses--IAV, IBV, PIV 1 - 4, RSV, RV, metapneumoviruses, and coronaviruses NL63, 229E and OC43--in the presence of IPC represented by equine arteritis virus using analogous PCR test-system provided by medical center of Leiden university. Results of tests for detection of IAV, IBV, RSV, PIV 1 - 4, and RV, analyzed by both systems, agreed in 94%. Multiplex format of RT-PCR performing significantly reduces time and cost of the test, which make it suitable and effective instrument of epidemiological studies.

[Real-time multiplex PCR for rapid detection of enteroviruses, adenoviruses and hepatitis A virus in clinical specimens].

Real-time multiplex polymerase chain reaction (PCR) with internal positive control (IPC) was developed for simultaneous detection of adenoviruses (AV), enteroviruses (EV) and hepatitis A virus (HAV). Primes and probes labeled with different fluorophores (FAM, R6G, ROX, and Cy5) and able to detect up to four viral RNAs (DNAs) with high specificity in a single tube in real-time PCR were designed. Sensitivity and specificity of the method was estimated using cultural strains of 8 serotypes of EV, 5 serotypes of AV and 2 clinical specimens containing HAV. Sensitivity of the method for detection of polioviruses types 1, 2, and 3 (Sabin vaccine strains) was 0.5--1 TCID50 per reaction mixture. Thirty clinical specimens were analyzed by the multiplex PCR with and without IPC, and by mono-specific PCR. Comparison of these methods with cultural one revealed results agreement in 86.7% in case of multiplex PCR with IPC and in 100% in case of multiplex PCR without IPC and mono-specific PCR. This method can be used for rapid diagnostics of enteric viral infections as well as for determination of viral contamination level of water. As intermediate results of the study the methods for quantitative assessment of HAV, AV, and EV nucleic acids were developed which are convenient tools for the control of antiviral therapy effectiveness.

[siRNA-mediated inhibition of respiratory syncytial virus replication].

The effect of siRNA against respiratory syncytial virus (RSV) was investigated in the cultured cells. MA104 cells were inoculated by RSV and transfected by different variants of siRNA against RSV phosphoprotein (P) mRNA or non-specific siRNA as a control. The inhibition of RSV was assessed by microscopically studying the cells, titrating the virus, and estimating viral RNA quantity in the culture supernatants by real-time polymerase chain reaction (PCR). The most potent variants of siRNA caused an up to 8-day delay of microscopically detectable syncytium generation to 8 days, an up to 280-fold decrease in viral titers, and an up to 40-fold reduction in viral RNA quantity in the supernatants, as compared to the controls (p < 0.001). RSV mRNA is a suitable target for siRNA-mediated RSV replication inhibition, promising an advance in the treatment of RSV infection.

[Rhinovirus diseases: pathogenesis, diagnostics and treatment].

Contemporary data on human rhinovirus diseases and their pathogenesis are presented. Special attention is paid to complications which may be caused by rhinovirus infections in allergy-susceptible patients. Furthermore, approaches to the diagnostics and treatment of rhinovirus diseases are described. In particular, the advantages of molecular methods for the diagnostics of rhinovirus infection (based on the PCR) in comparison with cultural and immunochemical methods are pointed out. New investigations aimed at the development of specific antirhinovirus preparations--capsid-binding (Pleconaril), blocking the binding of the virus with cell receptors (ICAM, soluble) and inhibiting rhinovirus protease 3C--have been considered.