[Molecular epidemiological analysis of SARS-CoV-2 genovariants in Moscow and Moscow region].

INTRODUCTION: SARS-CoV-2, a severe acute respiratory illness virus that emerged in China in late 2019, continues to spread rapidly around the world, accumulating mutations and thus causing serious concern. Five virus variants of concern are currently known: Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1), Delta (lineage B.1.617.2), and Omicron (lineage B.1.1.529). In this study, we conducted a molecular epidemiological analysis of the most prevalent genovariants in Moscow and the region. The aim of the study is to estimate the distribution of various variants of SARS-CoV-2 in Moscow city and the Moscow Region. MATERIALS AND METHODS: 227 SARS-CoV-2 sequences were used for analysis. Isolation of the SARS-CoV-2 virus was performed on Vero E6 cell culture. Sequencing was performed by the Sanger method. Bioinformatic analysis was carried out using software packages: MAFFT, IQ-TREE v1.6.12, jModelTest 2.1.7, Nextstrain, Auspice v2.34. RESULTS: As a result of phylogenetic analysis, we have identified the main variants of the virus circulating in Russia that have been of concern throughout the existence of the pandemic, namely: variant B.1.1.7, which accounted for 30% (9/30), AY.122, which accounted for 16.7% (5/30), BA.1.1 with 20% (6/30) and B.1.1 with 33.3% (10/30). When examining Moscow samples for the presence of mutations in SARS-CoV-2 structural proteins of different genovariants, a significant percentage of the most common substitutions was recorded: S protein D614G (86.7%), P681H/R (63.3%), E protein T9I (20.0%); M protein I82T (30.0%), D3G (20.0%), Q19E (20.0%) and finally N protein R203K/M (90.0%), G204R/P (73.3 %). CONCLUSION: The study of the frequency and impact of mutations, as well as the analysis of the predominant variants of the virus are important for the development and improvement of vaccines for the prevention of COVID-19. Therefore, ongoing molecular epidemiological studies are needed, as these data provide important information about changes in the genome of circulating SARS-CoV-2 variants.

[Antiviral properties of synthetic histidine derivatives containing membranotropic volumetrical carbocycles in their molecule against SARS-CoV-2 virus in vitro].

INTRODUCTION: Currently, low molecular-weight compounds are being developed as potential inhibitors of CoVs replication, targeting various stages of the replication cycle, such as major protease inhibitors and nucleoside analogs. Viroporins can be alternative protein targets. The aim of this study is to identify antiviral properties of histidine derivatives with cage substituents in relation to pandemic strain SARS-CoV-2 in vitro. MATERIALS AND METHODS: Combination of histidine with aminoadamantane and boron cluster anion [B10H10]2 (compounds IIV) was carried out by classical peptide synthesis. Compound were identified by modern physicochemical methods. Antiviral properties were studied in vitro on a monolayer of Vero E6 cells infected with SARS-CoV-2 (alpha strain) with simultaneous administration of compounds and virus. RESULTS: Derivatives of amino acid histidine with carbocycles and boron cluster were synthesized and their antiviral activity against SARS-CoV-2 was studied in vitro. Histidine derivatives with carbocycles and [B10H10]2 have the ability to suppress virus replication. The solubility of substances in aqueous media can be increased due to formation of hydrochloride or sodium salt. DISCUSSION: 2HCl*H-His-Rim (I) showed some effect of suppressing replication of SARS-CoV-2 at a viral load of 100 doses and concentration 31.2 g/ml. This is explained by the weakly basic properties of compound I. CONCLUSION: The presented synthetic compounds showed moderate antiviral activity against SARS-CoV-2. The obtained compounds can be used as model structures for creating new direct-acting drugs against modern strains of coronaviruses.

In Vitro Antiviral Activity of a New Indol-3-carboxylic Acid Derivative Against SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012-2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 µM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 106 TCID50/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility - high activity (IC50 = 1.06 µg/mL) and high selectivity index (SI = 78.6) - this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

[High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera].

The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNt and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.

High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera.

The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNT and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.

[Characteristics of imported cases of Dengue fever and hemorrhagic Dengue fever in 2009-2019].

INTRODUCTION: In Russia, the approved morbidity statistics system is represented by the International Classification of Diseases of the 10th revision (ICD-10). This classification provides two forms of dengue fever (DF): dengue fever (A90) and hemorrhagic dengue (A91). Official statistics on the ratio of forms of DF is not published in open sources and this lack of information about the real ratio of the forms of DF makes it difficult to objectively assess the factors that determine the severity of this disease. THE AIM: compare the clinical and epidemiological features of dengue fever and hemorrhagic dengue fever in patients hospitalized in 2009-2019 to the City Infectious Clinical Hospital No. 1, Moscow. MATERIALS AND METHODS: A retrospective cohort study. We analyzed the patient database and reviewed 391 medical records of patients with diagnosed dengue fever. We compared gender, age characteristics, travel geography including information about previous visits of patients to endemic regions and dengue virus serotype. To determine the primary and re-infection rate, an analysis of IgG for the dengue virus was carried out on days 1-5 of the disease. To compare indicators, 95% confidence intervals for proportions, medians, and interquartile ranges were calculated. The significance of differences between independent samples for assessing qualitative characteristics was carried out using the criteria χ2, the odds ratio. To assess the quantitative characteristics, the Mann-Whitney test was used. Differences were considered statistically significant at p ≤ 0.05. RESULTS: The proportion of patients with dengue fever was 14.9% of all hospitalized with febrile illnesses that developed after international travel. Hemorrhagic dengue fever (DHF) was diagnosed in 15.7% of patients with dengue fever. DHF developed significantly more often in women, as well as in those who had history of repeated visits to endemic regions. However, DHF was also diagnosed in 10.9% of first-time travelers to tropical countries. We did not find significant differences in the rates of DHF development depending on age and dengue virus serotype. In a number of patients who had not previously traveled to endemic regions, IgG to the dengue virus were detected, which may indicate a previous infection with related flaviviruses. CONCLUSION: It has been established that in the regions most visited by Russians, there is a circulation of all serotypes of the dengue virus with an annual change in the predominant serotype.

[L-Lysine-α-Oxidase in vitro Activity in Experiments on Models of Viruses Sindbis, Forest-Spring Encephalitis, Western Nile, Tyaginya and Dhori].

The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses.

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

AIM: Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. MATERIALS AND METHODS: During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. RESULTS: A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. CONCLUSION: Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

[Use of RT-PCR and enzyme immunoassays for the specific diagnosis of Crimean hemorrhagic fever].

The objective of the investigation was to evaluate the efficiency of the RT-PCR kit "AmplySens CHF" produced by InterlabService of the Central Research Institute of Epidemiology and that of the ELISA kits made by the D. I. Ivanovsky Research Institute of Virology for the specific diagnosis of Crimean hemorrhagic fever (CHF). Examination of sera from CHF patients from the Astrakhan Region showed that positive RT-PCR results were observed in 95.2 and 37.5% on days 4-8 and 9-13 after disease onset, respectively; but they were absent on days 13-17. Positive ELISA-IgM results were found in 93% on disease days 6 to 16. A high percentage (78.9%) of positive IgG samples was seen only on days 9-16. Thus, RT-PCR has a marked efficiency in diagnosing CHF until day 8 of illness while ELISA-IgM has it on day 8 or later. ELISA-IgG can be considered to be a confirming rather than compulsory test. The findings suggest that the RT-PCR kit "AmplySens CHF" produced by InterlabService of the Central Research Institute of Epidemiology and that of the ELISA kits made by the D. I. Ivanovsky Research Institute of Virology have a pronounced sensitivity and specificity and a high efficiency when concurrently used to verify CHF in patients.

[A case of Crimean-Congo hemorrhagic fever in the Anapa District, Krasnodar Territory].

The paper gives information on the first case of Crimean-Congo hemorrhagic fever detected in a female resident of the Anapa District, Krasnodar Territory, in 2005 in the past 57 years.

[PHOSPHAN microplate technology-based microarray for the determination of IgG antibodies against West Nile and Crimean-Congo hemorrhagic fever viruses].

The paper demonstrates it possible to work out a phosphorescence analysis (PHOSPHAN) microplate technology-based microarray for concurrently examining human sera and detection of their specific IgG antibodies against two heterological West Nile and Crimean-Congo hemorrhagic fever viruses. The sensitivity and specificity of the microarray were comparable with those of enzyme immunoassay with separate sample testing. The advantages of PHOSPHAN were associated with the microplate format of an immunoassay and its enhanced multiplexity, which may contribute to the lower cost of clinical sample testing.

[Results of examination of healthy residents in the southern region of Russia and patients feverish to pappataci fever virus antibodies].

Sera from patients with acute seasonal fevers and apparently healthy individuals living in the Astrakhan Region, Krasnodar Territory, or Rostov Region were examined in two modifications of enzyme immunoassay for detection of IgM and IgG antibodies to Neapolitan and Sicilian pappataci fever viruses. IgM antibodies to Sicilian pappataci fever virus were detected in a patient from the Volodarsky District, Astrakhan Region, who had been admitted for the unverified diagnosis of Q fever. A donor residing in the Novorossiysk District, Krasnodar Territory, was found to have IgA antibodies to Neapolitan pappataci fever virus. The findings show it expedient to conduct further investigations of the serodiagnosis and seroepidemiology of pappataci fevers in the southern Russian region where mosquitoes of the genus Phlebotomus inhabit.

[In vitro activity of Russian ribavirin on the models of Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses].

The high activity of ribavirin made by effective biotechnology in Russia was established in in vitro experiments using the models Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses, which suggests that it is promising in using the drug in the treatment of infection with these viruses.

[ELISA and PCR test systems used to detect Crimean-Congo hemorrhagic fever virus].

Studying the sensitivity and specificity of enzyme-linked immunosorbent assay (ELISA) for the indication of Crimean-Congo hemorrhagic fever (CCHF) virus antigens and those of reverse transcription and polymerase chain reaction (RT-PCR) for the detection of CCHF virus RNA, and those of a intercerebral infection method in newborn albino mice systems for the determination of viral infectious activity established that the sensitivity of ELISA was 1-2 orders of magnitude less than that of RP-PCR. The latter proved to be better in studying the sera sampled from patients with CCHF. The results of studying the samples of H. marginatum ticks, the CCHF virus vectors by ELISA and RT-PCR were similar.

[Morphological and immunohistochemical changes in tissues of the lung, myocardium and kidney in experimental West Nile fever].

An experimental infection of mice with West Nile virus (WNV) showed pronounced dystrophic changes in tissues of the kidneys and myocardium as well as expression of WNV antigens in cells of the lungs, kidneys and myocardium, which can denote tropism of WNV to tissues of the lungs, kidneys and myocardium.

[Hepatotropic action of the virus of West Nile fever].

An experimental infection of mice with West Nile Virus (WNFV) showed pronounced dystrophic changes in the hepatic parenchyma and expression of WNV antigens in the endothelium of hepatic capillaries and in hepatocyte cytoplasm, which testifies to the tropic action of WNFV to hepatic tissue.

[ West Nile fever among residents of the Astrakhan region in 2002]. / Zabolevaemost' i zarazhaemost' naseleniia Astrakhanskoi oblasti likhoradkoi Zapadnogo Nila v 2002 g.

Thirty-three persons infected with West Nile fever were detected in 2002 in the Astrakhan Region; the diagnosis was confirmed serologically and the maximal number of the infected was registered in August, same year. The indices of the specific humoral immunity varied from 3.3% to 27.1%. A monitoring determined the highest infection risk among the residents of the Volga middle delta.

Morphofunctional characteristics of antigen-presenting cells in lymph node in mice with experimental West Nile fever.

Pronounced transformation of cells in mesenteric lymph nodes, mainly in the thymus-independent zone and sinuses, was detected in albino mice experimentally infected with West Nile fever (strain 986). Maximum antigen-presenting activity was exhibited by activated macrophages, minimum activity--by dendritic cells of lymphoid follicles.

[The isolation of Dhori viruses (Orthomyxoviridae, Thogotovirus) and Crimean-Congo hemorrhagic fever virus (Bunyaviridae, Nairovirus) from the hare (Lepus europaeus) and its ticks Hyalomma marginatum in the middle zone of the Volga delta, Astrakhan region, 2001]. / Isoliatsiia virusov Dkhori (Orthomyxoviridae, Thogotovirus) i Krymskoi-Kongo gemorragicheskoi likhoradki (Bunyaviridae, Nairovirus) ot zaitsa (Lepus europaeus) i sobrannykh s nego kleshei Hyalomma marginatum v srednei sone del'ty Volgi, Astrakhanskaia oblast', 2001 g.

In August, 2001, in the middle zone of the delta of the Volga River, the Astrakhan region, during investigation of the natural foci of West Nile fever and Crimean--Congo hemorrhagic fever (CCHF), the material from the hare (Lepus europaeus, Pallas, 1778 (Lagomorpha, Leporidae) and collected from it the ticks Hyalomna marginatum Koch 1844, was obtained. 4 strains of Dhori virus (Orthomyxoviridae, Thogotovirus) and 2 strains of CCHF virus (Bunyaviridae, Nairovirus) were isolated. This is the first isolation of Thogotovirus genus virus from the wild vertebrates. Considering the overlap of the Dhori virus and CCHF virus areas, similar ecology and the isolation both viruses from the same pool of the ticks, the necessity for the use of the test-system for indication of the viruses, differential diagnosis and accumulation of the data concerning the role of Dhori virus in the human and farm animals pathology is discussed.

[Analysis of new variants of West Nile fever virus]. / Analiz novykh variantov virusa likhoradki Zapadnogo Nila.

The complete nucleotide sequences for 6 strains of the West Nile fever virus were determined. For the first time the complete nucleotide sequences of the Indian isolate and Krsn190 strain, that is the most far phylogenetically from all isolates known at present time were established. The scheme for separation of virus variants into 4 groups and criteria for determination the group to which the isolate belongs are suggested.