[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.

Phylogenetic reconstruction of the initial stages of the spread of the SARS-CoV-2 virus in the Eurasian and American continents by analyzing genomic data.

Samples from complete genomes of SARS-CoV-2 isolated during the first wave (December 2019-July 2020) of the global COVID-19 pandemic from 21 countries (Asia, Europe, Middle East and America) around the world, were analyzed using the phylogenetic method with molecular clock dating. Results showed that the first cases of COVID-19 in the human population appeared in the period between July and November 2019 in China. The spread of the virus into other countries of the world began in the autumn of 2019. In mid-February 2020, the virus appeared in all the countries we analyzed. During this time, the global population of SARS-CoV-2 was characterized by low levels of the genetic polymorphism, making it difficult to accurately assess the pathways of infection. The rate of evolution of the coding region of the SARS-CoV-2 genome equal to 7.3 × 10-4 (5.95 × 10-4-8.68 × 10-4) nucleotide substitutions per site per year is comparable to those of other human RNA viruses (Measles morbillivirus, Rubella virus, Enterovirus C). SARS-CoV-2 was separated from its known close relative, the bat coronavirus RaTG13 of the genus Betacoronavirus, approximately 15-43 years ago (the end of the 20th century).

[History of the discovery and study of tick-borne encephalitis in Russia: three Far Eastern expeditions (1937-1939)].

The review provides a brief historical outline of the discovery and study of tick-borne encephalitis (TBE) during three expeditions to the Far East (19371939). As a result of the Far Eastern expeditions, the TBE virus was discovered, numerous strains were isolated, a vector-borne transmission pathway was established, the main features of epidemiology, clinic and pathomorphology of the disease were described, serotherapy was tested, first inactivated "brain" vaccine against TBE was developed and its effectiveness was proved. The history of the discovery and study of TBE is marked by the heroism of researchers and tragic events illness of some participants, death during the development of the first vaccine, arrest of L.A. Zilber and two performers.

Genetic diversity and geographical distribution of the Siberian subtype of the tick-borne encephalitis virus.

The tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is currently subdivided into three main subtypes-the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberian (TBEV-Sib). The TBEV-Sib is the most common subtype and found in all regions where TBEV was detected, except for Central and Western Europe. Currently, four genetic lineages have been described within TBEV-Sib. In this study, detailed analysis of TBEV-Sib genetic diversity, geographic distribution, phylogeography and divergence time of different TBEV-Sib genetic lineages based on E gene fragments, complete genome sequences, and all currently available data in the GenBank database was performed. As a result, a novel Bosnia lineage within the TBEV-Sib was identified. It was demonstrated that the Zausaev lineage is the most widely distributed among the TBEV-Sib lineages, and was detected in all studied regions except the Far East. The Vasilchenko lineage was found from Western Siberia to the Far East. The Baltic lineage is presented from Europe to Western Siberia. The Obskaya lineage was found only in Western Siberia. TBEV strains from a newly described Bosnia lineage were detected in Bosnia, the Crimean peninsula, Kyrgyzstan and Kazakhstan. The greatest divergence of the TBEV-Sib genetic variants was observed in Western Siberia. Within the TBEV-Sib, the Obskaya lineage diverged from the common ancestor the earliest, after that the Bosnia lineage was separated, then the Baltic lineage, and the Zausaev and Vasilchenko lineages diverged most recently.

DEFINITION AND COMPARATIVE ANALYSIS OF THE GENOMIC STRUCTURE OF SIBERIAN STRAINS OF TICK-BORNE ENCEPHALITIS VIRUS OF THE EUROPEAN SUBTYPE.

Tick-borne encephalitis virus (TBEV) is classified into three subtypes: Far Eastern (TBEV-FE), European (TBEV-EU) and Siberian (TBEV-SIB). In Russia, these are also called genotypes 1, 2 and 3, respectively. Geographically, TBEV-EU dominates in Central and Northern Europe, but its representatives are also found to the east - along the southern part of the forest zone of extratropical Eurasia - up to Eastern Siberia and South Korea. However, the strains isolated outside Europe remain poorly investigated. In the proposed study, eight full genomes of the Siberian isolates of TBEV-EU were determined and 13 complete genomes were compared. The analysis of 152 full-genome TBEV sequences showed that the TBEV-EU has a higher degree of stability of the genome-coding region in the entire Eurasian area (3.1% of differences) compared to TBEV-FE (6.6%) and TBEV-SIB (7.8%). At the same time, the maximum differences are observed not between European and Siberian strains, as one could expect, but between the representatives from Europe - TBEV strains Mandl-2009 from Norway and Hypr from the Czech Republic. The studied strains from Siberia form the compact genetic cluster of 42 TBEV-EU strains and are divided into two subclusters - West Siberian and East Siberian variants. These variants differ in the combinations of amino acid substitutions in all proteins except NS2B. The West Siberian variant mostly circulates in the territory of Altai, and the closest relative of its representatives is Absettarov strain from the European part of Russia. The strains similar to the East Siberian variant of the European subtype were recorded in the Altai (strain 84.2, 2007) and in Belarus (N256, about 1940).

A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties.

This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine.

Comparative analysis of complete genome sequences of European subtype tick-borne encephalitis virus strains isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the Asian part of Russia.

Tick-borne encephalitis virus (TBEV) is divided into three subtypes: European (TBEV-Eu), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE) subtypes. The geographical range of TBEV-Eu dominates in Europe, but this subtype is present focally across the whole non-tropical forested Eurasian belt, through Russia to South Korea. However, the TBEV-Eu strains isolated outside Europe remain poorly characterized. In this study, full-genome sequences of eight TBEV-Eu isolates were determined. These strains were isolated from Ixodes persulcatus ticks, long-tailed ground squirrel (Spermophilus undulatus), and human blood in the natural foci of Western and Eastern Siberia, Russia. A phylogenetic analysis of all available TBEV-Eu genomic sequences revealed that strains from Siberia were closely related to other strains from Europe and South Korea. The closest relation was identified between the Siberian strains and strains from Zmeinogorsk (Western Siberia, Russia) and strain Absettarov (Karelia, Russia), and were most divergent from strains from the Czech Republic and Norway. TBEV-Eu strains isolated in Eastern Siberia were more closely related phylogenetically to strains from South Korea, but strains from Western Siberia grouped together with the strains from Europe, suggesting two genetic TBEV-Eu lineages present in Siberia.

[Detection of potential sites of recombination in the Tick-borne encephalitis virus by the methods of comparative genomics].

The results of the bioinformatic search for the potential sites of the recombination in the genome-wide structures of the tick-borne encephalitis virus (TBEV) through a series of software techniques were presented in this work. The genomes of the 55 TBEV strains were assayed, 21 of them showed the presence of the recombination sites. Recombinant strains belonged to the Far Eastern (19 strains) and European (2 strains) genotypes. 22 sites of the recombination attributed were identified to five types based on position, strain, and regional characteristics. The parental strains were identified based on the genotypic and geographical parameters, which do not contradict the possibility of the formation of the recombinants. Nearly two-thirds of the sites are located in the regions of NS4a and NS4b genes, which are the "hot spots" of the recombination, most of them being concentrated in the gene NS4. It was shown that the recombination processes did not occur at the level of the genotypes (European genotype) or certain groups within the genotype (Far East) and were typical of the peripheral populations.

Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia.

The Altai Republic is a highly endemic area as far as tick-borne encephalitis (TBE) is concerned. The aim of the research was to study the effect of altitude on the risk of tick-borne encephalitis infection in the Altai Republic. The paper analyzes the following data: the study of ixodid ticks collected from the vegetation in 116 sites at the 200-2383m elevation above sea level in 2012-2014, TBE virus prevalence of these vectors, tick-bite incidence rate, and TBE incidence rate of the population. Species identification of 4503 specimens has shown that the most common species are Dermacentor nuttalli (45.3%), Ixodes persulcatus (33.1%), Dermacentor silvarum (9.4%), Dermacentor reticulatus (8.9%), and Haemaphysalis concinna (5.0%). A total of 2997 adult ixodid ticks were studied for the presence of the TBE virus; 2163 samples were examined by ELISA, while 834 specimens were tested by PCR. The TBE virus prevalence of Dermacentor spp. ticks in both reactions was significantly higher than of Ixodes persulcatus ticks (p<0.001). The work shows that the altitude is an important factor in the development of the epidemiological situation of tick-borne encephalitis: the higher the elevation of the area above sea level, the smaller the range of vectors. There is also a change of a leading species: in middle altitude (800-1700m above sea level) the virus is transmitted by ticks of D. nuttalli along with I. persulcatus, and in high mountains (above 1700m above sea level) D. nuttalli becomes an absolute dominant species. However, these species of ticks are less effective vectors than I. persulcatus. With the increase of altitude the tick-bite incidence rate decreases (r=-0.78, p<0.05), and TBE incidence also reduces (r=-0.67, p<0.05).

[Genotypes 4 and 5 of the tick-borne encephalitis virus: features of the genome structure and possible scenario for its formation].

On the basis of the comparison of complete genome structures of 32 strains and gene E fragments (160 ndt) of 643 strains and RNA isolates of tick-borne encephalitis (TBE) virus, we confirmed our previously expressed assumption (Zlobin V.I. et al, 2001) of existence, along with the three major genotypes, of genotypes 4 (strain 178-79) and 5 (strain 886-84). "Mosaic" structure of the polyprotein in the two strains was established. It manifests itself in particular in the sequences of 14 positions (C-3, E-206, NS1-54, NS-285, NS2A-100, NS2A-127, NS2A-174, NS2A-175, NS2A-225, NS3-376, NS4B-28, NS4B-96, NS5-18, NS5-671) containing the amino acids strictly conserved for each of the three major genotypes and is consistent with a uniform pattern of distribution of nucleotide substitutions that are specific for genotypes 1, 2 and 3. Possible scenario of the origin of TBE genotypes 4 and 5 was suggested.

[Detection of recombinations in tick-borne encephalitis virus by using computer analysis of viral genomes].

Computer programs were used to search for tick-borne encephalitis (TBE) virus recombinants among the isolates whose complete nucleotide sequences are deposited in the GenBank database. The application of RDP, Chimaera, Maximum chi2, and TOPAL programs has revealed recombinant sites in a number of sequences, which indicates that TBE virus has recombinations and that the programs are suitable for their detection.

[Detection of tick-borne encephalitis virus in Ixodes ticks collected in a natural focus of Gornyi Altai].

Enzyme immunoassay of tick-borne encephalitis virus (TBE) in the samples of Ixodes ticks collected in the outskirts of the settlement of Manzherok, Maiminsk District, Republic of Altai, revealed TBE antigen in 16.9 +/- 1.9% of the talga ticks. Real-time reverse transcription-polymerase chain reaction (RT-PCR) with specific fluorescent probes and phylogenetic analysis of the nucleotide sequences of RT-RCR products corresponding to 5'-terminal fragment of the E gene of TBE, all the virus strains isolated from the ticks collected in Gomyi Altai were referred to as the Siberian genetic type that was dominant in virus-carrying ticks in the majority of endemic areas of Russia and near abroad. Viral load assays using the real-time RT-PCR with the probes indicated the threshold cycles Ct = 25.34-28.98, which, with regard to the efficiency of RNA identification and reverse transcription, was equal to about 10(4)-10(5) viral RNA copies per tick.

[Genetic characteristics of the causative agent of tick-borne encephalitis in Mongolia].

A patient with diagnosed meningoencephalitis and a history of tick bite died in Mongolia in 2008. The purpose of this paper is to characterize the virus causing the ill person's death. The virus was identified using the phylogenetic analysis of the 520-bp fragment of the tick-borne encephalitis virus (TBEV) genome, which codes the fragment of TBEV protein E between 52-223 amino acids. TBEV RNA was detected in the samples of medulla oblongata, cerebral cortex, and pia mater of brain, but not in the cerebellar tissue. The study virus fragment was genetically closest to the representatives of the Far East subtype. Its closest relative was virus 740-84 (GenBank EU878282) isolated from large-toothed redback voles (Clethrionomys glareolus) in Buryatia and greatly differed from the Far East virus Soffin. Two amino acid substitutions (H86R and VI7A) were detected within the study protein E fragment. The paper is the first to describe the causative agent of tick-borne encephalitis on the territory of Mongolia and to discuss the evolution and pathogenicity of TBEV.

Genotyping and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes.

A panel of deoxyoligonucleotide probes for studying the genetic variability and genotyping of Tick-borne encephalitis virus (TBEV) strains by molecular hybridization of nucleic acids (MHNA) was created. This panel allows to estimate the genetic structure of individual TBEV strains, as the targets for probes are both variable and genotype (subtype)-specific sequences of all TBEV genes. With the help of this panel using the method of molecular hybridization of nucleic acids 268 archived TBEV isolates were investigated and the distribution of its genotypes and subgenotypes of genotype 3 was made more precise in the territory of Eurasia. The conclusion made earlier has confirmed that five genotypes of TBEV co-circulate in Eastern Siberia. It is generally recognized that the Far Eastern (TBE-FE), European (TBE-Eu), and Siberian (TBE-Sib) genotypes are widespread and epidemiologically important. The fourth genotype is presented by only one isolate, TBE178-79, originated from Irkutsk region, Russia. The fifth genotype includes 10 isolates, 1 of them, TBE886-84, was found earlier and recognized as unique [Zlobin et al. (2001b): Vopr Virusol 1:12-16 (Russian)].

[RT-PCR detection of deformed wing virus in the honey bee Apis mellifera L. in the Moscow Region].

Deformed wing virus (DWV) was first detected in the honey bee Apis mellifera by reverse transcriptase-polymerase chain reaction (RT-PCT) in the Moscow Region. Molecular phylogenetic analysis of the detected nucleotide sequence of the virus fragment VP2-VP1 of DWV demonstrated that the Russian virus sequence is united in the common cluster with all earlier revealed nucleotide sequences of DWV in the Genbank worldwide, which confirms the previous conclusions that this virus has recently distributed in the honey bee by Varroa destructor mite. It has been shown that the level of homology for all DWV nucleotide sequences is 98%, except for nucleoside sequence of 7D isolate from Turkey (96% homology), 96% homology with Kakugo virus and 84-86% homology with Varroa destructor virus 1; there is a preponderance of insignificant nucleotide substitutions, mainly transitions, which supports the evolutionary propinquity of 3 viruses.

[Human bocavirus].

Human bocavirus (HBoV) is a newly identified parvovirus associated with acute respiratory infections in young children in different parts of the world. It is not inconceivable that this virus is also capable of causing acute gastroenteritis and asymptomatically persisting in infected children. HBoV is the third widespread human respiratory virus after respiratory syncytial virus and rhinovirus. Polymerase chain reaction remains the most reliable of HBoV detection in clinical samples. Phylogenetic analysis shows the presence of at least 2 circulating variants (genotypes) of HBoV.

[The genetic variability and genotyping of tick-borne encephalitis virus with deoxyoligonucleotide probes].

A panel of genotype-specific molecular probes has been designed, which is used to indicate and differentiate tickborne encephalitis (TBE) virus. It assesses the individual genetic structure of each strain since the targets for the probes are the variable sequences of all 10 virus genes, which are specific for each of three genotypes. The molecular nucleic acid hybridization by means of the panel was used to study 273 TBE virus strains. Isolated from a Eurasian area; along with the representatives of three genotypes, the virus strains, the genomic structures of which do not fit in the established concept on three genotypes, circulate in Eastern Siberia.

[The enter of viruses family Picornaviridae in residents macrophages].

Viruses enter in cells through clathrin- and dinamin-mediated uptake route-endocytosis, caveolae-mediated local destruction of cell plasma membranes, and macropinocytosis. The non-enveloped viruses to which Picornaviridae famiy is attributed are important human and animal pathogens. The aim of this study was to examine the mechanisms of penetration of viruses of this family (polio-, echo 11-, entero 71- and coxsackie B1-viruses) into resident macrophages. After attachment to the plasma membrane of macrophages the enterovirus 71 and coxsackievirus B1 penetrated into macrophages by invagination of the plasma membrane and formation of intracytoplasmic vesicules - caveoles. The poliovirus entered macrophages both by caveols formation and local destruction of plasma membranes of the host cells. Macropinocytos of polioviruses was observed after 45 min contact. The echovirus 11 entered in host macrophages by local destruction of their plasma membranes during first 15 min. Then the formation of endocytosed vesicles with included viruses was observed. The echovirus 11 went out of endocytosed vesicles by local destruction of membrane vesicles.

[Molecular epidemiology of tick-borne encephalitis].

The review presents information on the development of studies into the molecular epidemiology of tick-borne encephalitis (TBE) in Russia and foreign countries. The existence of three major virus genotypes has been established by various techniques, such as genomic fragment sequencing, molecular hybridization using genotype-specific probes, and restriction fragment length polymorphism test. Each of the genotypes prevails in different parts of a natural habitat; the Ural-Siberian genotype (a Siberian subtype) is most commonly encountered. The genetic differences between the strains belonging to different genotypes are great and comparable with differences between some mammalian flaviviruses transmitted by ticks (viruses of a TBE complex). Further studies of the molecular epidemiology of TBE are of importance in understanding the evolution of the causative agent, improving the taxonomy and the classification of flavivuruses, and designing highly effective methods for the specific diagnosis, prevention, and treatment of the disease.