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Being diverse and widely distributed globally, bats are a known reservoir of a series of emerging zoonotic viruses. We studied fecal viromes of twenty-six bats captured in 2015 in the Moscow Region and found 13 of 26 (50%) samples to be coronavirus positive. Of P. nathusii (the Nathusius' pipistrelle), 3 of 6 samples were carriers of a novel MERS-related betacoronavirus. We sequenced and assembled the complete genome of this betacoronavirus and named it MOW-BatCoV strain 15-22. Whole genome phylogenetic analysis suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV. Unexpectedly, the phylogenetic analysis of the novel MOW-BatCoV/15-22 spike gene showed the closest similarity to CoVs from Erinaceus europaeus (European hedgehog). We suppose MOW-BatCoV could have arisen as a result of recombination between ancestral viruses of bats and hedgehogs. Molecular docking analysis of MOW-BatCoV/15-22 spike glycoprotein binding to DPP4 receptors of different mammals predicted the highest binding ability with DPP4 of the Myotis brandtii bat (docking score -320.15) and the E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets and are commonly found in areas of human habitation. As this novel bat-CoV is likely capable of infecting hedgehogs, we suggest hedgehogs can act as intermediate hosts between bats and humans for other bat-CoVs.

The Bunyamwera serological group includes a number of geographically widespread viruses that are related but not identical and have serological cross-reactivity. As the first group members were obtained in the pre-sequencing era, their classifications (group attribution, species differentiation) were originally based on serological reactions. At the same time, the accuracy of the typing in each case depended on the variety of viruses that the researcher had as a comparison panel. With the advent of sequencing techniques, it has become customary to use identity thresholds (nucleotide or amino acid composition) as demarcation criteria for the interspecific differentiation of viral species. Identity thresholds are determined by the International Committee on Taxonomy of Viruses (ICTV) and are regularly reviewed. Similar criteria were established for the Orthobunyavirus genus, which includes members of the Bunyamwera serological group. On the basis of these criteria, the species attributions of some members of the serological group need to be clarified. For this purpose, we analyzed sequences (available in NCBI GenBank) of viruses belonging to the Bunyamwera serological group in order to clarify their phylogenetic positions on the basis of the current demarcation criteria established by the ICTV.

Acute febrile illnesses occur frequently in Guinea. Acute fever itself is not a unique, hallmark indication (pathognomonic sign) of any one illness or disease. In the infectious disease context, fever's underlying cause can be a wide range of viral or bacterial pathogens, including the Ebola virus. In this study, molecular and serological methods were used to analyze samples from patients hospitalized with acute febrile illness in various regions of Guinea. This analysis was undertaken with the goal of accomplishing differential diagnosis (determination of causative pathogen) in such cases. As a result, a number of pathogens, both viral and bacterial, were identified in Guinea as causative agents behind acute febrile illness. In approximately 60% of the studied samples, however, a definitive determination could not be made.

Introduction: Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity., Material and Methods: L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice., Results: A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with β-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization., Discussion: It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation., Conclusion: The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.

Coronavirus disease 2019 (COVID-19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay (COVID-19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection with an armored positive control and internal controls constructed from synthetic MS2-phage-based RNA particles. The COVID-19 Amp assay limit of detection was 10 3  copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID-19 Amp and World Health Organization (WHO)-based assay. Discordance in nine samples was observed (negative by the WHO-based assay) and discordant samples were retested as positive according to the results obtained from the Vector-PCRrv-2019-nCoV-RG assay. The developed COVID-19 Amp assay has high sensitivity and specificity, includes virus particles-based controls, provides the direct definition of the SARS-CoV-2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT-qPCR., (© 2020 Wiley Periodicals LLC.)

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae. Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

This chapter reports a library preparation protocol for efficient high-throughput sequencing of double-stranded RNA viruses. The protocol consists of four main steps, viz., enzyme treatment, precipitation using lithium chloride, full-length amplification of cDNAs, and tailing adapters for high-throughput sequencing. This protocol will be useful for all double-stranded RNA viruses and for all of the high-throughput sequencing platforms.

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 10 3 copies/ml to 10 5 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Paramushir virus belongs to Sakhalin virus genogroup within Orthonairovirus genus and is one of the poorly studied viruses with unknown pathogenicity. At the moment, only one nearly complete sequence of Paramushir virus genome, isolated in 1972, is available. Two new strains of PARV were isolated in 2015 from a sample collected at the Tyuleniy Island in the Okhotsk Sea and sequenced using a combination of high throughput sequencing and specific multiplex PCR. Both strains are closely related to the early sequenced PARV strain LEIV-1149 K. The signs of intersegment reassortment and probable recombination were revealed, which point to a high variability potential of Paramushir virus and may lead to the formation of strains with novel properties, different from those of the predecessors. The new data regarding Paramushir virus can promote a better understanding of the diversity and relations within Orthonairovirus genus and help define intragenic demarcation criteria, which have not yet been established., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Filoviruses are important etiological agents of emergent diseases with high mortality rates. Traditionally, filovirus fever diseases have primarily been a burden of African countries; however, global interconnectedness has increased the probability of the worldwide spread of filoviruses. Therefore, national healthcare organizations need tools for managing filovirus risk, including diagnostic kits based on real-time reverse transcription PCR (RT-qPCR), as this is the most suitable method for diagnosing filovirus fever diseases. Here we describe a real-time RT-qPCR assay for filovirus detection. This assay is a further development of our previously reported EBOV (Zaire)-Fl kit. Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-qPCR assays for the detection of filoviruses were developed and evaluated using armored RNA phage particles (ARs) as positive controls. The limit of detection of the assay was 5x10 2 copies/ml of the AR-positive control for the FiloA-Fl set and 5x10 3 copies/ml of the AR-positive control for the FiloB-Fl set. Our assay provides a rapid and sensitive tool for detecting filoviruses. The high specificity and sensitivity of the assay make it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases and their etiological agents.

Rabies virus is endemic to Russia, among other countries. It is therefore critical to develop a high-quality and high-precision diagnostic procedure for the control and prevention of infection. The main objective of the research presented here was to develop a reliable RT-qPCR assay for rabies diagnostics. For this purpose, a RABV strains from various biological and geographical origins were used. In addition, rabies-positive and rabies-negative samples, as well as nucleic acids from other viruses and DNA extracted from the brain tissues of mice, dogs, cats, bats and humans, were studied using the developed assay. The analytical sensitivity of the assay, as assessed using armored recombinant positive control dilutions, was 10 3 copies/ml, and the sensitivity measured using characterized strains was between 0.1 LD50/ml and 1.0 LD50/ml. A broad range of RNA from RABV strains circulating in different regions of Russia, as well as RNA from RABV-positive primary brain samples from 81 animals and two humans, was detected using the developed assay. No false-positive or false-negative results were obtained. Given that high analytical and diagnostic sensitivities and a high specificity were verified for this assay, it has high potential as a screening test that may be suitable for the epizootiological monitoring of animals and for the fast postmortem diagnosis of rabies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This article presents the results of a comprehensive survey of the burden of tick-borne infectious diseases (TBIDs) in the Altai region of Russia. Official data for TBID incidence were analyzed and 201 samples from patients with suspected TBID were studied. Furthermore, questing ticks and ticks recovered from humans were examined to estimate prevalence of TBID-causative agents. The Altai region was determined to have a heightened risk for TBIDs in Russia. The most epidemiologically significant tick-borne illness in this area is spotted fever group rickettsiosis, while nationally in Russia, the leading TBID is Lyme borreliosis. The prevalence of mixed infection was 12.4% among the studied cases. Additionally, the prevalence of poorly studied pathogens - Kemerovo virus (KEMV) and Rickettsia tarasevichiae - in ticks from the Altai region was determined., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Human adenovirus 7 (hAdv7) 19BOVLB/Volgograd/Rus/2014 was isolated from the autopsy material from an adult with fatal pneumonia in Volgograd, Russia, in March 2014. Whole-genome sequencing of the virus isolate was performed., (Copyright © 2016 Yatsyshina et al.)

Background: Rabies is prevalent in 150 countries and is definitely found in Russian Federation. The average registered incidence of rabies infection among animals in Russia is 3000 cases per year. At least 500,000 cases of animal bites and scratches are registered in the Russia every year, but only 2-4 cases of rabies infection in humans are reported per year. This relatively low incidence of rabies infection among humans is the result of a well-organized program of rabies surveillance and control as well as the readily available vaccination and immunoglobulin therapies. However, physician awareness of rabies infection in patients with acute encephalopathy is low, and some cases of rabies remain undiagnosed., Objectives: A retrospective study of autopsy materials from patients who died of encephalitis of unknown etiology in the Astrakhan region of Russia in 2003., Study Design: A broad-range polymerase chain reaction (PCR) analysis followed by high throughput sequencing were used for the diagnosis., Results: Two cases of rabies were detected and subsequently confirmed using a fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA) and a mouse inoculation test. Two strains of rabies virus were isolated and characterized using virological methods. The entire genome of each strain was sequenced., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In early February 2014, an outbreak of the Ebola virus disease caused by Zaire ebolavirus (EBOV) occurred in Guinea; cases were also recorded in other West African countries with a combined population of approximately 25 million. A rapid, sensitive and inexpensive method for detecting EBOV is needed to effectively control such outbreak. Here, we report a real-time reverse-transcription PCR assay for Z. ebolavirus detection used by the Specialized Anti-epidemic Team of the Russian Federation during the Ebola virus disease prevention mission in the Republic of Guinea. The analytical sensitivity of the assay is 5 × 10(2) viral particles per ml, and high specificity is demonstrated using representative sampling of viral, bacterial and human nucleic acids. This assay can be applied successfully for detecting the West African strains of Z. ebolavirus as well as on strains isolated in the Democratic Republic of the Congo in 2014., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.

The study of 146 patents with liver cirrhosis of different classes using clinical, endoscopic, morphological and immunohistochemical methods revealed characteristic colonic changes. The destructive and atrophic changes in colonic mucosa were associated with increased morphometric parameters of serotonin- and chromogranin A-positive epithelyocytes. Capillary dilatation in colonic mucosa developed earlier than rectal varicose veins and may be an earlier sign of portal colonopathy.

Research Objective: to define a role of structural and functional changes of a colon, expression indicators of epithelial cells of mucous coat of colon that produce serotonin and hromogranin-A at in encephalopathy development at liver cirrhoses. We have examined 146 patients with liver cirrhosis of various classes using the clinical, endoscopic, morphologic and immunohistochemical methods. It is established that with increase of a class of a cirrhosis frequency of revealing of a hepatic encephalopathy, structural changes, degree dysbacteriosis of a colon. Progressing of a hepatic encephalopathy at liver cirrhoses associates with development destructive and atrophic changes of a mucous membrane of a colon and a dysbacteriosis. The increase in the morphometric rates of epithelial cells of mucous coat of colon that produce serotonin and hromogranin-A at liver cirrhoses plays a part in development of a manifesting of a hepatic encephalopathy.

We have found out the clinical presentations and peculiarities of endoscopic and morphologic view of pathologies of mucous membrane of gastroduodenal zone caused by liver cirrhosis. We have examined 74 patients with liver cirrhosis of viral and nonviral etiology using the clinical, endoscopic, morphologic and immunohistochemical methods.We have found that during liver cirrhosis morphometric rates of epithelial cells of mucous coat of stomach that produce somatostatin and endothelin-1 decrease and morphometric rates of epithelial cells that produce nitrogen oxide synthase increase. We have also found out that during liver cirrhosis proliferate activity decrease and apoptosis of epithelial cell of mucous coat of stomach increase.

Clinical presentation, endoscopic picture and morphological changes of gastric and duodenal mucosa were evaluated in 75 patients with chronic hepatitis of different (viral and non-viral) etiology. Endoscopic, morphological, and immunohistochemical studies revealed deterioration of morphometric characteristics of somatostatin-immunopositive epitheliocytes in gastric mucosa and their improvement in epitheliocytes producing endothelin-1 and nitric oxide synthase. These changes were associated with decreased proliferative activity and enhanced apoptosis of epitheliocytes in gastric mucosa.

The dynamics of T and B cell immunity in spleens from rat exposed to whole-body chronic irradiation with dose rates of 12.9 cGy/day (range 1-10 Gy) and 3.0 cGy/day (range 0.57-2.04 Gy) were investigated. gamma-irradiation with a dose-rate of 12.9 cGy/day was shown to produce a wave-like suppression of the T lymphocyte mitogenic response. Irradiation with a dose-rate of 3.0 cGy/day caused a decrease in immune response of T lymphocytes 48 days after onset of exposure (total dose 1.4 Gy). It was also shown that chronic irradiation with a dose-rate of 3.0 cGy/day produced significant changes in the DNA of T lymphocytes. Our results show that the radiation-induced suppression of immune functions and damage to DNA structure were partially eliminated when animals were fed a daily diet supplemented with a natural antioxidant, ubiquinone Q-9. The inhibiting effect of chronic irradiation was more pronounced in B lymphocytes because of their higher radiosensitivity.

The dynamics of T- and B-immunity state of spleens from rats, exposed to chronic gamma-radiation at a dose rate of 0.129 Gy per day was investigated. It was shown that during 77 days of irradiation (total absorbed dose 10 Gy) the level of T-lymphocyte mitogenic activation experiences wavy inhibition, which correlates with the rate of lymphopenia within the same period of exposure.