[Molecular and genetic characteristics of the multicomponent flavi-like Kindia tick virus (Flaviviridae) found in ixodes ticks on the territory of the Republic of Guinea].

INTRODUCTION: Ixodes ticks are vectors for pathogens of many infectious diseases. Recently, during the study of Rhipicephalus geigyi ticks collected from livestock in the Republic of Guinea, a new multicomponent flavi-like RNA virus, called Kindia tick virus (KITV), was discovered with an unusual mechanism for the implementation of genetic information. The aim of the work is to detect and study the genetic diversity of KITV in ixodes ticks collected in the territory of the Kindia province of the Republic of Guinea. MATERIAL AND METHODS: In 2021, 324 specimens of ticks of the species Amblyomma variegatum, Rh. geigyi, Rh. annulatus, Rh. decoloratus, Rh. senegalensis were collected from cattle. The detection of viral RNA was carried out in individual samples of ticks by RT-PCR, followed by the determination of the nucleotide sequence and phylogenetic analysis. RESULTS AND DISCUSSION: KITV detection rates in ticks of the species Rh. geigyi was 12.2%, Rh. annulatus 4.4%, Rh. decoloratus 3.3%. However, the KITV genetic material has not been identified in Am. variegatum ticks, which are one of the dominant species in West Africa. For all virus isolates, a partial nucleotide sequences of each of the four viral segments (GenBank, OK345271OK345306) were determined. The phylogenetic analysis showed a high level of identity (98.599.8%) for each of the four segments of the viral genome with those previously found in the Republic of Guinea. The obtained KITV isolates are most genetically close to Mogiana tick virus, which was previously detected in South America in Rh. microplus ticks and significantly differed from other multicomponent viruses circulating in Europe and Asia, including the Russian Federation. CONCLUSION: KITV genetic material was found in three species of ixodid ticks collected from livestock in a number of prefectures of the Republic of Guinea. The infection rate in ticks was 3.312.2%. The continuation of research in this direction remains relevant.

[Structural Motifs and Spatial Structures of Helicase (NS3) and RNA-dependent RNA-polymerase (NS5) of a Flavi-like Kindia tick virus (unclassified Flaviviridae)].

INTRODUCTION: Kindia tick virus (KITV) is a novel segmented unclassified flavi-like virus of the Flaviviridae family. This virus is associated with ixodes ticks and is potentially pathogenic to humans. The main goal of this work was to search for structural motifs of viral polypeptides and to develop a 3D-structure for viral proteins of the flavi-like KITV. MATERIALS AND METHODS: The complete genome sequences for KITV, Zika, dengue, Japanese encephalitis, West Nile and yellow fever viruses were retrieved from GenBank. Bioinformatics analysis was performed using the different software packages. RESULTS: Analysis of the KITV structural proteins showed that they have no analogues among currently known viral proteins. Spatial models of NS3 and NS5 KITV proteins have been obtained. These models had a high level of topological similarity to the tick-borne encephalitis and dengue viral proteins. The methyltransferase and RNA-dependent RNA-polymerase domains were found in the NS5 KITV. The latter was represented by fingers, palm and thumb subdomains, and motifs A-F. The helicase domain and its main structural motifs IVI were identified in NS3 KITV. However, the protease domain typical of NS3 flaviviruses was not detected. The highly conserved amino acid motives were detected in the NS3 and NS5 KITV. Also, eight amino acid substitutions characteristic of KITV/2018/1 and KITV/2018/2 were detected, five of them being localized in alpha-helix and three in loops of nonstructural proteins. CONCLUSION: Nonstructural proteins of KITV have structural and functional similarities with unsegmented flaviviruses. This confirms their possible evolutionary and taxonomic relationships.

[Prevalence and molecular genetic characteristics of parenteral hepatitis B, C and D viruses in HIV positive persons in the Novosibirsk region].

INTRODUCTION: Parenteral viral hepatitis (B, C, D) and HIV share modes of transmission and risk groups, in which the probability of infection with two or more of these viruses simultaneously is increased. Mutual worsening of the course of viral infections is important issue that occurs when HIV positive patients are coinfected with parenteral viral hepatitis. The aim of the study was to determine the prevalence of HCV, HBV and HDV in HIV positive patients in the Novosibirsk region and to give molecular genetic characteristics of their isolates. MATERIALS AND METHODS: Total 185 blood samples were tested for the presence of total antibodies to HCV, HCV RNA, HBV DNA and HDV RNA. The identified isolates were genotyped by amplification of the NS5B gene fragment for HCV, the polymerase gene for HBV and whole genome for HDV. RESULTS: The total antibodies to HCV were detected in 51.9% (95% CI: 44.758.9), HCV RNA was detected in 32.9% (95% CI: 26.639.5) of 185 studied samples. The distribution of HCV RNA positive cases completely repeated the distribution of HCV serological markers in different sex and age groups. The number of HCV infected among HIV positive patients increases with age. HCV subgenotypes distribution was as follows: 1b (52.5%), 3а (34.5%), 1а (11.5%), 2а (1.5%). 84.3% of detected HCV 1b isolates had C316N mutation associated with resistance to sofosbuvir and dasabuvir. The prevalence of HBV DNA in the studied samples was 15.2% (95% CI: 10.721.0). M204I mutation associated with resistance to lamivudine and telbivudine was identified in one HBV isolate. Two HDV isolates that belonged to genotype 1 were detected in HIV/HBV coinfected patients. CONCLUSION: The data obtained confirm the higher prevalence of infection with parenteral viral hepatitis among people living with HIV in the Novosibirsk region compared to the general population of that region. The genetic diversity of these viruses among HIV infected individuals is similar to that observed in the general population.

[Development of a method for detection of specific antibodies to E protein of yellow fever virus (Flaviviridae: Flavivirus) by enzyme immunoassay].

INTRODUCTION: Yellow fever (YF) remains one of the most common natural focal infectious diseases in the world. In connection with the increasing tourist flow to countries endemic for YF, the discovery of stable populations of Aedes aegypti and Ae. albopictus which are the main vectors of the yellow fever virus (YFV), in the southern regions of Russia, and the fact that in medical institutions in our country it is possible to obtain a live attenuated vaccine against YF, but there is no way to evaluate the effectiveness of vaccination, the question arises of the development and implementation of diagnostic kits for detecting antibodies (AB) to the pathogen by enzyme immunoassay (ELISA).The aim of this study was to develop a method for detecting specific IgG antibodies to the E protein of YFV by ELISA and assessing its diagnostic characteristics. MATERIALS AND METHODS: A specific cDNA was synthesized by reverse transcription on an RNA template of YFV isolated on a cell culture of Aedes albopictus clone C6/36, and a fragment of the genome coding the YFV E protein was amplified and subsequently cloned into the plasmid pET160 (Thermo Fisher Scientific, USA). The resulting gene fragment was used as a DNA template to obtain a recombinant analog of the third domain of the YFV E protein in Escherichia coli cells (BL-21(DE3)). Next, the immunogenicity of the obtained antigen was evaluated and the analysis conditions were optimized. RESULTS: The optimal conditions for the production of the obtained recombinant E protein of YFV were determined, its specificity was confirmed by immunological methods (Western blot and ELISA), sorption buffers and blocking solutions were selected, and sensitivity and specificity of detection of antibodies to YFV using the recombinant antigen were assessed. CONCLUSION: A method for the detection of specific IgG antibodies to the YFV E protein by ELISA was developed. This diagnostic kit can be used both to study the protective properties of the YF vaccine and to detect imported cases of infection in non-endemic areas.

[Genetic Variability of Tick-Borne Encephalitis Virus Genome 5'-UTR from Northern Eurasia].

This paper reports the analysis of the nucleotide sequences of the 5'-untranslated region (5'-UTR) of tick-borne encephalitis virus (TBEV) genomic RNA isolated from 39 individual taiga ticks collected in several regions of Northern Eurasia. The sequences of 5'-UTRs of the Siberian and Far East TBEV genotypes were 89% and 95% identical to the prototype strains (Zausaev and 205), respectively. The detected nucleotide substitutions were typical for these two TBEV genotypes, which made possible unambiguous identification. Both conservative and variable motifs were detected in the 5'-UTR RNA. The B2, C1, and C2 elements of the Y-shaped 5'-UTR structure and the presumable viral RNA-dependent RNA-polymerase binding site were the most variable. The A2, CS A, CS В elements as well as the start codon were conservative. Interestingly, five substitutions in the 5'-UTR C1 variable element of the TBEVs isolated in different geographical regions were strictly conservative, while 11 different substitutions were detected in this element among the laboratory TBEV variants. A little less that a third of all nucleotide substitutions were mapped outside the main elements of the Y-shaped structure. In general, nucleotide substitutions were localized to stem structures, not being found in the hairpin regions of the TBEV 5'-UTR. The results indicated significant variability of the genomic RNA 5'-UTR in the TBEV laboratory strains and field isolates obtained from different geographical regions. It has been suggested that genetic variability of 5'-UTR is characteristic of the TBEV genome 5'-UTR organization and may serve as a structural basis for virus efficient replication in various avian, mammalian, and ixodic tick cells.

Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR.

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

Detection of Ehrlichia spp. and Theileria spp. in Hyalomma anatolicum ticks collected in Tajikistan.

The objectives of our study were to survey the prevalence of genetic markers for Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. in Hyalomma anatolicum ticks collected in southwestern Tajikistan and to perform sequencing and phylogenetic analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. and fragments of the 18S rRNA gene of Theileria spp. detected in H. anatolicum ticks. Hyalomma anatolicum ticks collected in the Tursunzade and Rudaki districts of Tajikistan were tested for DNA of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. by PCR with specific primers. The amplified fragments were sequenced and analyzed. DNA of Ehrlichia spp. (3.3 %) and Theileria spp. (3.3 %) was detected only in H. anatolicum ticks collected from the Rudaki district, and DNA of Ehrlichia spp. (0.7 %) was found in H. anatolicum ticks from the Tursunzade district. Sequence analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. revealed high similarity to Ehrlichia spp. The Tajik isolates of Theileria spp. were genotyped as Theileria annulata based on the analysis of 18S rRNA gene sequences. The phylogenetic analysis demonstrates that Ehrlichia spp. isolates are highly similar to Ehrlichia spp. circulating in China and Brazil. The isolate Tajikistan-5 is closely related to the putative novel species Ehrlichia mineirensis. The Tajik isolates of Theileria spp. were clustered with T. annulata isolates from Turkey, Iran, Pakistan, and China by phylogenetic analyses.

[Development and assessment of a reagent kit for RNA detection of CrimeanCongo hemorrhagic fever virus with using reverse transcription loop-mediated isothermal amplification method.]

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector¼ (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.

Cowpox in a human, Russia, 2015.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

[On-microchip PCR for detection of influenza A viruses subtypes, circulating in the human population].

A oligonucleotide microchip was developed for revealing Influenza A viruses subtypes, circulating in human population: pandemic H1N1 swine influenza viruses, seasonal H1N1, H2N2, H3N2, H5N1, H9N2, H7N9. Typing of influenza virus was performed by on-microchip PCR. We used immobilized primers-probes selected for the neuraminidase gene that allows determining both subtype of neuraminidase and subtype of hemagglutinin.

Imported cases of dengue fever in Russia during 2010-2013.

OBJECTIVE: To confirm dengue infection among Russian tourists returned from Southeast and Mexico in 2010-2013 with clinical signs of infection. METHODS: Blood and serum samples from patients were collected. NS1 antigen and human IgM/IgG antibodies to dengue virus were identified using commercial tests manufactured by "Standard Diagnostics, INC.", Korea. ELISA test was used for the quantitative analyses of human IgM/IgG antibodies to dengue virus ("Orgenics Ltd.", Israel). Viral RNA was detected using commercial real-time PCR tests manufactured by "Genome Diagnostics Pvt. Ltd.", India and "Vector", Russia. Genotypes of revealed dengue viruses were determined employing nucleotide sequencing and phylogenetic analysis of 5'-UTR of the viral genome. RESULTS: A total of 98 collected blood samples were analyzed. Fifty samples were positive for at least one of four markers of dengue infection. IgM to dengue virus were revealed in 38 samples, in 25 samples IgM were combined with IgG. NS1 antigen was detected in 43 samples. 22 serum samples were positive for dengue virus RNA. The majority of samples (12 patients) from tourists returned from Thailand were positive for genotype 1 of dengue virus, 2nd and 4th genotype were identified each in 1 patient. CONCLUSIONS: Due to laboratory confirmed cases of imported dengue fever in Russia, the differential diagnosis of dengue is strictly recommended for tourists returning from endemic areas.

[THE HIGHLY EFFECTIVE DETECTION OF DNA RICKETTSIA USING TECHNIQUE OF POLYMERASE CHAIN REACTION IN REAL-TIME].

The article considers development of highly effective technique of detection of genetic material of ricketsia based on polymerase chain reaction in real-time using original primers to the most conservative sites of gene of citrate synthase (gItA). The analytical sensitivity of the developed polymerase chain reaction in real-time test permits to detect from 80 genome equivalents in analyzed sample during three hours. The high specificity of test-system is substantiated by detection of nucleotide sequences of amplificated fragments of gene gltA. The approbation ofthe polymerase chain reaction in real-time test is carried out on collection of 310 ticks of species I. persulcatus, I. pavlovskyi, D. reticulatus. It is demonstrated that the developed alternate ofprimers and probe permits with high degree of sensitivity and specifcity to detect DNA of different species of ricketsia widespread on territory of Russia (R. sibirica, R. raoultii, R. helvetica, R. tarasevichiae). The proposed polymerase chain reaction in real-time test can be appliedfor isolation of fragment of gene gltA with purpose for detecting nucleotide sequence and subsequent genetic typing of ricketsia. The application ofthe proposed technique can facilitate task of monitoring hot spots of ricketsiosis.

[The role of birds in the maintenance of tick-borne infections in the Tomsk anthropurgic foci].

The role of birds in the focus of tick-borne infections was studied from 2006 to 2011. The frequency index of ticks carried by ground dwelling birds is about 49.7%. The index of their abundance is 3.8. The larvae of ticks have been found on birds in 43.8% of cases. Nymphs and adult ticks have been found in 39.9 and 16.3%, respectively. It was revealed that Ixodex pavlovskyi was transferred and dominated in the urban microfoci because of its ornithophily. The markers of infectious agents have been recorded in 42 of 60 bird species under study.

[The hepatitis A virus: structural and functional organization of the genome, its molecular diagnostic value and cultivation].

The analysis of recently published data on hepatitis A virus (HAV) genome clinical features, molecular diagnostic value and cell culture propagation are reviewed. The growing need in the study of the genetic diversity of HAV isolates and the search of its possible new antigenic variants are underlined. The results of the cultivation of different HAV strains are analyzed for possible application in vaccine and diagnostic kit production.

[Etiological structure of acute respiratory viral infections morbidity in Novosibirsk and Novosibirsk region in epidemic season 2011-2012].

INTRODUCTION: ARI occupying the first place in the structure of total human morbidity. The aim of the study was to investigate the species diversity of the viruses causing AR among residents of the Novosibirsk region during epidemic season (October to April). MATERIALS AND METHODS: 164 nasopharyngeal swabs were collected and analyzed. Viral RNA/DNA, cDNA synthesis and PCR were carried out employing "RIBO-prep" "eReverta-L", "AmpliSens Influenza virus A/B-FL" and "AmpliSens ARI-screen-FL" kits (CRI of Epidemiology). RESULTS: Etiological agent of the disease was found in 69(43%) samples. Monoinfection was found in 58 (35%). In 14 (9%) samples were detected serogroup I coronaviruses, in 13 (8%) rhinoviruses, in 7 (4%) respiratory syncytial virus, in 6 (4%) parainfluenza virus type 1, in 5 (3%) parainfluenza virus type 3. Adenoviruses and bocavirus were identified in 3 (2%) samples. Parainfluenza virus type 2 and 4, metapneumovirus, serogroup Il coronaviruses (HKU1 and OC43) were presented in 2 (1%) samples. In 11 (7%) samples was found mixed infection. CONCLUSION: The majority of common colds were caused by serogroup I coronaviruses (NL63 and 229E), rhinoviruses and mixed infections. The peak of species variability of viruses caused acute respiratory infections was determined in age group of children 2-4 years old. In older age groups the species variability of analyzed viruses was decreased, rhinovirus infection becomes prevalent.

[Development and validation of the real-time PCR assay for the identification of viral agents causing acute respiratory infections in humans].

The real time PCR assay targeting influenza A and B virus, 5 subtypes of influenza A virus (seasonal H1N1, pandemic H1N1 (2009), seasonal H3N2, pathogenic for human subtypes of avian influenza H5 and H7), respiratory syncytial virus, and adenovirus was developed. The analytical sensitivity of the developed assay was 1 x 10(3) genome equivalents per ml. The diagnostic sensitivity of the method was 1 x l0(3)-10(4) viral particles per ml. Experiments with human DNA/cDNA and viral cDNA showed a markedly high diagnostic specificity of the developed PCR assay. In the assay of the developed PCR test, 50 nasopharyngeal swab specimens were tested. The etiology was identified in 33 samples.

Development and verification of real-time PCR assay for identification of viral agents causing acute respiratory infections in human beings.

A multiplex polymerase chain reaction (PCR) for identification of four viruses causing acute respiratory diseases in human beings was developed. The analytical sensitivity of developed RT-PCR for identification of adenovirus, respiratory-syncytial virus, flu viruses types A and B, and actual subtypes of type A flu virus (seasonal and pandemic variants H1N1, seasonal H3N2, and viruses of bird flu that are pathogenic to human beings H5 and H7) was 1 × 103 genome equivalents per milliliter. Diagnostic sensitivity for flu virus type A and B, and also subtypes H1 (seasonal H1N1, pandemic variant of H1N1 of year 2009), H3, H5 was 1 × 103-104 viral particles per milliliter. The method developed has high specificity and does not have positive signal in experiments with DNA/cDNA of human beings and viral DNA. We have studied 50 samples using the developed set. Etiology was defined in 33 samples.

[Outbreak of acute enterovirus intestinal infection in Sakhalin region in August 2010].

The investigation of cases of acute intestinal infections in the Sakhalin region of Russia in August, 2010 is described. Epidemiological and molecular biological studies were conducted. After initial PCR screening and determining the nucleotide sequences of the positive samples the following enteroviruses were found: Coxsackie A2 - 42 samples (45%), Coxsackie A4--31 sample (34%), Enterovirus 71--6 samples (6,5%), Coxsackievirus B5--6 samples (6,5%), Coxsackie B3--4 samples (4%) and Coxsackie B1--4 samples (4%). The phylogenetic analysis of sequences showed that the closest analogues for the nucleotide sequences of these genotypes were previously identified in Japan, Korea and China in 2000-2010.

[Evaluation of immunogenicity and safety of 2 immunizations with allantoic intranasal live influenza vaccine Ultragrivac].

AIM: Evaluate reactogenicity, safety and immunogenicity in phase 2 clinical trials of 2 immunization schedules with Ultragrivac--an allantoic intranasal life influenza vaccine based on A/17/ duck/Potsdam/86/92 [17/H5] reassortant strain. MATERIALS AND METHODS: 4 groups of volunteers participated in the study: group 1--40 individuals were vaccinated twice with a 10 day interval; group 2--40 individuals were vaccinated twice with a 21 day interval; group 3 (control)--10 individuals received placebo twice with a 10 day interval; group 4 (control)--10 individuals received placebo twice with a 21 day interval. Local (secretory IgA), cellular and humoral immune response were evaluated. Humoral immunity was evaluated by the intensity of increase of geometric mean antibody titers against 2 influenza virus strains A/17/duck/Potsdam/86/92 [17/H5] and A/chicken/Suzdalka/Nov-1 1/2005 (H5N1), and by the level of significant (4 times or more) antibody seroconversions after the vaccination. RESULTS: After the use of Ultragrivac the level of secretory IgA in the nasal cavity of vaccinated volunteers in the groups with revaccination intervals of 10 and 21 days increased significantly. The second immunization with 10 or 21 day intervals significantly increased postvaccinal humoral immune response. Humoral immune response induction after 2 vaccinations with 10 day interval was no less effective than with 21 day interval. CONCLUSION: Ultragrivac allantoic intranasal live influenza vaccine is areactogenic, harmless for vaccinated individuals, safe for those around, and has immunogenic properties against not only homologous virus A(H5N2), but also against influenza strain A(H5N1).

[Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009].

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.