Epidemiology of Viral Hepatitis in the Indigenous Populations of the Arctic Zone of the Republic of Sakha (Yakutia).

The indigenous populations of the Arctic regions of Russia experience the lowest coverage of health-related services. We assessed the prevalence of hepatitis A, B, C, D and E viruses (HAV, HBV, HCV, HDV and HEV) among 367 healthy adult Native people of the Arctic zone of Yakutia. The HAV seroprevalence was above and increased with age. The anti-HEV IgM and IgG antibody detection rates were 4.1% and 2.5%, respectively. The average HBsAg detection rate was 4.6%, with no positive cases identified in participants aged under 30 years, confirming the effectiveness of the newborn vaccination program that began in 1998. Anti-HDV antibodies were detected in 29.4% of HBsAg-positive cases. The anti-HCV and HCV RNA detection rates peaked in the age cohort of 50-59 years (10.8% and 3.9%). No statistically significant gender differences in the prevalence of different viral hepatitis were observed. The time-scaled phylogenetic analysis demonstrated that all HBV genotype A and D strains isolated in this study were autochthonous and had an estimated most common recent ancestor (MCRA) age of around the 11th to 14th century. Unlike HBV, the HCV strains of subtypes 1b, 2a and 2k/1b were introduced from other regions of Russia in the 1980s and 1990s. The HCV 1b sequence analysis revealed a series of transmission events. In conclusion, these data emphasize the urgent need for expanded viral hepatitis screening and care programs in the indigenous populations of the Arctic zone of Yakutia.

Unlocking the Viral Universe: Metagenomic Analysis of Bat Samples Using Next-Generation Sequencing.

Next-generation sequencing technologies have revolutionized the field of virology by enabling the reading of complete viral genomes, extensive metagenomic studies, and the identification of novel viral pathogens. Although metagenomic sequencing has the advantage of not requiring specific probes or primers, it faces significant challenges in analyzing data and identifying novel viruses. Traditional bioinformatics tools for sequence identification mainly depend on homology-based strategies, which may not allow the detection of a virus significantly different from known variants due to the extensive genetic diversity and rapid evolution of viruses. In this work, we performed metagenomic analysis of bat feces from different Russian cities and identified a wide range of viral pathogens. We then selected sequences with minimal homology to a known picornavirus and used "Switching Mechanism at the 5' end of RNA Template" technology to obtain a longer genome fragment, allowing for more reliable identification. This study emphasizes the importance of integrating advanced computational methods with experimental strategies for identifying unknown viruses to better understand the viral universe.

Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages.

The Omicron strain is currently the main dominant variant of SARS-CoV-2, with a large number of sublineages. In this article, we present our experience in tracing it in Russia using molecular diagnostic methods. For this purpose, different approaches were used; for example, we developed multiprimer panels for RT-PCR and Sanger and NGS sequencing methods. For the centralized collection and analysis of samples, the VGARus database was developed, which currently includes more than 300,000 viral sequences.

SARS-CoV-2 Variants Monitoring Using Real-Time PCR.

According to the temporary recommendations of the 2021 World Health Organization (WHO), in addition to whole-genome sequencing, laboratories in various countries can also screen for known mutations utilizing targeted RT-PCR-based mutation detection assays. The aim of this work was to generate a laboratory technique to differentiate the main circulating SARS-CoV-2 variants in 2021-2022, when a sharp increase in morbidity was observed with the appearance of the Omicron variant. Real-time PCR methodology is available for use in the majority of scientific and diagnostic institutions in Russia, which makes it possible to increase the coverage of monitoring of variants in the territories of all 85 regions in order to accumulate information for the Central Services and make epidemiological decisions. With the methodology developed by the Central Research Institute of Epidemiology of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (FSSCRP Human Wellbeing) (CRIE), more than 6000 biological samples have been typed, and 7% of samples with the Delta variant and 92% of samples with the Omicron variant have been identified as of 25 August 2022. Reagents for 140,000 definitions have been supplied to regional organizations.

Molecular Epidemiology of mcr-1-Positive Escherichia coli and Klebsiella pneumoniae Isolates: Results from Russian Sentinel Surveillance (2013-2018).

BACKGROUND: The dissemination of mobile colistin resistance (mcr) genes is a serious healthcare threat because polymyxins represent "last-line" therapeutics for multi-drug-resistant Gram-negative pathogens. This study aimed to assess the prevalence of colistin resistance and mcr genes and characteristics of clinical Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) isolates and plasmids carrying these genes in Russia. METHODS: A total of 4324 Eco and 4530 Kpn collected in the frame of sentinel surveillance in 2013-2018 were tested for susceptibility to colistin and other antibiotics using the broth microdilution method. mcr genes were screened by real-time PCR. Phylogeny, genomic features and plasmids of mcr-positive isolates were assessed using whole-genome sequencing and subsequent bioinformatic analysis. RESULTS: Colistin resistance was detected in 2.24% Eco and 9.3% Kpn. Twenty-two (0.51%) Eco and two (0.04%) Kpn from distant sites carried mcr-1.1. Most mcr-positive isolates co-harbored ESBLs and other resistance determinants to various antibiotic classes. The mcr-positive Eco belonged to 16 MLST types, with ST359 being most common; Kpn belonged to ST307 and ST23. mcr-1.1 was carried mainly in IncI2 (n = 18) and IncX4 (n = 5) plasmids highly similar to those identified previously in human, animal and environmental isolates. CONCLUSION: This study demonstrated a dissemination of "typical" mcr-bearing plasmids among diverse Eco and Kpn genotypes and across a wide geographic area in Russia. Given the frequent association of mcr with other resistance determinants and potential clinical impact, the continual surveillance of this threat is warranted.

Effective Approaches to Study the Genetic Variability of SARS-CoV-2.

Significant efforts are being made in many countries around the world to respond to the COVID-19 pandemic by developing diagnostic reagent kits, identifying infected people, determining treatment methods, and finally producing effective vaccines. However, novel coronavirus variants may potentially reduce the effectiveness of all these efforts, demonstrating increased transmissibility and abated response to therapy or vaccines, as well as the possibility of false negative results in diagnostic procedures based on nucleic acid amplification methods. Since the end of 2020, several variants of concern have been discovered around the world. When information about a new, potentially more dangerous strain of pathogen appears, it is crucial to determine the moment of its emergence in a region. Eventually, that permits taking timely measures and minimizing new risks associated with the spreading of the virus. Therefore, numerous nations have made tremendous efforts to identify and trace these virus variants, which necessitates serious technological processes to sequence a large number of viral genomes. Here, we report on our experience as one of the primary laboratories involved in monitoring SARS-CoV-2 variants in Russia. We discuss the various approaches used, describe effective protocols, and outline a potential technique combining several methods to increase the ability to trace genetic variants while minimizing financial and labor costs.

Utilizing the VirIdAl Pipeline to Search for Viruses in the Metagenomic Data of Bat Samples.

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.

Association of CASR, CALCR, and ORAI1 Genes Polymorphisms With the Calcium Urolithiasis Development in Russian Population.

Kidney stone disease is an urgent medical and social problem. Genetic factors play an important role in the disease development. This study aims to establish an association between polymorphisms in genes coding for proteins involved in calcium metabolism and the development of calcium urolithiasis in Russian population. In this case-control study, we investigated 50 patients with calcium urolithiasis (experimental group) and 50 persons lacking signs of kidney stone disease (control group). For molecular genetic analysis we used a previously developed gene panel consisting of 33 polymorphisms in 15 genes involved in calcium metabolism: VDR, CASR, CALCR, OPN, MGP, PLAU, AQP1, DGKH, SLC34A1, CLDN14, TRPV6, KLOTHO, ORAI1, ALPL, and RGS14. High-throughput target sequencing was utilized to study the loci of interest. Odds ratios and 95% confidence intervals were used to estimate the association between each SNP and risk of urolithiasis development. Multifactor dimensionality reduction analysis was also carried out to analyze the gene-gene interaction. We found statistically significant (unadjusted p-value < 0.05) associations between calcium urolithiasis and the polymorphisms in the following genes: CASR rs1042636 (OR = 3.18 for allele A), CALCR rs1801197 (OR = 6.84 for allele A), and ORAI1 rs6486795 (OR = 2.25 for allele C). The maximum OR was shown for AA genotypes in loci rs1042636 (CASR) and rs1801197 (CALCR) (OR = 4.71, OR = 11.8, respectively). After adjustment by Benjamini-Hochberg FDR we found only CALCR (rs1801197) was significantly associated with the risk of calcium urolithiasis development. There was no relationship between recurrent course of the disease and family history of urolithiasis in investigated patients. Thus we found a statistically significant association of polymorphism rs1801197 (gene CALCR) with calcium urolithiasis in Russian population.

Refining pairwise sequence alignments of membrane proteins by the incorporation of anchors.

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution, and in the generation of homology-based models. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly matched by a standard pairwise sequence alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the aligned regions either side of the newly matched positions. Previous studies have introduced restraints as a means to impose the matching of positions during sequence alignments, originally in the context of genome assembly. Here we introduce position restraints, or "anchors" as a feature in our alignment tool AlignMe, providing an aid to pairwise global sequence alignment of alpha-helical membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of anchors can be used to modify alignments, while still maintaining the reproducibility and rigor of the rest of the alignment. Anchored alignments can be generated using the online version of AlignMe available at www.bioinfo.mpg.de/AlignMe/.

Complete Genome Sequence, Molecular Characterization and Phylogenetic Relationships of a Novel Tern Atadenovirus.

Discovery and study of viruses carried by migratory birds are tasks of high importance due to the host's ability to spread infectious diseases over significant distances. With this paper, we present and characterize the first complete genome sequence of atadenovirus from a tern bird (common tern, Sterna hirundo) preliminarily named tern atadenovirus 1 (TeAdV-1). TeAdV-1 genome is a linear double-stranded DNA molecule, 31,334 base pairs which contain 30 methionine-initiated open reading frames with gene structure typical for Atadenovirus genus, and the shortest known inverted terminal repeats (ITRs) within the Atadenovirus genus consisted of 25 bases. The nucleotide composition of the genome is characterized by a low G + C content (33.86%), which is the most AT-rich genome of known avian adenoviruses within Atadenovirus genus. The nucleotide sequence of the TeAdV-1 genome shows high divergence compared to known representatives of the Atadenovirus genus with the highest similarity to the duck atadenovirus 1 (53.7%). Phylogenetic analysis of the protein sequences of core genes confirms the taxonomic affiliation of the new representative to the genus Atadenovirus with the degree of divergence from the known representatives exceeding the interspecies distance within the genus. Thereby we proposed a novel TeAdV-1 to be considered as a separate species.

Isolation and characterization of Wad Medani virus obtained in the tuva Republic of Russia.

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.

Ngari virus (Orthobunyavirus, Peribunyaviridae) in ixodid ticks collected from cattle in Guinea.

Ngari virus is a mosquito-borne virus belonging to the genus Orthobunyavirus (Peribunyaviridae family). This virus is pathogenic to humans and causes severe illness. Ngari virus is present in several African countries, including Madagascar. Here, we report the detection of Ngari virus in ixodid ticks collected from cows in Guinea. A tick survey was conducted in March-November of 2018 in six regions of Guinea. The sample comprised 710 pools, with a total of 2067 ticks belonging to five species collected from 197 cows. At the initial stage, we screened a subsample of tick pools of vector-borne viruses with a multiplex genus-specific primer panel. In the second stage of the study, we narrowed the search and screened all the samples by qPCR for the detection of Ngari virus. All positive samples were sequenced with primers flanking Ngari virus-specific fragments on the S and M segments. We found Ngari virus in 12 pools that were formed from engorged ticks collected from livestock in three villages of the Kindia and Kankan regions. Sequencing of the S and M segments confirmed that the detected viruses belong to Ngari virus, and the viruses were most similar to the strain Adrar, which was isolated in Mauritania. We detected viral RNA in ticks of the following species: Amblyomma variegatum, Rhipicephalus geigyi, and Rh. (Boophilus) spp. There is no evidence that ixodid ticks are competent vectors of the Ngari virus. Most likely, the ticks obtained the virus through blood from an infected host. The study of engorged ticks can be recommended as a simpler approach for the wide screening of the Ngari virus and subsequent testing of cattle and mosquitos in those locations where the PCR-positive ticks were collected.

Lethal Outcome of Leptospirosis in Southern Russia: Characterization of Leptospira Interrogans Isolated from a Deсeased Teenager.

This article describes a lethal case of leptospirosis that occurred in Southern Russia. The Leptospira strain was isolated and characterized using a microscopic agglutination test, MALDI-TOF mass spectrometry, targeted PCR, and high-throughput sequencing. We show that molecular and mass-spectrometry methods can be an alternative to conventional methods of leptospirosis diagnostics and Leptospira study, which require highly qualified staff and can be performed only at specialized laboratories. We also report the first whole genome of L. interrogans isolated in Russia.

Current Trends in Diagnostics of Viral Infections of Unknown Etiology.

Viruses are evolving at an alarming rate, spreading and inconspicuously adapting to cutting-edge therapies. Therefore, the search for rapid, informative and reliable diagnostic methods is becoming urgent as ever. Conventional clinical tests (PCR, serology, etc.) are being continually optimized, yet provide very limited data. Could high throughput sequencing (HTS) become the future gold standard in molecular diagnostics of viral infections? Compared to conventional clinical tests, HTS is universal and more precise at profiling pathogens. Nevertheless, it has not yet been widely accepted as a diagnostic tool, owing primarily to its high cost and the complexity of sample preparation and data analysis. Those obstacles must be tackled to integrate HTS into daily clinical practice. For this, three objectives are to be achieved: (1) designing and assessing universal protocols for library preparation, (2) assembling purpose-specific pipelines, and (3) building computational infrastructure to suit the needs and financial abilities of modern healthcare centers. Data harvested with HTS could not only augment diagnostics and help to choose the correct therapy, but also facilitate research in epidemiology, genetics and virology. This information, in turn, could significantly aid clinicians in battling viral infections.

Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group.

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.

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products.

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Sequencing and genetic characterization of two strains Paramushir virus obtained from the Tyuleniy Island in the Okhotsk Sea (2015).

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.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

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.

Targeted sequencing reveals complex, phenotype-correlated genotypes in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods. METHODS: We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients. RESULTS: By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%). CONCLUSION: NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.

Reconstructed Serine 288 in the Left Flipper Region of the Rat P2X7 Receptor Stabilizes Nonsensitized States.

Serine 275, a conserved residue of the left flipper region of ATP-gated P2X3 receptors, plays a key role in both agonist binding and receptor desensitization. It is conserved in most of the P2X receptors except P2X7 and P2X6. By combining experimental patch-clamp and modeling approaches, we explored the role of the corresponding residue in the rat P2X7 receptor (rP2X7) by replacing the phenylalanine at position 288 with serine and characterizing the membrane currents generated by either the wild-type (WT) or the mutated rP2X7 receptor. F288S, an rP2X7 mutation, slowed the deactivation subsequent to 2 and 20 s applications of 1 mM ATP. F288S also prevented sensitization (a progressive current growth) observed with the WT in response to a 20 s application of 1 mM ATP. Increasing the ATP concentration to 5 mM promoted sensitization also in the mutated rP2X7 receptor, accelerating the deactivation rate to typical WT values. YO-PRO1 uptake in cells expressing either the WT or the F288S P2X7 receptor was consistent with recorded membrane current data. Interestingly, in the human P2X7 (hP2X7) receptor, substitution Y288S did not change the deactivation rate, while the Y288F mutant generated a "rat-like" phenotype with a fast deactivation rate. Our combined experimental, kinetic, and molecular modeling data suggest that the rat F288S novel phenotype is due to a slower rate of ATP binding and/or unbinding and stabilization of nonsensitized receptor states.