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.

Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions.

BACKGROUND: Ferns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes. RESULTS: We characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN). CONCLUSIONS: IRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative.

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.

AlignMe--a membrane protein sequence alignment web server.

We present a web server for pair-wise alignment of membrane protein sequences, using the program AlignMe. The server makes available two operational modes of AlignMe: (i) sequence to sequence alignment, taking two sequences in fasta format as input, combining information about each sequence from multiple sources and producing a pair-wise alignment (PW mode); and (ii) alignment of two multiple sequence alignments to create family-averaged hydropathy profile alignments (HP mode). For the PW sequence alignment mode, four different optimized parameter sets are provided, each suited to pairs of sequences with a specific similarity level. These settings utilize different types of inputs: (position-specific) substitution matrices, secondary structure predictions and transmembrane propensities from transmembrane predictions or hydrophobicity scales. In the second (HP) mode, each input multiple sequence alignment is converted into a hydrophobicity profile averaged over the provided set of sequence homologs; the two profiles are then aligned. The HP mode enables qualitative comparison of transmembrane topologies (and therefore potentially of 3D folds) of two membrane proteins, which can be useful if the proteins have low sequence similarity. In summary, the AlignMe web server provides user-friendly access to a set of tools for analysis and comparison of membrane protein sequences. Access is available at http://www.bioinfo.mpg.de/AlignMe.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.

Gene regulation by PAX6: structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204.

PURPOSE: Pax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action. METHODS: Mutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells. RESULTS: PAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site. CONCLUSIONS: These studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.

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.

Crystal structure of human Karyopherin ß2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2.

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin ß2 or Kapß2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapß2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapß2 is conserved throughout eukaryotes. Kap104, the Kapß2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapß2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapß2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapß2·PY-NLS structures.

The role of trimerization in the osmoregulated betaine transporter BetP.

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum.

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.

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.

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.

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.

Role of the ectodomain serine 275 in shaping the binding pocket of the ATP-gated P2X3 receptor.

ATP-activated P2X3 receptors expressed in nociceptive sensory neurons play an important role in pain signaling. Basic properties of this receptor subtype, including very strong desensitization, depend on the rate of dissociation of the agonist from the binding site. Even though the rough structure of the ATP binding site has been proposed on the basis of the X-ray structure of the zebrafish P2X4 receptor and mutagenesis studies, the fine subunit-specific structural properties predisposing the receptor to tight capture of the agonist inside the binding pocket have not been elucidated. In this work, by exploring in silico the functional role for the left flipper located in the ectodomain region, we identified within this loop a candidate residue S275, which could contribute to the closure of the agonist-binding pocket. Testing of the S275 mutants using the patch-clamp technique revealed a crucial role for S275 in agonist binding and receptor desensitization. The S275A mutant showed a reduced rate of onset of desensitization and accelerated resensitization and was weakly inhibited by nanomolar agonist. Extracellular calcium application produced inhibition instead of facilitation of membrane currents. Moreover, some full agonists became only partial agonists when applied to the S275A receptor. These effects were stronger with the more hydrophobic mutants S275C and S275V. Taken together, our data suggest that S275 contributes to the closure of the agonist-binding pocket and that effective capture of the agonist provided by the left flipper in calcium-dependent manner determines the high rate of desensitization, slow recovery, and sensitivity to nanomolar agonist of the P2X3 receptor.

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor.

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of ß,γ-meATP, α,ß-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.

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.

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.