Viral Diversity in Samples of Freshwater Gastropods Benedictia baicalensis (Caenogastropoda: Benedictiidae) Revealed by Total RNA-Sequencing.

Previously, the main studies were focused on viruses that cause disease in commercial and farmed shellfish and cause damage to food enterprises (for example, Ostreavirusostreidmalaco1, Aurivirus haliotidmalaco1 and Aquabirnavirus tellinae). Advances in high-throughput sequencing technologies have extended the studies to natural populations of mollusks (and other invertebrates) as unexplored niches of viral diversity and possible sources of emerging diseases. These studies have revealed a huge diversity of mostly previously unknown viruses and filled gaps in the evolutionary history of viruses. In the present study, we estimated the viral diversity in samples of the Baikal endemic gastropod Benedictia baicalensis using metatranscriptomic analysis (total RNA-sequencing); we were able to identify a wide variety of RNA-containing viruses in four samples (pools) of mollusks collected at three stations of Lake Baikal. Most of the identified viral genomes (scaffolds) had only distant similarities to known viruses or (in most cases) to metagenome-assembled viral genomes from various natural samples (mollusks, crustaceans, insects and others) mainly from freshwater ecosystems. We were able to identify viruses similar to those previously identified in mollusks (in particular to the picornaviruses Biomphalaria virus 1 and Biomphalaria virus 3 from the freshwater gastropods); it is possible that picorna-like viruses (as well as a number of other identified viruses) are pathogenic for Baikal gastropods. Our results also suggested that Baikal mollusks, like other species, may bioaccumulate or serve as a reservoir for numerous viruses that infect a variety of organisms (including vertebrates).

Inferring Evolutionary Timescale of Omsk Hemorrhagic Fever Virus.

Until 2020, there were only three original complete genome (CG) nucleotide sequences of Omsk hemorrhagic fever virus (OHFV) in GenBank. For this reason, the evolutionary rate and divergence time assessments reported in the literature were based on the E gene sequences, but notably without temporal signal evaluation, such that their reliability is unclear. As of July 2022, 47 OHFV CG sequences have been published, which enables testing of temporal signal in the data and inferring unbiased and reliable substitution rate and divergence time values. Regression analysis in the TempEst software demonstrated a stronger clocklike behavior in OHFV samples for the complete open reading frame (ORF) data set (R2 = 0.42) than for the E gene data set (R2 = 0.11). Bayesian evaluation of temporal signal indicated very strong evidence, with a log Bayes factor of more than 5, in favor of temporal signal in all data sets. Our results based on the complete ORF sequences showed a more precise OHFV substitution rate (95% highest posterior density (HPD) interval, 9.1 × 10-5-1.8 × 10-4 substitutions per site per year) and tree root height (416-896 years ago) compared with previous assessments. The rate obtained is significantly higher than tick-borne encephalitis virus by at least 3.8-fold. The phylogenetic analysis and past population dynamics reconstruction revealed the declining trend of OHFV genetic diversity, but there was phylogenomic evidence that implicit virus subpopulations evolved locally and underwent an exponential growth phase.

Dates and Rates of Tick-Borne Encephalitis Virus-The Slowest Changing Tick-Borne Flavivirus.

We evaluated the temporal signal and substitution rate of tick-borne encephalitis virus (TBEV) using 276 complete open reading frame (ORF) sequences with known collection dates. According to a permutation test, the TBEV Siberian subtype (TBEV-S) data set has no temporal structure and cannot be applied for substitution rate estimation without other TBEV subtypes. The substitution rate obtained suggests that the common clade of TBEV (TBEV-common), including all TBEV subtypes and louping-ill virus (LIV), is characterized by the lowest rate (1.87 × 10-5 substitutions per site per year (s/s/y) or 1 nucleotide substitution per ORF per 4.9 years; 95% highest posterior density (HPD) interval, 1.3-2.4 × 10-5 s/s/y) among all tick-borne flaviviruses previously assessed. Within TBEV-common, the TBEV European subtype (TBEV-E) has the lowest substitution rate (1.3 × 10-5 s/s/y or 1 nucleotide substitution per ORF per 7.5 years; 95% HPD, 1.0-1.8 × 10-5 s/s/y) as compared with TBEV Far-Eastern subtype (3.0 × 10-5 s/s/y or 1 nucleotide substitution per ORF per 3.2 years; 95% HPD, 1.6-4.5 × 10-5 s/s/y). TBEV-common representing the species tick-borne encephalitis virus diverged 9623 years ago (95% HPD interval, 6373-13,208 years). The TBEV Baikalian subtype is the youngest one (489 years; 95% HPD, 291-697 years) which differs significantly by age from TBEV-E (848 years; 95% HPD, 596-1112 years), LIV (2424 years; 95% HPD, 1572-3400 years), TBEV-FE (1936 years, 95% HPD, 1344-2598 years), and the joint clade of TBEV-S (2505 years, 95% HPD, 1700-3421 years) comprising Vasilchenko, Zausaev, and Baltic lineages.

Genomic Determinants Potentially Associated with Clinical Manifestations of Human-Pathogenic Tick-Borne Flaviviruses.

The tick-borne flavivirus group contains at least five species that are pathogenic to humans, three of which induce encephalitis (tick-borne encephalitis virus, louping-ill virus, Powassan virus) and another two species induce hemorrhagic fever (Omsk hemorrhagic fever virus, Kyasanur Forest disease virus). To date, the molecular mechanisms responsible for these strikingly different clinical forms are not completely understood. Using a bioinformatic approach, we performed the analysis of each amino acid (aa) position in the alignment of 323 polyprotein sequences to calculate the fixation index (Fst) per site and find the regions (determinants) where sequences belonging to two designated groups were most different. Our algorithm revealed 36 potential determinants (Fst ranges from 0.91 to 1.0) located in all viral proteins except a capsid protein. In an envelope (E) protein, most of the determinants were located on the virion surface regions (domains II and III) and one (absolutely specific site 457) was located in the transmembrane region. Another 100% specific determinant site (E63D) with Fst = 1.0 was located in the central hydrophilic domain of the NS2b, which mediates NS3 protease activity. The NS5 protein contains the largest number of determinants (14) and two of them are absolutely specific (T226S, E290D) and are located near the RNA binding site 219 (methyltransferase domain) and the extension structure. We assume that even if not absolutely, highly specific sites, together with absolutely specific ones (Fst = 1.0) can play a supporting role in cell and tissue tropism determination.

Metagenomic Assessment of DNA Viral Diversity in Freshwater Sponges, Baikalospongia bacillifera.

Sponges (type Porifera) are multicellular organisms that give shelter to a variety of microorganisms: fungi, algae, archaea, bacteria, and viruses. The studies concerning the composition of viral communities in sponges have appeared rather recently, and the diversity and role of viruses in sponge holobionts remain largely undisclosed. In this study, we assessed the diversity of DNA viruses in the associated community of the Baikal endemic sponge, Baikalospongia bacillifera, using a metagenomic approach, and compared the virome data from samples of sponges and Baikal water (control sample). Significant differences in terms of taxonomy, putative host range of identified scaffolds, and functional annotation of predicted viral proteins were revealed in viromes of sponge B. bacillifera and the Baikal water. This is the evidence in favor of specificity of viral communities in sponges. The diversity shift of viral communities in a diseased specimen, in comparison with a visually healthy sponge, probably reflects the changes in the composition of microbial communities in affected sponges. We identified many viral genes encoding the proteins with metabolic functions; therefore, viruses in Baikal sponges regulate the number and diversity of their associated community, and also take a part in the vital activity of the holobiont, and this is especially significant in the case of damage (or disease) of these organisms in unfavorable conditions. When comparing the Baikal viromes with similar datasets of marine sponge (Ianthella basta), in addition to significant differences in the taxonomic and functional composition of viral communities, we revealed common scaffolds/virotypes in the cross-assembly of reads, which may indicate the presence of some closely related sponge-specific viruses in marine and freshwater sponges.

Delimitation of the tick-borne flaviviruses. Resolving the tick-borne encephalitis virus and louping-ill virus paraphyletic taxa.

The tick-borne flavivirus (TBFV) group contains at least 12 members where five of them are important pathogens of humans inducing diseases with varying severity (from mild fever forms to acute encephalitis). The taxonomy structure of TBFV is not fully clarified at present. In particular, there is a number of paraphyletic issues of tick-borne encephalitis virus (TBEV) and louping-ill virus (LIV). In this study, we aimed to apply different bioinformatic approaches to analyze all available complete genome amino acid sequences to delineate TBFV members at the species level. Results showed that the European subtype of TBEV (TBEV-E) is a distinct species unit. LIV, in turn, should be separated into two species. Additional analysis of TBEV and LIV antigenic determinant diversity also demonstrate that TBEV-E and LIV are significantly different both from each other and from the other TBEV subtypes. The analysis of available literature provided data on other virus phenotypic particularities that supported our hypothesis. So, within the TBEV + LIV paraphyletic group, we offer to assign four species to get a more accurate understanding of the TBFV interspecies structure according to the modern monophyletic conception.

The Mitochondrial Genome of a Freshwater Pelagic Amphipod Macrohectopus branickii Is among the Longest in Metazoa.

There are more than 350 species of amphipods (Crustacea) in Lake Baikal, which have emerged predominantly through the course of endemic radiation. This group represents a remarkable model for studying various aspects of evolution, one of which is the evolution of mitochondrial (mt) genome architectures. We sequenced and assembled the mt genome of a pelagic Baikalian amphipod species Macrohectopus branickii. The mt genome is revealed to have an extraordinary length (42,256 bp), deviating significantly from the genomes of other amphipod species and the majority of animals. The mt genome of M. branickii has a unique gene order within amphipods, duplications of the four tRNA genes and Cox2, and a long non-coding region, that makes up about two thirds of the genome's size. The extension of the mt genome was most likely caused by multiple duplications and inversions of regions harboring ribosomal RNA genes. In this study, we analyzed the patterns of mt genome length changes in amphipods and other animal phyla. Through a statistical analysis, we demonstrated that the variability in the mt genome length may be a characteristic of certain phyla and is primarily conferred by expansions of non-coding regions.

Phylogeography and Re-Evaluation of Evolutionary Rate of Powassan Virus Using Complete Genome Data.

In this paper, we revealed the genetic structure and migration history of the Powassan virus (POWV) reconstructed based on 25 complete genomes available in NCBI and ViPR databases (accessed in June 2021). The usage of this data set allowed us to perform a more precise assessment of the evolutionary rate of this virus. In addition, we proposed a simple Bayesian technique for the evaluation and visualization of 'temporal signal dynamics' along the phylogenetic tree. We showed that the evolutionary rate value of POWV is 3.3 × 10-5 nucleotide substitution per site per year (95% HPD, 2.0 × 10-5-4.7 × 10-5), which is lower than values reported in the previous studies. Divergence of the most recent common ancestor (MRCA) of POWV into two independent genetic lineages most likely occurred in the period between 2600 and 6030 years ago. We assume that the divergence of the virus lineages happened due to the melting of glaciers about 12,000 years ago, which led to the disappearance of the Bering Land Bridge between Eurasia and North America (the modern Alaskan territory) and spatial division of the viral areal into two parts. Genomic data provide evidence of the virus migrations between two continents. The mean migration rate detected from the Far East of Russia to North America was one event per 1750 years. The migration to the opposite direction occurred approximately once per 475 years.

Extended Evaluation of Viral Diversity in Lake Baikal through Metagenomics.

Lake Baikal is a unique oligotrophic freshwater lake with unusually cold conditions and amazing biological diversity. Studies of the lake's viral communities have begun recently, and their full diversity is not elucidated yet. Here, we performed DNA viral metagenomic analysis on integral samples from four different deep-water and shallow stations of the southern and central basins of the lake. There was a strict distinction of viral communities in areas with different environmental conditions. Comparative analysis with other freshwater lakes revealed the highest similarity of Baikal viromes with those of the Asian lakes Soyang and Biwa. Analysis of new data, together with previously published data allowed us to get a deeper insight into the diversity and functional potential of Baikal viruses; however, the true diversity of Baikal viruses in the lake ecosystem remains still unknown. The new metaviromic data will be useful for future studies of viral composition, distribution, and the dynamics associated with global climatic and anthropogenic impacts on this ecosystem.

Virome Analysis of Near-Bottom Coastal Water of Lake Baikal.

In recent years, Lake Baikal has undergone significant changes in the composition of coastal communities associated with the increasing anthropogenic influence and global climate changes. In this context, we carried out metagenomic sequencing of the DNA viral community of an integral near-bottom water sample from the littoral zone of the lake.

Metavirome datasets from two endemic Baikal sponges Baikalospongia bacillifera.

Sponges are ecologically important components of marine and freshwater benthic environments; these holobionts contain a variety of microorganisms and viruses. For the metagenomic characterization of potential taxonomic and functional diversity of sponge-associated dsDNA viruses, we surveyed two samples of Baikal endemic sponge Baikalospongia bacillifera (diseased and visually healthy). In total, after quality processing, we have obtained 3 375 063 and 4 063 311 reads; of these 97 557 and 88 517 sequences, accounting for ca. 2.9 and 2.2% of datasets, have been identified as viral. We have revealed approximately 28 viral families, among which the bacteriophages of the Myoviridae, Siphoviridae and Podoviridae families, as well as the viruses of the Phycodnaviridae and Poxviridae families, dominated in the samples. Analysis of viral sequences using the COG database has indicated 22 functional categories of proteins. Viral communities of visually healthy and diseased Baikal sponges were significantly different. The metagenome sequence data were deposited to NCBI SRA as BioProject PRJNA577390.

Bioinformatic tools for tRNA gene analyses in mitochondrial DNA sequence data.

The data presented here are related to the research article entitled "Hidden cases of tRNA genes duplication and remolding in mitochondrial genomes of amphipods" (Romanova et al., 2020) [1]. Correct tRNA gene sequence annotation in mitochondrial (mt) and nuclear genomes sometimes can be a challenging task because of the differential performances of tRNA annotation/prediction programmes. These programmes may cause false positive or false negative predictions. Moreover, additional difficulties with annotation may be caused by the presence of duplicated tRNA genes and those coding tRNAs with altered identities occurring as due to a mutation in their anticodon sequence (tRNA gene remolding/recruitment). We developed an R script automating the diagnosis of ancestor tRNA gene coding specificity regardless of anticodon sequence based on genetic distance comparison. Some of the predicted tRNA genes from the mt genomes of amphipods are presented. We also developed an R script for estimation of the best mode of sequence alignment, which was applied to determine the best alignment of tRNA genes in [1], but is also suitable for testing of any nucleotide alignment sets used in phylogenetic inferences.

Hidden cases of tRNA gene duplication and remolding in mitochondrial genomes of amphipods.

The evolution of tRNA genes in mitochondrial (mt) genomes is a complex process that includes duplications, degenerations, and transpositions, as well as a specific process of identity change through mutations in the anticodon (tRNA gene remolding or tRNA gene recruitment). Using amphipod-specific tRNA models for annotation, we show that tRNA duplications are more common in the mt genomes of amphipods than what was revealed by previous annotations. Seventeen cases of tRNA gene duplications were detected in the mt genomes of amphipods, and ten of them were tRNA genes that underwent remolding. The additional tRNA gene findings were verified using phylogenetic analysis and genetic distance analysis. The majority of remolded tRNA genes (seven out of ten cases) were found in the mt genomes of endemic amphipod species from Lake Baikal. All additional mt tRNA genes arose independently in the Baikalian amphipods, indicating the unusual plasticity of tRNA gene evolution in these species assemblages. The possible reasons for the unusual abundance of additional tRNA genes in the mt genomes of Baikalian amphipods are discussed. The amphipod-specific tRNA models developed for MiTFi refine existing predictions of tRNA genes in amphipods and reveal additional cases of duplicated tRNA genes overlooked by using less specific Metazoa-wide models. The application of these models for mt tRNA gene prediction will be useful for the correct annotation of mt genomes of amphipods and probably other crustaceans.

Estimate of the diversity of viral and bacterial assemblage in the coastal water of Lake Baikal.

In this study, we analysed the diversity and composition of double-stranded DNA viral and bacterial communities within the sample of surface coastal water of Southern Baikal through metagenomics and deep sequencing of the 16S ribosomal RNA gene, respectively. The 16S rRNA gene analysis has revealed 14 phyla and dominance of the 'Actinobacteria' (43.6%), 'Proteobacteria' (25.2%) and 'Bacteroidetes' (11.5%). The bacterial composition was similar to that obtained previously in Lake Baikal littoral zone. Out of 1 030 169 processed virome reads, 37.4% of sequences (385 421) were identified as viral; 15.1% were identified as nonviral and related to the domains Eukarya, Bacteria and Archaea; and 47.5% had no matches in the databases. The identified virotypes belonged to different families and were predicted to infect a wide range of organisms, from bacteria to mammals. Six families (Myoviridae, Poxviridae, Mimiviridae, Siphoviridae, Phycodnaviridae and Podoviridae) were dominant accounting for more than 90% of the identified sequences (48.3%, 17.4%, 8.3%, 6.8%, 5.8% and 4.1%, respectively). In contrast to other freshwater systems, high percentage of the Poxviridae and Mimiviridae was recorded in the water sample of Lake Baikal.