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.

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).

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.

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.

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.

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.

Reporting of New tick-borne encephalitis virus strains isolated in Eastern Siberia (Russia) in 1960-2011 and explaining them in an evolutionary context using Bayesian phylogenetic inference.

Tick-borne encephalitis virus (TBEV) is one of the main tick-borne viral pathogens of humans. Infection may induce signs of meningitis, encephalitis, paralysis and high fever. TBEV is well studied by molecular phylogenetic methods. The present-day implementation of Bayesian phylogenetic models allows population dynamics to be tracked, providing changes in population size that were not directly observed. However, the description of the past population dynamics of TBEV is rare in the literature. In our investigation, we provide data on the dynamics of viral genetic diversity of TBEV in Zabaikalsky Krai (Eastern Siberia, Russia) revealed by the Bayesian coalescent inference in a BEAST program. As a data set, we used the envelope (E) protein partial gene sequences (1308 nt) of 38 TBEV strains (including six "886-84-like" or Baikalian subtype strains (TBEV-B)), isolated in Zabaikalsky Krai (Eastern Siberia, Russia) in 1960-1963 and 1995-2011. To increase estimations reliability, we compared 9 model combinations by Path sampling and Stepping-stone sampling methods. It has been shown that the genetic diversity decline in the population history of TBEV in the 1950s coincides with the date of the beginning of wide dichlorodiphenyltrichloroethane forest dusting in Siberia. We assumed that the TBEV population on the territory of Siberia went through a genetic bottleneck. Also, we provide data estimating the divergence time of TBEV-B strains and indicate the specific evolution rate of an ancestor lineage of the Baikalian subtype, illustrated on a phylogenetic tree, and reconstructed under a relaxed clock model.

"886-84-like" tick-borne encephalitis virus strains: Intraspecific status elucidated by comparative genomics.

Tick-borne encephalitis virus (TBEV) can cause severe meningitis, encephalitis, and meningoencephalitis. TBEV represents a pathogen of high zoonotic potential and an emerging global threat. There are three known subtypes of TBEV: Far-Eastern, Siberian and European. Since 2001 there have been suggestions that two new subtypes may be distinguished: "178-79" and "886-84". These assumptions are based on the results of the envelope gene fragment sequencing (Zlobin et al., 2001; Kovalev and Mukhacheva, 2017) and genotype-specific probes molecular hybridization (Demina et al., 2010). There is only one full-genome sequence of "178-79" strain and two identical ones of "886-84" strain can be found in GenBank. For clarification of the intraspecific position of the "886-84-like" strains group we completely sequenced six previously unknown "886-84-like" strains isolated in Eastern Siberia. As a result of applying different bioinformatics approaches, we can confirm that "886-84-like" strains group is a distinct subtype of TBEV.