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.

Brown Rot Syndrome and Changes in the Bacterial Сommunity of the Baikal Sponge Lubomirskia baicalensis.

Mass mortality events have led to a collapse of the sponge fauna of Lake Baikal. We describe a new Brown Rot Syndrome affecting the endemic species Lubomirskia baicalensis. The main symptoms are the appearance of brown patches at the sponge surface, necrosis, and cyanobacterial fouling. 16S rRNA gene sequencing was used to characterize the bacterial community of healthy versus diseased sponges, in order to identify putative pathogens. The relative abundance of 89 eubacterial OTUs out of 340 detected has significantly changed between healthy and diseased groups. This can be explained by the depletion of host-specific prokaryotes and by the appearance and proliferation of disease-specific OTUs. In diseased sponges, the most represented OTUs belong to the families Oscillatoriaceae, Cytophagaceae, Flavobacteriaceae, Chitinophagaceae, Sphingobacteriaceae, Burkholderiaceae, Rhodobacteraceae, Comamonadaceae, Oxalobacteraceae, and Xanthomonadaceae. Although these families may contain pathogenic agents, the primary causes of changes in the sponge bacterial community and their relationship with Brown Rot Syndrome remain unclear. A better understanding of this ecological crisis will thus require a more integrative approach.

Molecular identification and phylogeny of Dermacentor nuttalli (Acari: Ixodidae).

Dermacentor nuttalli is an epidemiologically important tick in Palearctic Asia which transmits several infectious diseases including tularemia, North Asian tick-borne rickettsiosis, Lyme disease and tick-borne encephalitis. The genetic specificity and phylogeny of D. nuttalli from four geographic localities in Eastern Siberia were characterized using the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene and internal transcribed spacer 2 (ITS2). Low genetic diversity was observed in the populations of ticks distributed from South Siberia to North China. From 11 detected mt 16S haplotypes, one was found in all populations, whereas the others were restricted to specific localities. These results suggested that the genetic structure of D. nuttalli represents integrated populations with no geographic isolation across the distribution area. The phylogenetic reconstructions inferred from the mt 16S rRNA gene and ITS2 were in agreement and showed a distinct D. nuttalli clade within a monophyletic Eurasian lineage of Dermacentor sp.

Tick-borne encephalitis virus in Eastern Siberia: complete genome characteristics.

Tick-borne encephalitis (TBE) is one of the most important arboviral diseases across Eurasia. TBE virus (TBEV) is transmitted by tick bite and causes a potentially fatal neurological infection in humans. In the Russian Federation, TBE is endemic in most regions, with 3000-5000 cases of the disease annually. To characterise TBEV in Eastern Siberia, the complete genomes of five TBEV isolates from patients with different clinical manifestations were sequenced. The results show that the Siberian and Far Eastern subtypes of TBEV cause the disease in people in Eastern Siberia. Complete genome analysis revealed an unexpectedly high genetic variability within the Siberian subtype.

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus.

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20 ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.