Table 1.
TBEV isolates included in the characterization of 3′ non-coding region.
Table 2.
Primers and PCR conditions used for amplification of seven overlapping fragments (5′NCR-prM, E, E-NS3, NS3, NS3-NS5, NS4B-NS5, and NS5-3′NCR) to obtain complete genomic sequence.
Figure 1.
Genomic variations detected within the 3′NCR of the TBEV strains Saringe 2009 and Mandal 2009.
(A) Alignment of 3′NCR partial sequences from pcDNA3.1 clones of the Saringe 2009 virus together with Neudoerfl, Hypr, Mandal 2009, and the Toro 2003 replicon used as control. Nucleotides' positions refer to the strain Neudoerfl. Different quasispecies variants of Saringe 2009 are labeled V1–V17 and number of clones with identical sequences is provided within parenthesis. (B) Schematic presentation of the 3′NCRs of Neudoerfl, Saringe 2009, Toro 2003, Mandal 2009, and Hypr showing heterogeneity in the V 3′NCR. The unique sequence elements: cyclization motifs (CSA, CSb-1, and CSb-2), repetitive sequences (R1, R2, and R3), poly(A) tract, homopurine box (PU), homopyrimidine box (PY), and flavivirus-conserved CACAG box are highlighted. Figure is adapted from Wallner et al., (1995).
Figure 2.
Genomic variations detected within the 3′NCR of W-TBEV strains.
Alignment of 3′NCR partial sequences from pcDNA3.1 clones of the tick and human isolates of the TBEV to explore the existence of different variants within the individual viral pool. The two quasispecies variants of the Neudoerfl are labeled V1 and V2, respectively. Number of sequenced clones with identical sequences is mentioned within parenthesis. Nucleotide numbers correspond to the TBEV strain, Neudoerfl.
Figure 3.
Phylogenetic analysis based on the 3′end of the NS5 gene (543 nt).
Nucleotide sequences of 31 strains were analyzed by the Neighbor-joining method. The tree was inferred from 500 bootstrap replicates in MEGA5 [16], and the percentage of replicate trees is shown next to the branches [32]. The PV was used as outgroup and the tree was drawn to scale, with branch lengths corresponding to the substitutions per site. The TBEV strains sequenced in this study are indicated in bold. The positions of Mandal 2009 and Hypr are indicated by arrows.