Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus from Ticks Collected from Cattle in Kyrgyzstan, 2023

Abstract

Ticks are important vectors of the tick-borne encephalitis virus (TBEV). In Kyrgyzstan, the livestock farming trade and nomadic lifestyle enable tick-borne diseases to be imported from neighboring countries, but there are few relevant studies. In this study, we collected 40 ticks from cattle in Kyrgyzstan. Molecular marker analysis identified the ticks as Ixodes persulcatus (97.5%; n = 39) and Haemaphysalis punctata (2.5%; n = 1). Real-time PCR screening revealed two ticks to be positive for TBEV, but only one tick was amplified using nested PCR targeting the TBEV envelope (E) and non-structure 5 (NS5) gene. The obtained sequences belonged to the TBEV Siberian subtype and phylogenetic tree analysis results confirmed that the virus was related to the Bosnia strain. We also performed next-generation sequencing, which confirmed the TBEV Siberian subtype. Continuous research and surveillance of TBEV in Kyrgyzstan are required to provide further information on tick-borne diseases.

Keywords: Kyrgyzstan; next-generation sequencing; tick-borne encephalitis virus.

Figure 1

Map for study site in the Kemin region (red triangle), Chuy province, Kyrgyzstan. Adapted from [25].

Figure 2

Phylogenetic analysis for tick identification based on the partial nucleotide sequences of (A) COⅠ, (B) 12S rDNA, and (C) 16S rDNA. The maximum likelihood (ML) method was used with the Kimura 2-parameter model. The number on the branches indicates bootstrap percentages based on 1000 replications. The cut-off value for the consensus tree was 70%. The sequences identified in this study are indicated by red dots. The number of sequences (n) with a corresponding identical sequence is shown if the sequence was detected in more than one case. The bar indicates the number of nucleotide substitutions per site.

Figure 3

Phylogenetic analysis of TBEV (A) E and (B) NS5 gene-positive sequences. The maximum likelihood (ML) method was used with the Kimura 2-parameter model. The number on the branches indicates bootstrap percentages based on 1000 replications. The cut-off value for the consensus tree was 70%. The sequences identified in this study are indicated by red dots. The bar indicates the number of nucleotide substitutions per site.

Figure 4

Radial phylogenetic tree of TBEV sequences. The radial phylogenetic tree was constructed using IQ-tree v2.1.2 and the maximum likelihood (ML) with the General Time Reversible (GTR) model, embedded in Nextstrain CLI 7.2.0. The tree was divided into seven subtypes (Far Eastern, Baikalean-1, East Siberian, Siberian, European, Western European, and Himalayan), with labels indicating the corresponding GenBank accession numbers of the genomes. Within the tree, OR896869 was classified as the Siberian subtype, revealing a close relationship with the MH645616.1 (Bosnia) and KJ626343.1 (Kyrgyzstan) genome sequences.