The increase in tick-borne encephalitis (TBE) cases in Norway and Europe has led to a need for more research within the field. Tick-borne encephalitis virus (TBEV), a causative agent of TBE, belongs to the flaviviridae family. The main vector for transmission of the TBEV European subtype (TBEV-Eu) to humans is the castor bean tick Ixodes ricinus. TBE can cause mild to severe illness, and diagnosis of the disease is performed mostly during the phase after the virus is not detectable in the bloodstream. This study was designed to develop a method to detect and sequence the whole genome from high to low TBE-viral load in ticks and human samples from Norway. The model developed for detection and genomic polyprotein sequencing of the virus would facilitate the diagnosis of TBE as well as determine possible genetic diversity of the virus within Norway.
TBEV prevalence in questing ticks was estimated by molecular technologies on 883 ticks (750 nymphs, 73 males and 60 female adult ticks). Longer (2000 bp) and shorter (400 bp) overlapping primer pairs were designed based on partial TBE sequences from patient samples to sequence the TBEV polyprotein by Oxford Nanopore Technology (ONT). The assembling of sequences to reconstruct the polyprotein and phylogeographic relationship of the retrieved sequences with other TBEV-Eu strains available in GenBank was performed.
In samples from Larvik Site-4 in Norway, TBEV was detected in nymph pools with an overall estimated TBEV prevalence of 1% (7/75), and with a minimum infection rate of 27.4 % (20/73) in adult males and 28.3% (17/60) in adult females. There was a significant difference in prevalence between the different life stages of ticks; nymphs and adult ticks (chi-square test of independence, p-value <0.05). The positive tick samples had Ct values ranging from 18.3 to 42.9 and there was a borderline significant difference in the Ct value range between nymph and adult ticks (P= 0.07). ONT sequencing of the nymph and TBE patient samples with longer overlapping primer pairs retrieved the majority of the TBEV genomic polyprotein (~10,234 nucleotides) lacking the 5’ and 3’ non-coding regions (NCRs). The obtained TBEV sequences was closely related to TBEV Kumlinge 25-03 strain. Previously the whole genome sequence from another TBEV tick sample were published. This Mandal 2009 strain were in this study shown to be phylogenetic distant compared to the present TBEV sequences indicating the presence of different TBEV variants circulating in Norway.
Our results show that TBEV is found in ticks in a new location of Norway, suggesting an increased spread with high infection rate and prevalence. The established sequencing protocol can be implemented and refined further as a method to sequence the whole genome of TBEV in human and tick samples with medium to low virus load. The phylogeographic analysis to determine TBEV variants is necessary to understand disease severity and transmission dynamics.