The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Impact of nanopore-based metagenome sequencing on tick-borne virus detection.

Introduction: We evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays. Methods: Forty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach. Results: Eleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted segment read counts was observed between PCR-positive and PCR-negative samples. NS further enabled the initial description of Quaranjavirus sequences in ticks, where human and avian pathogenicity of particular isolates had been previously documented. Discussion: NS was observed to surpass broad-range and nested amplification in detection and to generate sufficient genome-wide data for investigating virus diversity. It can be employed for monitoring pathogens in tick vectors or human/animal clinical samples in hot-spot regions for examining zoonotic spillover.