In a recent study published in Scientific Reports, a group of researchers analyzed tick samples from countries in Eastern Europe and the Black Sea region using nanopore sequencing (NS) and targeted amplification, identifying prevalent viruses and pathogens. They assessed the spread of newly documented tick-borne viruses into Europe.
Study: The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region. Image Credit: KPixMining/Shutterstock.com
The increasing global frequency of tick-borne infections is related to expanding tick populations, environmental and climatic changes, and rising human exposure. These infections are major contributors to vector-borne diseases and cause significant public health challenges, stressing healthcare systems.
Ticks transmit a variety of pathogens, including diverse viruses like Crimean-Congo hemorrhagic fever virus (CCHFV), Tick-borne encephalitis virus (TBEV), and Severe fever with thrombocytopenia syndrome virus (SFTSV). Recent discoveries of viruses such as Jingmen Tick Virus (JMTV) and Haseki Tick Virus (HTV) underscore the dynamic nature of these pathogens.
The identification of new viruses, like Tacheng Tick Virus (TTV)1 in Europe, emphasizes the need for further research, vector surveillance, and advanced detection methods like NS to monitor and respond to these evolving tick-borne threats effectively.
About the study
Researchers in Georgia, Bulgaria, Poland, and Ukraine collected adult ticks over various years through drag/flagging at different sites, morphologically identified them, and stored them at ultra-low temperatures.
Samples from three countries were sent to the Walter Reed Biosystematics Unit (WRBU) in the United States of America (USA), while Ukrainian samples were processed locally. The ticks were imaged using a specialized machine for dorsal and ventral views.
The team then extracted nucleic acids from the ticks, employing a meticulous process involving homogenization, lysis, and purification. The purified supernatants were stored in extremely cold conditions.
To confirm the morphological identification of the ticks, the researchers used deoxyribonucleic acid (DNA) barcoding, focusing on a specific region of the mitochondrial genome.
The pooled nucleic acids underwent complementary DNA (cDNA) synthesis, cleanup, and quantification for NS. They prepared sequencing libraries using modules and kits, barcoding each sample. They used an automated workstation for library preparation, and the sequencing was conducted on a GridION device for an extended period.
To detect specific viral pathogens in single tick samples, the researchers utilized nested polymerase chain reaction (PCR), focusing on particular protein segments of the viruses and visualizing these amplifications' results through electrophoresis.
Data analysis involved a series of steps: base-calling, demultiplexing, trimming, and filtering of the raw reads. They removed host viz tick genome data and aligned the remaining data to a comprehensive database for virus identification.
Various software and tools were used for sequence handling, similarity searches, read mapping, alignment, and phylogenetic analysis, thoroughly assessing the viral presence in the tick samples.
In the present comprehensive study involving 1,337 ticks across 11 species, researchers screened these specimens in 217 pools. They discovered virus sequences in 46.5% of these pools, with 7.3% containing viruses known to infect humans.
Notably, nearly a third of the virus-positive pools showed probable co-infections. However, co-infections of human pathogens were not present in these pools, and the prevalence of tick species and virus detection varied by country, as detailed in their report.
The study identified 21 different virus taxa in the tick pools, and among these, TTV2, JMTV, and TTV1 were notable for their presence in 5.9%, 0.9%, and 0.4% of the collections, respectively.
These pathogens were found in ticks from Poland but none in those from Bulgaria. Researchers observed specific associations between certain viruses and tick species, with TTV2 being a unique case found in multiple tick species.
In a targeted approach, the team conducted PCR and NS on individual ticks from pools where viral pathogens were detected. This included 59 individual samples from 15 pools, and these efforts led to identifying TTV2 and closely related viruses in specific pools. They sequenced PCR-positive and selected negative pieces, revealing significant viral diversity.
Further analysis showed two distinct TTV2 clades, and sequencing revealed significant genetic diversity among TTV2 strains, with notable differences in nucleotide and amino acid alignments. Phylogenetic analysis confirmed these findings, placing the detected TTV2 and related sequences in a distinct cluster.
For TTV1, despite negative PCR results, NS detected the virus in individual ticks. The sequences showed considerable divergence from known TTV1 genomes. Phylogenetic trees grouped the detected TTV1 sequences with previously reported isolates.
JMTV was detected in two pools, but insufficient material limited targeted screening. Subsequent NS revealed very low virus abundance. Other viruses detected included Changping tick virus 1 and sequences related to Norwavirus, among others.
The study also found diverse virus sequences related to various viral families, indicating a broad range of viruses present in ticks.
In addition to the pooled NS findings, individual tick analysis revealed four additional viral taxa. These findings highlight the diversity and complexity of tick-borne viruses, underscoring the importance of continued surveillance and research in this area.