In a recently published study, Dr Scientific reportA A team of researchers analyzed tick samples from countries in Eastern Europe and the Black Sea region using Nanopore Sequencing (NS) and identified prevalent viruses and pathogens. They estimated the spread of the newly documented tick-borne virus in Europe.
Study: A wide range of tick-borne viruses originating in Eastern Europe and the Black Sea region. Image credit: KPixMining/Shutterstock.com
Background
The increasing global frequency of tick-borne infections is related to increasing tick populations, environmental and climate change, and increasing human exposure. These infections are major contributors to vector-borne diseases and pose significant public health challenges by straining healthcare systems.
Ticks transmit a variety of pathogens including Crimean-Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and thrombocytopenia syndrome virus (SFTSV), causing severe fever. Recent discoveries of viruses such as Zingmen tick virus (JMTV) and Haseki tick virus (HTV) underscore the dynamic nature of these pathogens.
The detection of new viruses such as Tacheng Tick Virus (TTV)1 in Europe emphasizes the need for further research, vector surveillance and improved detection methods such as NS to effectively monitor and respond to these evolving tick-borne threats.
About the study
Researchers in Georgia, Bulgaria, Poland, and Ukraine collected adult ticks over several years by trapping/trapping at different sites, morphologically identified them, and stored them at ultra-low temperatures.
Samples from all three countries were sent to the Walter Reed Biosystematics Unit (WRBU) in the United States, while Ukrainian samples were processed locally. Ticks were imaged using a special machine for dorsal and ventral views.
The team then extracted the nucleic acid from the ticks, employing a delicate process involving homogenization, lysis and purification. Purified supernatants were stored extremely cold.
To confirm morphological identification of ticks, researchers used Deoxyribonucleic acid (DNA) barcoding, focusing on a specific region of the mitochondrial genome.
Synthesis, purification and quantification of complementary DNA (cDNA) for pooled nucleic acid ns. They prepared sequencing libraries using modules and kits by barcoding each sample. They used an automated workstation for library preparation and sequencing was carried out on a Gridion device over an extended period of time.
To identify specific viral pathogens in single tick samples, researchers used nested polymerase chain reaction (PCR), focusing on specific protein segments of the virus and visualizing the results of this amplification by electrophoresis.
Data analysis involves a series of steps: base-calling, demultiplexing, trimming and filtering of the raw text. They remove the host such as Tick genome data and align the remaining data with a comprehensive database for virus detection.
A variety of software and tools were used for sequence handling, similarity search, read mapping, alignment and phylogenetic analysis to thoroughly assess viral presence in tick samples.
Results of the study
In the current comprehensive study involving 1,337 ticks across 11 species, researchers examined these samples in 217 pools. They discovered 46.5% of virus sequences in these pools, including 7.3% of viruses known to infect humans.
Notably, approximately one third of the virus-positive pool showed possible co-infection. However, co-infection with human pathogens was not present in these pools, and 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 pool and among these TTV2, JMTV and TTV1 were notable for their presence in 5.9%, 0.9% and 0.4% of collections respectively.
The pathogen was found in ticks from Poland but none from Bulgaria. Researchers have 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 the pool where the viral pathogen was detected. These included 59 individual samples from 15 pools, and these efforts led to the identification of TTV2 and closely related viruses in specific pools. They sequenced PCR-positive and selected negative fragments, revealing significant viral diversity.
Further analysis showed two distinct TTV2 clades, and sequencing revealed significant genetic diversity among TTV2 strains, with significant differences in nucleotide and amino acid alignments. Phylogenetic analysis confirmed these results, placing the detected TTV2 and related sequences in a distinct cluster.
For TTV1, NS detected virus in individual ticks, despite negative PCR results. The sequences showed significant divergence from the known TTV1 genome. Phylogenetic trees grouped the identified TTV1 sequences with previously reported isolates.
JMTV was detected in two pools, but insufficient material limited targeting. Later NS revealed very low virus abundance. Other viruses identified include sequences related to Changping tick virus 1 and Norvavirus.
The study also found sequences of different viruses belonging to different viral families, indicating a wide range of viruses present in ticks.
In addition to the pooled NS findings, analysis of individual ticks revealed four additional viral taxa. These findings highlight the diversity and complexity of tick-borne viruses, emphasizing the importance of continued surveillance and research in this area.