Our pooled and individual metagenome-based tick screening using NS revealed 25 virus taxa including four tick-borne pathogens in samples collected from countries from Eastern Europe and around the Black Sea. Known tick-borne viruses endemic in the region, such as CCHFV or TBEV, were not detected, owing to the low prevalence of these viruses within the tick populations in targeted sites or sampling bias. However, we identified recently described tick-borne pathogens TTV2, TTV1, JMTV (7.2% in pools) and HTV (10.2% in individuals) in the sample cohort and produced abundant virus genome data for further downstream analysis.
Among the tick-borne viral pathogens identified, TTV2 was the most frequent, detected in 5.9% of the tick pools (13/217) from several sites in three of the four countries of collection, including Eastern Europe (Poland and Ukraine) and the Asian Black Sea region (Georgia). Detections were further confirmed in individual targeted PCR and NS (Supplementary Table S2). Classified in the Uukuvirus tachengense species, TTV2 was initially described during a metagenomic investigation of arthropods from China and has since been detected in D. marginatus, Dermacentor nuttalli, Dermacentor silvarum, and Hyalomma asiaticum ticks9,16,17. Subsequently, it has been documented as the causative agent in a patient with tick-associated febrile disease from China, with implications of person-to-person transmission through droplets or direct contact with body fluids9. Further detections of TTV2 in Hyalomma / Dermacentor spp. from Kazakhstan (according to GenBank records) and D. reticulatus from Romania are reported18. TTV2 is also documented in D. marginatus and the prominent CCHFV vector, H. marginatum, from ecologically diverse locations in Asia Minor, with evidence for trans-stadial or horizontal virus transmission among ticks19,20. Interestingly, we observed two distinct TTV2 clades in our phylogenetic analysis, where one of the groups also included DRUV, an unclassified Ribovirus separately detected in 4.1% of the tick pools in the study. DRUV was recently described from Croatia in D. reticulatus pools21, but to date there is scant information. Genomes of both TTV2 and DRUV consist of L and S segments, while seemingly lacking the glycoprotein encoding M segment, even in cell culture grown isolates9,21. The presence of TTV2 and DRUV divergent clades and human exposure in Europe or around the Black Sea region require further investigation.
We further detected TTV1 in 0.4% of the pools, comprising D. reticulatus from Poland. Although no amplification was observed in virus specific targeted PCR, individual NS revealed several contigs of varying sizes, encompassing all virus genome segments. Subsequent phylogenetic analyses placed all contigs with the global TTV1 sequences as a separate group within the orthonairovirus genus (Fig. 2). This is the first documentation of TTV1 in Europe. Currently, TTV1 is classified as the sole taxon in the Tacheng orthonairovirus species (genus Orthonairovirus, family Nairoviridae)15. Initially described during virus discovery efforts in arthropods from China, TTV1 was also detected in febrile diseases associated with tick bites, and as a co-infecting agent in a case with Rickettsial fever and meningitis8,16,22. Virus excretion patterns in infected individuals suggested possible transmission by direct contact with body fluids or droplets, similar to CCHFV, as noted for TTV28,9. Virus exposure was further documented in local sheep, cattle and human populations in China, with Dermacentor spp. suggested as probable vectors9. Virus genomes were detected in D. marginatus, D. silvarum, D. nuttalli, and H. asiaticum ticks in China, and also identified from spleen tissues of great gerbils (Rhombomys opimus)9,23. Recently, the virus has been documented in pools of Hyalomma aegyptium from southern Turkey24. It remains to be determined whether the lack of detection in targeted amplification in our tick cohort reflects genome diversity or low sensitivity of the PCR.
JMTV is the best studied emerging tick-borne pathogen observed in our cohort. It was identified in 0.9% of the samples, comprising I. ricinus samples collected from Poland and Georgia (Table 2). Classified within the Flaviviridae family, JMTV and related viruses (Jingmenviruses) appear to be widely distributed, having been identified in a diverse spectrum of tick species as well as in bats, cattle and rodents from Asia, Africa, Europe and the Americas25. JMTV and Alongshan virus (another Jingmenvirus) have been documented to cause human infections and subsequent seroconversion, with further evidence for exposure in domestic animals and humans6,7. Co-infections with JMTV were reported among individuals with CCHFV in Kosovo, with possible impacts on disease outcome26. In Europe, JMTV-infected ticks were reported from Romania, Serbia and the Thrace region of Turkey, while Alonghsan virus detection is more pronounced in northern Europe25,26,27,28. Recently, the integration of the JMTV polymerase gene into I. ricinus genome was documented, in ticks of both sexes and I. ricinus embryo-derived tick cell line IRE/CTVM1929. The integrated fragment of the viral genome was reported to be variable in size and forming several genovariants, with an average of 1.5 copies per cell. Several tick species other than I. ricinus were further suggested to contain the integrated segments. In field collected I. ricinus samples, the integration event was observed with a prevalence of up to 34.3% in various regions of Russia29. The JMTV findings in our study strongly suggest integrated virus sequences being detected, rather than replicating viruses. We also have previously reported NS as capable of detecting all JMTV genome segments, surpassing the sensitivity of screening by nested PCR13. Documentation of virus integration in tick vectors also has ramifications for PCR-based screening, which may require additional genome targets or steps to exclude amplification of integrated fragments.
Our findings on HTV and DRPV1 demand further discussion. We detected DRPV1 in 6.4% and 23.1% of the pooled and individual D. reticulatus samples, respectively. This virus was first described in D. reticulatus from Croatia, sharing significant NS3–NS5 protein homologies with members of the Pestivirus genus21. It is closely related to other recently documented pestivirus-like viruses, Bole Tick Virus 4 (BTV4), Trinbago Virus (TBOV), as well as HTV21. HTV genomes were recently documented in retrospectively screened sera from individuals with febrile disease associated with tick bites, with prevalences of 0.9–9.3% in different regions of the Russian Federation, with and without TBEV or Borrelia spp.11. We detected HTV in 10.2% of the single D. reticulatus samples from Georgia and Poland and observed clustering of the sequences with HTVs as well as DRPV1 in phylogenetic analyses. Given these findings and limited sequence variation between complete HTV and DRPV1 polyprotein coding sequences, these viruses represent sub-lineages of the same taxon, observed in geographically segregated regions. The capacity of either virus to produce human symptomatic infections requires further investigation.
Finally, we detected Norwaviruses, distinct from Beiji nairovirus, in 10.1% of the pools, comprising I. ricinus ticks from Bulgaria and Poland. Norwavirus is a recently described genus in the family Nairoviridae, which currently includes Grotenhout Virus (GRHV) as the sole member of the Grotenhout norwavirus species15. Closely related viruses have been documented in northern Europe and central Asia, such as Pustyn Virus (PTV) and Norway Nairovirus 1 (NWNV1), but these strains currently remain taxonomically unclassified7,14,15. Similar to TTV2, norwaviruses lack the genomic segment M, which encodes for nairovirus glycoproteins. Beiji nairovirus (BJNV), a norwavirus-related virus, was documented as a potential agent causing tick-borne febrile diseases in China, supported by experimental inoculations7. The public health implications of norwaviruses currently remain underexplored.
In conclusion, our findings reveal several tick-borne viral pathogens, recently reported from various regions from Asia, to be present in various locations in Eastern Europe and Asian Black Sea region. In any of the sampling regions, actual prevalences of individual viruses is hard to assess, due to our cross-sectional collection and non-targeted screening strategy employed in the study. Moreover, local virus clades or integrated virus genomes might undermine targeted screening due to sequence variation (TTV1) or amplification of the integrated fragments (JMTV). Currently, the lack of standardized antibody testing further hinders investigation of previous exposures and serological diagnosis. Nevertheless, these viruses should be considered in the diagnostic assessment of symptomatic cases associated with tick bites and tested when possible. NS proves to be a useful tool for monitoring tick-associated pathogens in pooled and individual samples.
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Publish date : 2023-11-14 08:00:00
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Author : love-europe
Publish date : 2024-06-11 23:44:06
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