Metatranscriptomics reveals 89 tick virus genomes across Italy

Italian ticks carry a far richer array of viruses than previous national surveys suggested, according to a new metatranscriptomic study that sequenced ticks collected along a north-south gradient of the country. By reconstructing viral genomes directly from tick RNA, researchers assembled 89 uncultivated viral genomes corresponding to 31 viral operational taxonomic units, including 10 putative new species and 19 species reported in Italy for the first time. The work, published in the journal Discover Viruses, offers the first genome-scale characterization of tick-associated viral diversity in Italy, a country where several regions are known to be affected by tick-borne viral infections but where comprehensive virome data had been lacking.

The research team, led by Andrea Silverj and colleagues at institutions including the Edmund Mach Foundation, analyzed 22 tick transcriptomes from five tick species: Dermacentor marginatus, Haemaphysalis punctata, Ixodes ricinus, Rhipicephalus bursa and Rhipicephalus sanguineus. Samples came from three regions chosen to span both latitude and ecology: Trentino-Alto Adige in the north, representing alpine and subalpine habitats; Lazio in central Italy, with mixed rural and peri-urban Mediterranean landscapes; and Sicily in the south, with warmer climates and distinct vegetation. Ticks were collected in 2018 in Trentino-Alto Adige, in 2011-2012 in Lazio and in 2020 in Sicily, by dragging or flagging vegetation, and were identified microscopically by species, sex and life stage.

The sequencing effort was substantial. The researchers generated more than 1.1 billion paired-end 150-base-pair reads on an Illumina NovaSeq 6000 platform. After trimming adapters, removing low-quality reads, filtering out tick host sequences by alignment to the closest available reference genomes, and depleting ribosomal RNA, they assembled the remaining reads using two different assemblers, SPAdes and Trinity, to reduce methodological bias. The combined assembly produced 180 candidate contigs, which were filtered through a series of similarity searches against viral and non-viral databases, length thresholds and coverage checks. The final curated set contained 99 putative viral contigs, which were assembled into 89 uncultivated viral genomes, a number lower than the contig count because some viral species possess segmented genomes composed of multiple contigs.

To classify these genomes, the team combined BLAST-based similarity searches with phylogenetic analysis of the RNA-dependent RNA polymerase, or RdRp, protein and genome comparison tools that assess gene content and synteny. Viruses showing at least 80 percent identity over the whole genome, or at least 90 percent identity in the RdRp gene, to a known species were assigned to that species and labeled known viruses; the rest were treated as putative new viruses, a term the authors use strictly to mean viruses that could not be assigned to an existing classified taxon based on genetic distance, not simply viruses sequenced for the first time. The identified viruses belonged to thirteen known viral families as well as unclassified groups. Some families appeared host-specific: Chuviridae were found only in R. bursa, while Botourmiaviridae, Flaviviridae, Partitiviridae, Rhabdoviridae and an unclassified Tolivirales family were mainly or exclusively present in I. ricinus. Others, such as the unclassified Cirlivirales and Orthototiviridae, showed broader host ranges.

The most abundant viruses in terms of reads per kilobase per million were members of the Flaviviridae, in particular tick-borne encephalitis virus, or TBEV. TBEV accounted for almost all Flaviviridae reads and was detected in five I. ricinus samples, four of which came from Trentino-Alto Adige, plus one H. punctata sample from Sicily. The Trentino detections were independently validated: PCR screening of the same samples in a previous study produced coherent results, and samples that tested PCR-positive showed very high numbers of metatranscriptomic reads mapping to the assembled TBEV genomes, exceeding two million reads in three samples, while a weakly PCR-positive sample showed far fewer. The Sicilian detection, however, is suspect. The sample contained only 586 viral reads, well below the index-hopping contamination threshold the authors set at 0.1 percent of the most abundant viral reads in another library sequenced on the same flow cell, and the Sicilian genome was closely similar to and phylogenetically nested with a Trentino genome sequenced in the same batch. The authors therefore treat it as a likely false positive and call for further studies in Sicily to verify whether TBEV is actually present there.

The phylogenetic analysis of TBEV placed all five newly generated sequences, four from Trentino-Alto Adige and one from Sicily, within the European cluster of the species, one of five major genetic lineages that also include Far Eastern, Baikalian, Siberian and Himalayan groups. The new sequences form a clade with an Italian genome isolated in Friuli-Venezia Giulia in 2021, a German sequence collected in 2005 and a Finnish sequence from a sample collected in 1960, a grouping that reflects the virus’s slow substitution rate. The I. ricinus ticks positive for TBEV in this study came from a previous molecular screening of 2,500 samples, and the authors note that the frequency of infected ticks in nature is much lower than these targeted samples suggest.

Among the other findings, the study reports the first detection in Italy of Haseki virus, a Flavi-like virus recently discovered in Russia in Ixodes persulcatus ticks and in patients with fever and respiratory symptoms after tick bites, here found in H. punctata from Sicily. The researchers also detected a virus similar to Bole tick virus 3 in I. ricinus and R. bursa, the first report of this virus in Ixodes ticks, as it had previously been found only in Rhipicephalus and Hyalomma. Tacheng tick virus 5, a serotype whose variant was recently isolated in China from a hospitalized patient with multi-organ syndrome after a tick bite, was also recovered, with a nearly complete genome reconstructed. Four phleboviruses were identified at the contig level: Brown dog tick phlebovirus 2, Norway phlebovirus 1, Leuven phlebovirus and Tick phlebovirus. Although phleboviruses found in Italy are not clearly associated with human disease, the authors note their presence is noteworthy because some phleboviruses can undergo gene mutation and genetic reassortment that could produce new pathogenic strains.

Several detected viruses appear to be tick symbionts rather than threats. Grotenhout virus and Bronnoya virus, both members of the order Bunyavirales, are considered part of the core tick virome. Hubei toti-like virus 24, a double-stranded RNA virus in the family Orthototiviridae, was particularly abundant in D. marginatus samples, and the new sequences more than doubled the number of available genomes for this group. For Xinjiang tick totivirus 2, genetic distances suggested the sequences may represent at least three distinct viral species, supporting the need to expand formal viral taxonomy. The team also detected a plant virus, Apple Mosaic Virus, in an I. ricinus from Val di Non, an apple-producing area of Trentino-Alto Adige, likely a remnant from tick contact with plant surfaces or cuticular contamination, since the ticks were washed only in ethanol and water rather than aggressive treatments such as bleach that would better remove surface viruses.

Beyond viruses, the read-profiling analysis of non-tick, non-viral reads revealed well-known tick symbiotic bacteria, including Midichloria mitochondrii and Delftia acidovorans detected only in I. ricinus, Rickettsia species only in D. marginatus, and Coxiella species only in Rhipicephalus ticks, alongside Wolbachia pipientis. The authors caution that metatranscriptomic abundance estimates reflect transcriptional activity rather than actual genomic abundance, though high relative transcript levels strongly suggest the corresponding species were present.

The authors emphasize that several results should be interpreted with caution. The sampling design was unbalanced and temporally split: different tick species were collected in different regions in different years, which prevented formal tests of geographic or species effects on viral profiles and made beta-diversity analysis impossible. Alpha diversity comparisons were therefore treated as descriptive rather than inferential. Within those limits, R. sanguineus showed the highest alpha diversity among tick species, though differences were not statistically significant, while Sicily was the most diverse region, with the Lazio-Sicily pair the only significant individual contrast. Northern populations showed a higher presence of viruses of potential health concern, but the authors state this observation should be interpreted cautiously given the design. The study, whose data are publicly available through the NCBI Sequence Read Archive and GenBank, expands available reference genomes for several recently described tick viral species and, the authors argue, provides a foundation for future surveillance activities targeting tick-borne viruses in Italy.

Subject of Research: Viral diversity and novel virus discovery in Italian ticks using metatranscriptomics

Article Title: Virome diversity and novel virus discovery in Italian ticks through metatranscriptomics

Article References: Silverj, A., Alfano, N., Rosso, F., Tagliapietra, V., Toma, L., Di Luca, M., Torina, A., La Russa, F., Segata, N., Rota-Stabelli, O., & Rizzoli, A. (2026). Virome diversity and novel virus discovery in Italian ticks through metatranscriptomics. Discover Viruses, 3(1), Article 13. https://doi.org/10.1007/s44370-026-00047-y

Image Credits: AI Generated

DOI: 10.1007/s44370-026-00047-y

Keywords: ticks, virome, metatranscriptomics, tick-borne encephalitis virus, phlebovirus, Italy, viral genomes, phylogenomics, Ixodes ricinus, surveillance, uncultivated viral genomes, Haseki virus