Virome diversity of ticks feeding on domestic mammals in China

Abstract

Ticks are considered the second most common pathogen vectors transmitting a broad range of vital human and veterinary viruses. From 2017 to 2018, 640 ticks were collected in eight different provinces in central and western China. Six species were detected, including H.longicornis, De.everestianus, Rh.microplus, Rh.turanicus, Rh.sanguineous, and Hy.asiaticum. Sixty-four viral metagenomic libraries were constructed on the MiSeq Illumina platform, resulting in 13.44 ​G (5.88 ​× ​10⁷) of 250-bp-end reads, in which 2,437,941 are viral reads. We found 27 nearly complete genome sequences, including 16 genome sequences encoding entire protein-coding regions (lack of 3′ or 5′ end non-coding regions) and complete viral genomes, distributed in the arboviral family (Chuviridae, Rhabdoviridae, Nairoviridae, Phenuiviridae, Flaviviridae, Iflaviridae) as well as Parvoviridae and Polyomaviridae that cause disease in mammals and even humans. In addition, 13 virus sequences found in Chuviridae, Nairoviridae, Flaviviridae, Iflaviridae, Hepeviridae, Parvoviridae, and Polyomaviridae were identified as belonging to a new virus species in the identified viral genera. Besides, an epidemiological survey shows a high prevalence (9.38% and 15.63%) of two viruses (Ovine Copiparvovirus and Bovine parvovirus 2) in the tick cohort.

Highlights

• •640 ticks (6 tick species) were collected from central and western China, with diverse tick hosts.
• •Viruses discovered from these ticks were mainly assigned into 21 virus families, including 6 DNA virus families and 15 RNA virus families.
• •Virus genomes from Chuviridae, Rhabdoviridae, Nairoviridae, Phenuiviridae, Parvoviridae, and Polyomaviridae ​were fully characterized.

1. Introduction

Ticks (class Arachnida, subclass Acari) are highly specialized obligate hematophagous arthropods (Anderson and Magnarelli, 2008) spreading around the world from tropic to subarctic regions (Jia et al., 2020), with the oldest records dating back to the mid-late Cretaceous (Peñalver et al., 2017). Ticks are considered the second most common pathogen vectors after the mosquitoes (Parola and Raoult, 2001; Kernif et al., 2016), transmitting a broad range of pathogenic microorganisms, protozoa, and viruses to mammals, birds, reptiles, and amphibians (Sukumaran et al., 2006; Dehhaghi et al., 2019). There are over 900 species of ticks globally, and many of them are essential in pathogen replication and spread (Shi et al., 2018), especially members of Ixodidae. More than 21 species harbour and transmit pathogens known to contribute to human disease (Anderson and Magnarelli, 2008), mainly in the following genera: Ixodes, Haemaphysalis, Hyalomma, Dermacentor, Rhipicephalus, and Boophilus.

Tick-borne viruses (TBVs), presenting in numerous viral families, form a significant constituency of pathogens that can cause serious and even fatal illnesses. One of the first tick-borne viruses identified in people was the flavivirus (louping ill virus), the causative agent of louping ill in sheep, monkeys, mice, and even human (Rivers and Schwentker, 1934). Tick-borne encephalitis virus (TBEV) is a flavivirus that leads to tick-borne encephalitis (TBE) that has no effective treatment to date. Its transmission from western Europe to the eastern coast of Japan led to over 10,000 cases every year (Lindquist and Vapalahti, 2008). One hundred and fifty eight cases of Crimean-Congo hemorrhagic fever virus (CCHFV) infection in Africa, Asia, and Europe were published, with an overall case fatality rate of 32.4% (Tsergouli et al., 2020). Notably, the incidence of some tick-borne infections and transmissions in recent decades showed an increasing or fluctuating tendency due to contemporary urbanization, deforestation, climate change, and rapidly changing interactions between people, animals, and their respective habitats (Jaenson et al., 2012; Tijsse-Klasen et al., 2014). Bourbon virus is a newly re-emerging arbovirus belonging to the genus Thogotovirus. It was first documented in a patient with thrombocytopenia and leukopenia after tick bites in 2015 (Kosoy et al., 2015). In 2019, Alongshan virus (ALSV), which belongs to the Jingmenvirus group of the family Flaviviridae, was found in 86 patients from Inner Mongolia and Heilongjiang Provinces who represented fever, headache, and a history of tick bites (Wang et al., 2019b). In 2021, researchers identified a new orthonairovirus, Songling virus (SGLV), from 42 patients who reported being bitten by ticks in Heilongjiang Province in northeastern China, with the main clinical manifestations being headache, fever, depression, fatigue, and dizziness (Ma et al., 2021). Hence, investigating tick-borne virome in the natural environment is critical for improved control and prevention of large epidemics caused by TBVs.

Metagenomics is routinely performed to assess microorganisms in the natural world, where they are part of communities frequently dominated by as-yet uncultivated populations (Delogu et al., 2020). The rise of viral metagenomic analysis has transformed virus discovery and revealed a remarkable diversity of viruses sampled from ticks (Zhang et al., 2018a, 2019). For example, researchers found a large monophyletic group of newly discovered viruses by viral metagenomics and named the Chuviridae in 2015 (Li et al., 2015). Researchers identified five clades of RNA viruses in which RdRp domains are so divergent that they might be considered as new virus families or orders, and provisionally named them as “Yuevirus”, “Qinvirus”, “Zhaovirus”, “Weivirus” and “Yanvirus” (Shi et al., 2016a). An increasing number of viruses have been identified from different tick species in recent years, suggesting that the current knowledge of TBVs may be a drop in the bucket.

Central and western China is a suitable habitat for ticks because of the rich biological resources and the complex and diverse ecological environment: Xinjiang (northern Xinjiang region) is one of the essential natural foci in China; Qinghai has the source region of three rivers, one of the highest concentrations of high-altitude biodiversity in the world; Shaanxi is rich in biological resources with outstanding diversity, and Qinba Mountain area is known as the “biological gene pool”; Camels, the ship of the desert, are distributed mainly in Gansu Province; Yunnan has the abundant rainfall, lush vegetation, diverse wildlife, and livestock; Hebei is an eastern-coastal region with recently increasing cases of tick-borne death; Henan is a critical economic province in central China; Hubei is a central location with strong industry in terms of animal husbandry (Fig. 1). For instance, severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by a novel phlebovirus (SFTS virus, SFTSV) in the family Phenuiviridae (Li et al., 2019). The reported cases increased annually from 2010 to October 2016 and had a 5.3% national average mortality rate (Zhang et al., 2017). The highest numbers of reported cases occurred in Henan (37%), Hubei (12.6%), and Haemaphysalis longicornis is considered the most likely vector for transmission (Li et al., 2015). TBE cases have been reported in Xinjiang and Yunnan Province, and the primary vector is the members of genus Ixodes (Yoshii et al., 2017).

Fig. 1.

Map of tick collection. The depth of blue represents the number of ticks.

However, the virome profiling of the ticks in these provinces has not been fully characterized. This study collected six tick species from domestic animal surfaces from 2017 to 2018 in the eight provinces. We investigated the virome of ticks using viral metagenomics and the prevalence of important identified viruses among tick samples. The data will contribute to understanding viral diversity harboured by ticks and virus evolutionary history.

2. Materials and methods

2.1. Tick samples

The sampling sites were distributed in eight provinces in central and western China, including Hebei, Henan, Hubei, Hunan, Yunnan, Shaanxi, Gansu, Xinjiang, and Shanghai Municipality (Fig. 1). Ticks were either directly picked from sheep, cattle, dog, camel, and chicken or captured using a tick drag-flag method from grassland (Fig. 1). Table 1 describes the specific type of ticks. The samples were initially identified by experienced field biologists and further confirmed by analyzing sequences of the cytochrome oxidase I gene (COI), which encodes a subunit of the cox1 protein (Walker et al., 2003; Shi et al., 2016b, 2021; Getange et al., 2021). Six species from four genera (Haemaphysalis, Dermacentor, Rhipicephalus, Hyalomma) of ticks were detected from sampled ticks, including 390 Haemaphysalis longicornis (H.longicornis), 110 Hyalomma asiaticum (Hy.asiaticum), 40 Rhipicephalus microplus (Rh.microplus), 30 Dermacentor everestianus (De.everestianus), 60 Rhipicephalus turanicus (Rh.turanicus) and 10 Rhipicephalus sanguineous (Rh. sanguineous) (Table 1). Of these, H.longicornis ticks were widely distributed among Henan, Shaanxi, and Yunnan provinces, Rh.microplus, De.everestianus, Rh.turanicus, and Hy.asiaticum were limited in Hubei, Qinghai, Hebei, and Gansu provinces, respectively. The remaining ticks were found from Shanghai Municipality (Fig. 1). Most of the ticks were engorged (590 out of 640) and were collected as follows: H.longicornis ticks were collected on sheep, cattle, and camel; Rh.microplus ticks were gathered from cattle; De.everestianus ticks parasitized in sheep and grassland; Rh.turanicus ticks fed on sheep, dog, and chicken; Hy.asiaticum ticks lived on camels; Rh. sanguineous ticks were collected on grassland (Fig. 1, Table 1).

Table 1.

Pooling information of ticks before sequencing.

Year	Location	Host	Type	Species	Pools (10 per pool)	Total number
2017	Henan	Ovine	adults, nymph, larva	H. longicornis	319–321, 323–326	70
	Hubei	Bovine	adult, salivary ​gland, midgut, ovary	Rh. microplus	322, 359–361	40
2018	Qinghai	Grassland	1 adult	De. everestianus	328	30
		Ovine	2 adults		327, 329
	Hebei	Ovine	1 adult, 1 fat body, 1 ganglion, 1 salivary ​gland	Rh. turanicus	331, 362–364	60
		Chicken	1 adult		358
		Dog	1 adult		330
	Shanxi	Ovine	5 adults, 1 nymph, 1 salivary gland, 1 midgut, 1 ovary	H. longicornis	332–335, 337–338, 365–367	110
		Bovine	1 adult		336
		Dog	1 nymph		348
	Henan	Ovine	1 nymph, 1 larva, 3 adults, 1 salivary gland, 1 midgut, 1 ovary	H. longicornis	339–340, 342–344, 368–370	120
		Grassland	1 nymph, 2 larvae		341, 353–354
		Bovine	1 adult		347
	Gansu	Camel	2 adults, 1 egg, 1 larva, 1 salivary gland, 1 midgut, 1 ovary	Hy. asiaticum	345–346, 356, 371–373	60
	Yunnan	Bovine	2 adults, 2 egg, 2 larvae, 1 salivary gland, 1 midgut, 1 ovary	H. longicornis	349–352, 355, 357,374–376	90
	Xinjiang	Ovine	2 adults, 1 salivary ​gland, 1 midgut, 1 ovary	Hy. asiaticum	377–378, 380–382	50
	Shanghai	Grassland	1 adult	Rh. sanguineous	379	10

2.2. Tick sample pool preparation

The collected ticks were divided into sixty-four groups (Pool 319–382) of ten based on their location, species, and sampling date (Table 1). All samples were collected with disposable materials and shipped on dry ice. Each tick pool was initially washed with 75% alcohol and then washed with 1 ​mL of phosphate-buffered saline (PBS) to eliminate external contaminants (Zhang et al., 2018b; Zhao et al., 2020b; Shi et al., 2021). Every pool was suspended in 600 ​μL of Dulbecco's phosphate-buffered saline (DPBS), added steel ball to vigorously vortex oscillation for 5 ​min, and then frozen and thawed in liquid nitrogen, repeating the above steps for three times. The 500 ​μL supernatants were then collected from each pool after centrifugation (5 ​min, 15,000×g, 4 ​°C).

2.3. Viral metagenomic analysis

The 500 ​μL supernatant of every pool was purified through a 0.45-μm filter (Millipore, MA, USA) to remove eukaryotic and bacterial cell-sized particles. A 166.5 ​μL aliquot of each filtrate was mixed with 10 ​μL of a DNases mixture (Turbo DNase, Ambion, Vilnius, Lithuania; Baseline-ZERO, Epicentre,WI, USA), 20 ​μL of DNase I buffer (Ambion, Vilnius, Lithuania), 3 ​μL of benzonase (Novagen, Gujarat, India) and 0.5 ​μL of RNase A (Fermentas, Burlington, Canada) and incubated at 37 ​°C for 60 ​min to eliminate host genomic DNA and other free nucleic acids (Zhang et al., 2014, 2016, 2017). Total nucleic acids, including RNA and DNA protected from nuclease digestion within viral capsids, were then extracted using a QIAamp Viral RNA Mini Kit (QIAGEN HQ, Germany) (Zhao et al., 2022) following the manufacturer's instructions. For RNA viruses, a reverse transcription kit (SuperScript III Reverse Transcriptase, Invitrogen, CA, USA) was used for reversely transcribing RNA into cDNA, after which the product was denatured at 95 ​°C for 2 ​min and quickly placed on ice for about 2 ​min. Then the DNA polymerase I large fragment (Klenow, HQ, Germany) was added to synthesize the second strand of cDNA (double-strand DNA, dsDNA). For single-strand DNA (ssDNA) viruses, ssDNA was converted to dsDNA using the Klenow reaction and the product was utilized to construct libraries. Specifically, 12 ​μL nucleic acid extracts were added to the reaction system for synthesizing dsDNA (total reaction system: 20 ​μL) and the experiments were performed in the same tube. Sixty-four libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the MiSeq Illumina platform with 250 bp-ends with dual barcoding for each pool.

2.4. Bioinformatics analysis

Using vendor software from Illumina, paired-end reads of 250 bp generated by MiSeq sequencing were debarcoded (Zhang et al., 2014, 2016, 2017; Wang et al., 2019a). An in-house analysis pipeline running on a 32-node Linux cluster was used to process the data. Reads were considered duplicates if bases 5 to 55 were identical and only one random copy of duplicates was kept. Exact duplicate reads were removed using the Dedupe plugin v37.28 (Wagner et al., 2020). Clonal reads were removed, and low-sequencing-quality tails were trimmed using Phred quality score 10 (Q10) as the threshold. Adaptors were trimmed using VecScreen (https://www.ncbi.nlm.nih.gov/tools/vecscreen/) with the default parameters, which uses NCBI BLASTn with specific parameters designed for adapter removal. The cleaned reads were then compared to an in-house non-virus non-redundant (NVNR) protein database to remove false-positive viral hits using DIAMOND (Buchfink et al., 2015) BLASTx search with default parameters. The NVNR database was compiled using non-viral protein sequences extracted from an NCBI nr fasta file (based on annotation taxonomy, excluding the virus kingdom). Then, taxonomic classification for DIAMOND results was parsed using MEGAN (Bağcı et al., 2021) to perform the LCA-assignment algorithm according to default parameters. Gene assembly, prediction, and annotation were completed with Geneious software (Tanca et al., 2017).

2.5. Nested PCR

Gaps between contigs of the same viral genome that could not be merged using Geneious Prime were filled by Nested PCR based on the design of PCR primers bridging the gaps and then sequenced by Sanger method. Nested PCR was performed using rTaq DNA Polymerase (Takara, Kusatsu, Japan). The specific primers are shown in the Supplementary Table S1. At least three positive clones of each fragment were sequenced.

2.6. Phylogenetic analysis

The RdRp is the only conserved-sequence domain across all RNA viruses and was used for phylogenetic inference (Shi et al., 2016a). The RdRp predicted were aligned with their corresponding homologs of reference viruses using MAFFT version 7.450 (L-INS-i algorithm) (Katoh and Standley, 2013) and MUSCLE multiple sequence alignment program with default settings (Edgar, 2004). The final alignment lengths were 572 amino acids (aa), 698 aa, 553 aa, 525 aa, 1812 aa, 231 aa, 209 aa and 1156 aa for data sets of overall, Bunyavirales, Mononegavirales-Jingchuvirales, flavi-like (NS3 and NS5), Iflaviridae, Parvoviridae (structural and nonstructural protein) and Polyomavirinae data sets, respectively. All phylogenetic analysis was performed based on a Bayesian method implemented in MrBayes version 3.2.7 (Huelsenbeck and Ronquist, 2001; Ronquist et al., 2012). In the MrBayes analyses, we used two simultaneous runs of Markov chain Monte Carlo sampling, and the runs were terminated upon convergence (standard deviation of the split frequencies <0.01) (Li et al., 2015). The visualization and beautification of the phylogenetic trees were achieved by the iTOL online tool (https://itol.embl.de/) and Figtree version 1.4.43 (http://tree.bio.ed.ac.uk/software/figtree/).

2.7. Prediction of protein domains and functions

All protein prediction was conducted by Geneious software version 2019.2.3. Putative exon and intron were predicted by Netgenes2 (http://www.cbs.dtu.dk/services/NetGene2/). Functional domains were predicted using conserved domain database in NCBI (Lu et al., 2020).

2.8. Statistical analysis

The species rarefaction curve is also calculated and generated by MEGAN software v6.20.19 (Huson et al., 2007) in rarefaction window to evaluate sampling completion in each library. Alpha-diversity analyses, normalization and visual presentation were executed using Microbiomeanalyst (Dhariwal et al., 2017; Chong et al., 2020). Statistical differences were calculated by Kruskal-Wallis nonparametric tests, and a P ​< ​0.05 was considered statistically significant. The viral community structure and richness results were visualized in the heatmap, which was generated using R v3.6.3 package pheatmap (v1.0.12, https://cran.r-project.org/package¼pheatmap) and ggplot2 (v3.2.1, https://ggplot2.tidyverse.org), respectively.

2.9. Accession numbers

All genome sequences have been deposited into GenBank under accessions MZ244224–MZ244342. Quality-filtered sequence reads have been deposited in the sequence read archive (SRA) under BioProject ID PRJNA680460 and BioSample ID SAMN19003962. The specific information about per pool was shown in the Supplementary Table S2.

3. Result

3.1. Tick sample collection and taxonomic identification

From 2017 to 2018, a total of 640 ticks (specific tick type was shown in Table 1) were collected in central and western China, including 50 free ticks from grassland and 590 ticks fed on sheep, cattle, dog, camel and chicken. After observing by experts and sequencing the COI gene, 640 ticks were found representing six species: H.longicornis, Rh.microplus, De.everestianus, Rh.turanicus, Hy.asiaticum, and Rh. sanguineous. Sample information showed that H.longicornis was most prevalent (39/64, 60.9%) and was mainly distributed in Henan, Shaanxi and Yunan provinces. Rh.turanicus (6/64, 9.4%) was only collected in Hebei Province. Hy.asiaticum (6/64, 9.4%) was only identified in Gansu Province. Rh.microplus (4/64, 6.3%) was only found in the Hubei Province and Rh. sanguineous (1/64, 1.6%) was only found in Shanghai Municipality (Fig. 1).

3.2. Viral metagenomic analysis revealed virome profile of ticks

We performed viral metagenomic analysis on sampled ticks to study the virome, as well as virus biodiversity and evolution. Sixty-four viral metagenomic libraries were constructed and sequenced using the MiSeq Illumina platform, resulting in 13.44 ​G (5.88 ​× ​10⁷) of 250-bp-end reads. Sequence reads were compared to the GenBank non-redundant protein database using DIAMOND (Buchfink et al., 2015; Bağcı et al., 2021), generating 2,437,941 viral reads. The reads similar to viral sequences were classified into 21 viral families, including six DNA viral families and fifteen RNA viral families (Fig. 2). In the DNA virus group, most of the viral reads belong to the ssDNA families, which were dominated by Parvoviridae and Genomoviridae, and relatively low numbers of sequences were classified into other DNA families. The most predominant virus group of RNA viruses is ssRNA virus. The families of Chuviridae and Rhabdoviridae in the ssRNA (−) virus group occupied a dominant position, whereas the families of Hepeviridae and Flaviviridae did so in ssRNA (+) virus group. A small portion of sequences were assigned to Picobirnaviridae in dsRNA virus group.

Fig. 2.

A heatmap of clustering analysis. The heatmap shows the normalized reads counts on log10 scale. The maximum and minimum values are shown on the left side of the heatmap. The tick species and location are labeled with different colors. The gene type of viruses is also annotated at the right by the color bars.

The rarefaction curves illustrate this point. Rarefaction curves of every library yielded a horizontal asymptote, demonstrating that the sequencing depth might be enough to cover almost all known viral species in the samples, and the sequencing data were credible and rational (Supplementary Fig. S1). The species accumulation curves revealed that >150 viral species were present in the tick samples, and the curves tend to be relatively flat, suggesting the samples could essentially cover the common species (Fig. 3A). To estimate the overall richness and diversity of the viral communities, the alpha diversity indices in viral species level were analyzed: Shannon index (non-parametric quantitative species richness/evenness) (Fig. 3B). No significant differences in alpha diversity (Shannon indices) were observed between different tick species (P ​> ​0.05) (Fig. 3B). For the beta diversity, Bray-Curtis dissimilarities were calculated from the abundance of viral species and then used for principal coordinates analysis (PCoA) (Fig. 3C). Permutational multivariate analysis of variance (PERMANOVA) test on tick species resulted in P ​< ​0.001 and R ​= ​0.2 for the virome, further suggesting that the viromes in these tick species had different centroids. The tick species of Rh.microplus, Rh.turanicus, Hy.asiaticum were not grouped together. Although the tick species of De.everestianus and Rh.microplus were more closely grouped, the distance with H.longicornis ticks is close. The results of UPGMA clustering tree confirmed those of PCoA (Fig. 3D).

Fig. 3.

Taxonomic analyses of viral metagenomic reads on the species level. A Species accumulation curve of the individual library. The abscissa represents the number of libraries, and the ordinate represents the number of species found. The blue shading indicates the 95% confidence interval. B Comparison of virus alpha diversity (Shannon index) between different tick species. The horizontal bars within boxes represent medians. The tops and bottoms of boxes represent the 75th and 25th percentiles, respectively. C Principal coordinate analysis (PCoA) scatter plot. Circles show the 95% normal probability ellipse for each group. D UPGMA tree using Bray-Curtis distances among sampling pools. Legends are displayed at the left of the figure, and each legend represents a species of ticks.

The different pattern of tick virome between different sampling locations was also evident in log₁₀ normalized abundance of 56 viral species (rows) across the 8 locations as shown in Supplementary Fig. S2. Eight locations that had less than 50 reads were removed from the analysis. The virus names shown in the heatmap were from the taxonomic annotation of DIAMOND and MEGAN based on BLASTx. The virome of seven locations clearly clustered into two large clades according to the hierarchical clustering based on the Euclidean distance matrix, except for location Shaanxi, which formed a separate clade, characterized by a set of unique viruses. Henan and Gansu had some viruses in common, such as Genoa virus, Suffolk mivirus with a high abundance. Reads of Ungulate copiparvovirus 1 were highly abundant in Shaanxi, and only sporadically presented in other locations, suggesting a lower viral load in other locations.

After de novo assembly, reads mapping, and combining PCR to bridge gaps based on the viral sequence contigs from the virome, 63 complete or partial genomes belonging to Nairoviridae, Phenuiviridae, Chuviridae, Rhabdoviridae, Flaviviridae-like, Iflaviridae, parvoviridae and Polyomaviridae were generated from these ticks, which were fully characterized in the following sections.

3.3. Viruses within the family Chuviridae

Chuviridae is a novel discovery family (Li et al., 2015; Shi et al., 2016a) of RNA viruses that displays a wide variety of genomic organizations, including unsegmented, bi-segmented, and circular forms (Li et al., 2015; Siddell et al., 2019). The circular genomic form is a unique feature distinguishing chuviruses from some other RNA viruses (Li et al., 2015). Chuviruses present genome structural diversification, from a circular L-G-N order of genes (clade I, Fig. 4) to segmented or linear G-N-L order of genes (clade Ⅱ, Fig. 4) (Temmam et al., 2019). All chuviruses identified in this study accord with the L-G-N order of genes. We tentatively named four representative viruses Hebei mivirus 1 (HBMV1) strain HBD330MV, Bole tick virus 3 (BLTV3) strain GSC346MV, Wuhan mivirus (WHMV) strain YNB352MV, and Hebei mivirus 3 (HBMV3) strain HBG358MV. HBMV1 strain HBD330MV, with complete circular L-G-N genome (Supplementary Fig. S3), was identified in Rh.turanicus tick parasitizing dog from Hebei Province, sharing 88% amino acid (aa) identity with Mivirus sp. isolate TTP-Pool-7 (Supplementary Table S3). Meanwhile, two incomplete viral sequences, HBMV2 and Xinjiang mivirus 1 (XJMV1), sharing > 96% nucleotide (nt) identity with HBMV1, were found in pool 331 and pool 381. BLTV3 strain GSC346MV identified in Hy.asiaticum tick parasitizing camel in Gansu Province presents 97% aa identity with BLTV3 strain BL199 (Supplementary Table S3). In another pool 345 with the same sampling site, host, and species, we found a sequence sharing > 99% nt identity to BLTV3. Furthermore, three sequences were identified in H.longicornis ticks in pool 350, 352, and 355, which fed on camel in Yunnan Province. Of these, WHMV, with L-G-N genome form, shares a high aa identity (97%) to Wuhan mivirus isolate Thailand tick chuvirus 1. The remaining one, Hebei Mivirus 3 strain HBG358MV(HBMV3), demonstrates low aa identity (50%) to Umea virus isolate OTU2.IU18 and forms a separated branch, showing it may be a novel member of Mivirus (Fig. 4). The phylogenetic tree showed that HBMV1, BLTV3, WHMV identified in this study all fell in clade Ⅱ, with bi-segmented or circular L-G-N form (Fig. 4, Supplementary Fig. S3).

Fig. 4.

Phylogenetic relationship of Mononegavirales and Jingchuvirales. Phylogenetic tree based on RdRp (698 aa) was constructed based on a Bayesian method implemented in MrBayes. Nodes with bootstrap values ​> ​70 are noted. The red and blue names indicate sequences obtained in this study.

3.4. Viruses belonging to Rhabdoviridae

Rhabdoviridae is a family of RNA viruses that infect hosts as diverse as plants, vertebrates and arthropods (Walker et al., 2015, 2018). The genome of Rhabdoviruses encode five structural proteins, which are nucleocapsid protein (N), large multi-functional RdRp (L), polymerase-associated phosphoprotein (P), matrix protein (M), and transmembrane glycoprotein (G) (King et al., 2012; Walker et al., 2018). Here, we characterized three rhabdoviruses, which were provisionally named Bole tick virus 2 (BLTV2) strain GSC346rhabdoV1, Taishun tick virus (TSTV) strain GSC346rhabdoV2, and Rhipicephalus associated rhabdo-like virus (RARV) strain YNB352rhabdoV, respectively. BLTV2 and TSTV included the complete coding region while RARV lacked the extreme terminal sequences of nucleocapsid protein. BLTV2 strain GSC346rhabdoV1 and TSTV strain GSC346rhabdoV2 identified in this study share 98% and 95% identity at aa level with BLTV2 strain 15-CY and TSTV strain 17-L2, respectively (Supplementary Table S3). These four viruses may have the same host, all being detected in Hy.asiaticum. The genome of BLTV2 possessed the classical rhabdovirus genome organization of N-P-M-G-L (Supplementary Fig. S3). In contrast, the genome of TSTV consists of four ORFs. Of these, two encode RdRp and NP, both of which are homologous with rhabdovirus. RARV strain YNB352rhabdoV were found in H.longicornis tick parasitic cattle from Dali of Yunnan Province, and shared 98% aa identity to the RARV (YN-rhabdoV1). Except RdRp and NP, the functions of the remaining two ORFs from YN-rhabdoV1 are undiscovered for no homologous known protein has been found (Shi et al., 2021). Analysis of phylogenetic tree based on the conserved domains of RdRp revealed that BLTV2, TSTV, and RARV formed three separated tick-borne branches (Fig. 4). Notably, these viruses are highly divergent to classical rhabdoviruses as well as to each other (Fig. 4). Although tick-borne clade Ⅰ has a similar genome structure to alphanemrhavirus, the hosts are different, which are Spirurian nematodes and ticks, respectively. Meanwhile, viruses in tick-borne clade Ⅰ–Ⅲ were far distant from alphanemrhavirus in evolution (Fig. 4). Furthermore, numerous tick-borne rhabdoviruses were found worldwide. However, they are still unassigned to an identified genus and fall in several distinct clades that likely constitute new genera in the family (Moming et al., 2018; Tokarz et al., 2018; Temmam et al., 2019). BLTV2 strain 15-CY in tick-borne clade Ⅱ, and TSTV strains 17-L2 and YN-rhabdoV1 in tick-borne clade I (Fig. 4) were assigned to unclassified rhabdovirus (Moming et al., 2018; Temmam et al., 2019). So, we provisionally incorporated BLTV2, TSTV, and RARV into unclassified rhabdovirus and considered them as a new genus within Rhabdovirdae.

3.5. Viruses belonging to Nairoviridae

The genome of the members of the family Nairoviridae contain three negative-sense, single-stranded RNA segments, which are designated large (L), medium (M), and small (S). Nairoviruses are maintained in arthropods or transmitted by ticks among birds, bats, even humans (Lasecka and Baron, 2014; Garrison et al., 2020). To date, four viruses have been considered as human pathogens in this genus, including Crimean-Congo hemorrhagic fever virus (CCHFV), Dugbe virus (DUGV), Nairobi sheep disease virus (NSDV), and Kasokero virus (KASV) (Liu et al., 2020). In the present study, we determined two complete or nearly complete genomes of nairovirus. BLASTx search also revealed several viral sequences representing partial genomic segments of NSDV. The two viruses were identified from H.longicornis ticks fed on sheep from Pingqiao of Henan Province and Baicun of Shaanxi Province, respectively, and were provisionally named Henan tick virus (HNTV) strain HNO321nairoV and Shaanxi tick virus 2 (SXTV2) strain SXO338nairoV. HNTV strain HNO321nairoV presents 77%, 66%, and 63% aa identity with the L and S segments of Wenzhou tick virus strain TS1-2 and the M segment of Songling virus strain YC585, respectively (Supplementary Table S3). SXTV2 strain SXO338nairoV shares 74%, 62%, and 59% aa similarity with the L, M, S segment of three different viruses, which were Burana virus strain 760, Songling virus (SGLV) strain YC585, Wenzhou tick virus strain TS1-2, respectively. Moreover, we obtained seven segments representing partial genomic segments of L and M of NSDV in H.longicornis ticks collected on sheep from Poxian of Shaanxi Province. We attempted to get a complete sequence by nested PCR but failed. Fortunately, NSDV strain 337nairoc-L-5 included part of the conserved domain of RdRp by Search for Conserved Domains in NCBI. HNTV and SXTV2 identified in this study were phylogenetically grouped into Tamdy genogroup and formed a district clade with Burana virus and Wenzhou tick virus (Fig. 5). SGLV and Tacheng tick virus 1 (TcTV1), identified from homo sapiens, have priority phylogenetically over HNTV and SXTV2, suggesting these four viruses shared a common evolutionary origin. Importantly, SGLV strain HLJ1202 was reported to be associated with human febrile illness, with the main clinical manifestations being headache, fever, depression, fatigue, and dizziness (Ma et al., 2021). TcTV1, a Tamdy orthonairovirus, was found to be associated with febrile illness in a woman with a history of tick bites (Liu et al., 2020). Furthermore, NSDV strain 337nairoc-L-5 fell into the bunch of NSDV, sharing 96% aa identity with NSDV isolated from Hubei. NSDV is a type of zoonotic and tick-borne virus that can cause over 90% mortality in small ruminants, which emerges in Africa and Asia (Gong et al., 2015; Krasteva et al., 2020). Ganjam virus, a variant of NSDV, is associated with febrile illness in humans and livestock (Marczinke and Nichol, 2002; Yadav et al., 2011). This demonstrates that NSDV strain 337nairoc-L-5 is probably a putative pathogen that may lead to zoonotic disease and its pathogenicity need to be further explored.

Fig. 5.

Phylogenetic relationship of Bunyavirales. Phylogenetic tree based on RdRp (572 aa) was constructed based on a Bayesian method implemented in MrBayes. Nodes with bootstrap values ​> ​70 are noted. The orange and purple names indicate sequences obtained in this study.

3.6. Viruses in the family Phenuiviridae

Phenuiviridae is a family of segmented negative, single-strand RNA viruses which contains 19 genera. Uukuvirus is a genus in the family of Phenuiviridae and contains a clade of tick-borne phenuiviruses being separated from the Phlebovirus and Banyangvirus (Bandavirus) clades. Among currently known emerging tick-borne phleboviruses, two viruses [severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus] have been documented to infect humans and cause multiple organ damage (Dong et al., 2021). In the current study, two viruses were identified in tick pools, and BLASTx search indicated these sequences were mostly related to the Uukuvirus genus in Phenuiviridae, temporarily named Bole tick virus 1 (BLTV1) strain GSC346phenuiV and Rhipicephalus associated phlebovirus 1 (RAPV1) strain YNB351phenuiV. RAPV1 has L and S segments and misses M segments. The transcripts of L and S segments lack approximately 7 and 76 aa in the 5′ terminal respectively. It was found from H.longicornis ticks parasitized in cattle from Dali of Yunnan Province, sharing 98% aa identity with RAPV1 isolate YNTV3 which is closest to Uukuniemi virus, forming a separated branch with a group of novel putative phleboviruses (Shi et al., 2021). BLTV1 has incomplete L segment and was discovered in Hy.asiaticum ticks fed on camels in Gansu Province, presenting the aa identity of 97% with BLTV1 strain 17-L2, which was originally identified in Xinjiang Province (Supplementary Table S3). The transcripts of an incomplete L segment lack about 100 aa in the 5′ terminal. And, an S segment of BLTV1 is so short (327 ​nt) that we were unable to proceed with phylogenic analysis. Phylogenetic tree shows that the viruses found in our study and their corresponding similar sequences formed a well-supported monophyletic group closely related to the “classic” Uukuviruses (Fig. 5), suggesting that they may share the common ancestor. However, a branch of the “classic” Uukuviruses and another branch including viruses in this study are genetically far from each other (Fig. 5), suggesting these viruses may represent a novel genus in the family Phenuiviridae. Interestingly, the clade of Tacheng tick virus 2 (TcTV2) identified from homo sapiens, seemed prior to the viruses found in our study as well as their corresponding similar sequences (Fig. 5). Most importantly, TcTV2 can result in infections in patients with febrile illness if they have a recent history of tick bites (Dong et al., 2021), suggesting BLTV1 and RAPV1 identified in this study may have the potential to infect people.

3.7. Flavi-like viruses

Flaviviruses, the positive-stranded and non-segmented RNA viruses, contain a single and long ORF, flanked by 5′- and 3′-terminal non-coding regions that are translated into three structural proteins (C, M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Wu et al., 2005; Simmonds et al., 2017). NS3 and NS5 are homologous among all members of Flaviviridae, which contain conserved domains (RNA helicase and RdRp) and are encoded at similar locations in the genome (Moradpour and Penin, 2013; Wang et al., 2017; Zhao et al., 2017). A set of four segmented positive-stranded RNA viruses related to flavivirus were identified in diverse arthropods, such as Jingmen tick virus (JMTV) strain YN-flaviV and strain SY84 identified in R. microplus ticks in Hubei and Yunnan provinces (Li et al., 2015; Shi et al., 2021). Here, we determined the complete genomic sequence of a new strain, Xinjiang tick virus 1 (XJTV) strain XJS381flaviV, in the ticks collected from sheep in Xinjiang Province, which showed 75%–91% aa identity with Yanggou tick virus strain YG (Supplementary Table S3). The genome of XJTV contains four segments, among which segment 1 and segment 2 encode proteins associated with NS5 and NS3 (Supplementary Fig. S3). Segment 3 codes two proteins related to capsid protein and membrane protein. Segment 4 has two ORFs, predicted to encode glycoprotein 1 and glycoprotein 2. Moreover, another complete viral genome was identified, provisionally named Bole tick virus 4 (BLTV4) strain GSC346flaviV, presenting 98% aa similarity with BLTV4 strain BLP-1. The genomic and proteomic structures of BLTV4 strain BLP-1 resemble the prototype of the family Flaviviridae, which comprises the host signalase at N-terminal, a serine protease and an RNA helicase in the central part, as well as an RdRp toward the C-terminal end of the polyprotein (Shi et al., 2016b). BLTV4 strain GSC346flaviV identified in this study contains a serine protease and an RNA helicase in the central portion and RdRp at the C-terminal end of the polyprotein (Supplementary Fig. S3), as seen in previously described BLTV4 strain BLP-1. Phylogenetic tree based on NS3 (Fig. 6A) and NS5 (Fig. 6B) protein demonstrated XJTV strain XJS381flaviV clustered with a Jingmen virus group of four segmented flavirus-like viruses fed on ticks, separating a district branch from other clade related to insects or arthropods. Interestingly, based on the NS3, BLTV4 strain GSC346flaviV fell between the flavivirus clade and pestivirus clade (Fig. 6A), but it formed a separate monophyletic group with a great distance from the members of Flaviviridae based on the NS5 (Fig. 6B). Notably, a newly discovered segmented virus belongs to Jingmen virus group, Alongshan virus (ALSV), was found (Fig. 6). ALSV infection was confirmed in 86 patients who presented with fever, headache, and a history of tick bites (Wang et al., 2019b). Furthermore, ALSV can infect multiple human cell lines, induced pathologic changes in mice, and caused inflammatory responses in patients (Wang et al., 2019b). Genetically, ALSV and XJTV showed a close relationship based on NS3 and NS5 protein (Fig. 6), indicating XJTV may potentially infect humans.

Fig. 6.

Phylogenetic relationship of Flaviviridae. A Phylogenetic tree based on NS3-like protein (553 aa). B Phylogenetic tree based on NS5-like protein (525 aa). Nodes with bootstrap values ​> ​70 are noted. The orange and yellow names indicate sequences obtained in this study. The hosts of virus are annotated with corresponding iron.

3.8. Viruses belong to Iflaviridae

Iflaviridae is a family with monopartite, positive-stranded, non-segmented RNA genome, infecting arthropod hosts, with the majority infecting insects. The coding regions contain capsid proteins, arranged in the order VP2-VP4-VP3-VP1 and nonstructural proteins, which comprise an RNA helicase, a 3C-like cysteine protease and an RdRp (Valles et al., 2017). We found two complete sequences in Rh.turanicus ticks fed on chicken in Xingtai of Hebei Province, with 92% nt identity. Because they present 88% aa similarity with Hubei tick virus 2 strain tick109131 (Supplementary Table S3), we momentarily named it Hubei tick virus 2 (HBTK1) strain HUBG358iflaV1 and Hubei tick virus 2 (HBTK2) strain HUBG358iflaV2. The genomes of the two viruses both encode a 3105-aa polyprotein, which has similar conserved motifs from C-terminal to N-terminal (rhv-like, CRPV-capsid, RNA helicase, RdRp) (Supplementary Fig. S3). Phylogenetic tree showed that these viruses well grouped with their relatives in the family of Iflaviridae (Fig. 7). According to International Committee on Taxonomy of Viruses (ICTV), aa sequence identity of the capsid proteins of strains in one species is above 90% (Valles et al., 2017), suggesting these viruses are the members of the genus Iflavirus, but not a novel species.

Fig. 7.

Phylogenetic relationship of Picornavirales. Phylogenetic tree based on RdRp (1812 aa) was constructed based on a Bayesian method implemented in MrBayes. Nodes with bootstrap values ​> ​70 are noted. Viruses from the genus Flavivirus, unclassified Flaviviridae, unclassified Riboviria are marked in yellow, blue and purple, respectively. The sequences found in this study are labeled in red.

3.9. Viruses showing sequence similarity to Parvoviridae

Excluding RNA viruses, the overwhelming majority of reads are annotated to parvoviruses. Parvoviridae is a family with linear, single-stranded DNA genomes, comprising three subfamilies, Parvovirinae, Densovirinae and Hamaparvovirinae. Members of the subfamily Parvovirinae infect vertebrates (mammals, birds and reptiles) (Cotmore et al., 2019). This study found two complete sequences in H.longicornis ticks fed on sheep from Yimen of Shaanxi Province, momentarily named Ovine Copiparvovirus (OVPV4) strain SXO335parvoV1 and Bovine parvovirus 2 (BVPV2) strain SXO335parvoV2. The genome of OVPV4 consists of two ORFs. One ORFs encodes the nonstructural protein (NS1), sharing 60% aa identity with Copiparvovirus strain 101/BR/2018 (Supplementary Table S3). Another encodes the capsid protein (VP1), sharing 73% aa identity with Copiparvovirus strain 101/BR/2018. Likewise, the genome of BVPV2 consists of two ORFs sharing 58% and 57% aa similarity with the nonstructural protein and structural protein of BVPV2 isolate ujs2665, respectively. Moreover, we identified an incomplete parvovirus genome (Ovine Copiparvovirus strain HNO325parvoV3) lacking 5′ terminal end in structural protein and two segments (2208 bp and 1508 bp) encoding nonstructural protein in H.longicornis ticks fed on sheep. Phylogenetic analysis with other viruses of Parvovirinae subfamily shows the above sequences fell with the genus Copiparvovirus (Fig. 8). Based on replication initiator proteins, the branch of BVPV2 descended into Ungulate copiparvovirus 1 and Ungulate copiparvovirus 3, while other sequences fell in between Pinniped copiparvovirus 1 and Ungulate copiparvovirus 5 (Fig. 8). However, the genetic relationships between viruses in this study and other viruses are too far (Fig. 8), suggesting that they belong to novel species in the Copiparvovirus genus. This was also identified by the latest accepted proposal of ICTV for parvovirus classification, which announced that viruses within a species in genus Copiparvovirus are monophyletic and encode replication initiator proteins (called NS1 or Rep1, 68, or 78) showing >85% aa sequence identity (King et al., 2012). Unlike the above tree, OVPV1 (strain HNO325parvoV1) and OVPV4 (strain SXO335parvoV1) grouped with Ungulate copiparvovirus 1 and Ungulate copiparvovirus 3 based on structural protein (Fig. 8), indicating two proteins in OVPV may undergo recombination in the evolutionary process. In addition, due to the strong pathogenicity of this virus family, epidemiological screening was carried out for these complete viruses. The detection rate of OVPV4 and BVPV2 are 9.38% and 15.63%.

Fig. 8.

Phylogenetic relationship of Parvovirinae. Phylogenetic trees based on the conserved domain of structural (left) and nonstructural (right) proteins (231 aa and 209 aa). Nodes with bootstrap values ​> ​70 are noted. The sequences found name in this study are labeled in orange. The hosts of virus are annotated with corresponding iron.

3.10. Viruses belonging to Polyomaviridae

Polyomaviridae is a family of viruses with circular dsDNA genomes that are so far the only members known to perform oncogenic roles in their natural host (Polyomaviridae Study Group of the International Committee on Taxonomy of et al., 2016; Moens et al., 2017). The taxonomy of family Polyomaviridae has undergone several revisions (Johne et al., 2011). Now, the family includes four genera whose members have restricted host range, infecting mammals and birds, even fish (Fig. 9). We obtained a full-length circular polyomavirus genome sequence (Supplementary Fig. S3) in H.longicornis ticks fed on sheep in Shihe of Henan Province by nest-PCR, temporarily named Tick polyomavirus 1 (TPyV1) strain HNO339polyomaV. The specific primer sequences are shown in Supplementary Table S1. TPyV1 has a typical polyomavirus (PyV) organization, with an early region encoding the small T and large T-antigens (t-Ag and T-Ag) and a late region encoding the structural proteins VP1 and VP2/3 (Fagrouch et al., 2012). In addition, TPyV1 is also capable of encoding an agnoprotein. According to ICTV, if two polyomaviruses exhibit < 15% observed genetic distance for T-Ag coding sequence, the existence of a new species could be proved (Polyomaviridae Study Group of the International Committee on Taxonomy of et al., 2016). Although TPyV1 has 88% aa identity with Sheep polyomavirus 1 (SPyV1) isolate VH4-S14, they were drawn from different host species. Meanwhile, the evolutionary analysis based on T-Ag, VP1, and VP2 all indicated that TPyV1 formed a branch with SPyV1 and fell into the genus Betapolyomavirus (Fig. 9, Supplementary Fig. S4). Although TPyV1 was found in ticks (positive rate: 1.56%), the ticks in this pool are collected from sheep, suggesting TPyV1 probably comes from sheep.

Fig. 9.

Phylogenetic relationship of Polyomavirinae based on L-Ag (1156 aa). The tree of genus Betapolyomavirus is at the bottom right. Different hosts are marked by different colors. Nodes with bootstrap values ​> ​70 are noted.

4. Discussion

Central and western China are rich in biological resources, complex and diverse ecological environments, and developed animal husbandry, providing major breeding sites and suitable habitats for ticks and convenient conditions for tick-borne infectious diseases. In order to explore the composition of tick virus communities, and discover novel viruses, this study was conducted in central and western China. Free ticks and parasitic carcass ticks were collected and the genetic characteristics of the tick virus community and virus sequences were studied by viral metagenomics and bioinformatics techniques. However, similar studies in China are limited in the range of tick collection, such as only Hubei (Xu et al., 2021), Yunnan (Shi et al., 2021), Heilongjiang (Meng et al., 2019).

In the world, the most widely distributed tick genus was Dermacentor (in 574 counties), followed by Haemaphysalis (570), Ixodes (432), Rhipicephalus (431), Hyalomma (298) (Zhao et al., 2021), while it is a little bit different from China. Statistics between 1960 and 2017 showed the distribution of different ticks in China (Jia et al., 2020). The broadest distributed species is H.longicornis (long-horned tick) (Zhao et al., 2020a), followed by Rh.sanguineous (brown dog tick), Rh.microplus (southern cattle tick), Rh.turanicus, Hy.asiaticum and De.everestianus ticks. The distribution was consistent with the identification in this study. Moreover, we increased the new location of two species: Rh.turanicus ticks in Hubei Province and Rh.sanguineous ticks in Shanghai Municipality (Fig. 1). Furthermore, we found that tick species have a certain specificity of locations since the ticks collected from an exact location almost belong to the same species (Fig. 1). In addition to alpha diversity analysis by tick species, we also analyzed by collection time (Supplementary Fig. S5), location (Supplementary Fig. S6), host (Supplementary Fig. S7), tick developmental stages (Supplementary Fig. S8) and the results showed no significant differences between groups.

Some tick species are not only distributed widely but also have multiple viral pathogens. The following tick families are the vectors of mostly disease-causing viruses: Ixodes, Haemaphysalis, Hyalomma, Dermacentor, Rhipicephalus and Boophilus (Labuda and Nuttall, 2004). H.longicornis is found to harbour the highest variety of tick-borne agents (Zhao et al., 2021). At least 30 human pathogens were linked with H.longicornis, including six species of virus: SFTSV, Jingmen tick virus, Bocavirus, NSDV, Lymphocytic choriomeningitis virus, Tick-borne encephalitis virus (Zhao et al., 2020a). Moreover, H.longicornis (long-horned tick) in our study was widely distributed in three provinces and contained the most abundant viruses. We found three chuviruses, five parvoviruses, four bunyaviruses, one rhabdovirus, one polyomavirus and one hepe-like virus (Supplementary Table S3). Regrettably, no SFTSV positivity was detected in tick specimens. Although H.longicornis is considered as the major vector for STSFV, there are limited data on the detection rate of this virus. Li et al. collected 119 ticks belonging to H.longicornis from goats, and identified 1/119 of ticks carried SFTSV (Li et al., 2020). Shao et al. sampled a total of 2522 ticks from five cities of Shandong Province, with SFTSV infection rate of 2.5% (63/2522) (Shao et al., 2020). Rhipicephalus is also known to associate with many viral pathogens, such as Thogoto virus (Orthomyxoviridae), Wad Medani virus (Reoviridae), CCHFV and NSDV (Bunyaviridae, genus Nairovirus), Kismayo virus and Chim virus (Bunyaviridae, genus Phlebovirus), and Kandam virus (Flaviviridae), all of which can cause disease in livestock and humans (Xia et al., 2015). Rh.microplus (southern cattle tick), Rh.turanicus, and Rh.sanguineous (brown dog tick) are tick species from genus Rhipicephalus found in this research. The alpha and beta diversity suggests the notable discrepancy of viral species not only based on the different tick species but also due to the different geographical contexts, climatic conditions, food resources, selective pressures from immune response and microbiota interaction, and population to a much greater extent (Ramirez et al., 2012; Jupatanakul et al., 2014). Besides, the richness and quality of the sample may be affected by storage, shipping, and sampling size. We found that the virus abundance and diversity in the intact tick library samples are significantly higher than those in the tick tissue samples (Supplementary Fig. S9). This shows that the virus can also be detected in tick tissue.

In the negative single-stranded viruses, we identified nine chuviruses, three rhabdoviruses, two nairoviruses, a segment of NSDV and two L segments, an S segment in Phenuiviridae. All viruses in Chuviridae fall in clade Ⅱ of Mivirus and are with a circular L-G-N form. However, HBMV3 only has a complete polyprotein, so its genome form is uncertain. And it demonstrates low identity (50% aa) to Umea virus isolate OTU2.IU18 and forms a separated branch individually, showing it may be a novel member of Mivirus. The tree of Rhabdoviridae fell into two big clusters that are very far apart (Fig. 4), indicating that Rhabdoviridae is an abundant family and may derive novel family from it. The tick-borne clade is a part of a cluster that is the farthest away from a clade of Chuviridae. Nevertheless, the tick-borne clade did not belong to any genus of Rhabdoviridae. Although tick-borne clade Ⅰ has a similar genome structure to the members of the genus Alphanemrhavirus, the evolutionary relationships between them were far. Furthermore, the viruses in this study had different genome structures (Supplementary Fig. S1). The genome of BLTV2 has the classical rhabdovirus genome organization of N-P-M-G-L. In contrast, the others only consist of four structural proteins, suggesting genome contraction has likely occurred frequently throughout the evolution of the family. We provisionally incorporated three viruses into unclassified rhabdovirus and considered them as a new genus within Rhabdovirdae. Two nairoviruses filled in major phylogenetic “gaps” that exist in Tamdy group in the genus Orthonairovirus (Fig. 5). Importantly, SGLV and TcTV1 have priority phylogenetically over these two nairoviruses, indicating HNTV and SXTV2 likely have the pathogenic potential. But the detection rate of these two viruses is only 1.56%. Unfortunately, we only found some RdRp segments of NSDV, which probably leads to severe zoonotic disease. Two phenuiviruses and their corresponding similar sequences formed a well-supported monophyletic group closely related to the “classic” Uukuviruses (Fig. 5).

In the positive single-stranded viruses, we identified a segmented flavi-like virus, a non-segmented flavi-like virus and two iflaviruses. XJTV fell into Jingmenvirus group and formed a well-supported monophyletic group with viruses collected from ticks (Fig. 6). Interestingly, BLTV4 fell between the flavivirus clade and pestivirus clade based on the NS3, while it formed a separate monophyletic group that had a great distance from the members of Flaviviridae based on the NS5 (Fig. 6), suggesting that it may share a single common ancestor with pestivirus and has the extent of variation in genome. The two iflaviruses have similar genomic structure, conserved motifs, and a close phylogenetic relationship to members of the genus Iflavirus (Fig. 7), indicating they may be new members of the genus Iflavirus, family Iflaviridae. Interestingly, viruses in the only genus Iflavirus and unclassified viruses in Iflaviridae intersect and formulate several distinct clades (Fig. 7). These will likely be separated taxonomically into different genera in the future as more virus sequences become available.

In DNA viruses, we found two complete parvoviruses (OVPV4 strain SXO335parvoV1 and BVPV2 strain SXO335parvoV2), a near-complete sequence and three segments of replication protein. Based on replication protein, all sequences fell with the genus Copiparvovirus (Fig. 8), but we cannot identify the specific genus of these viruses. In particular, OVPV fell between Pinniped copiparvovirus 1 and Ungulate copiparvovirus 5 based on replication protein, while it is between Ungulate copiparvovirus 2 and Ungulate copiparvovirus 3 based on capsid protein (Fig. 8), indicating two proteins in OVPV may undergo recombination in the evolutionary process. Importantly, the prevalence of OVPV4 and BVPV2 is at a high level: 10.9% (7/64) and 35.9% (23/64), respectively. Moreover, both of them were found from more than one tick species, suggesting they are relatively common. Nevertheless, to date, no viruses from this genus have been isolated, and their molecular biology remains to be explored, so it is not clear whether they are associated with disease (King et al., 2012). Furthermore, we acquired a circular genome in ticks, TPyV1, which shared 88% aa identity with SPyV1. As is well-known, some polyomaviruses can drive tumorigenicity, especially SV40, murine polyomavirus (MPyV) (Carter et al., 2013) and Merkel cell polyomavirus (MCPyV) (Feng et al., 2008). Additionally, PyVs can cause acute, fatal infections in birds, and these viruses primarily establish lifelong infections that can develop into fatal infections in immunosuppressed hosts in mammals (Krumbholz et al., 2009). The best-studied human polyomaviruses, BK polyomavirus and JC polyomavirus, belong to Betapolyomaviridae and are associated with nephropathy and progressive multifocal leukoencephalopathy, respectively. Although TPyV1 belongs to the genus Betapolyomavirus, it and the above human PyVs are clustered into two branches (Fig. 9). The host of PyV was to date mammals, birds and fish. However, this was the first time to find PyV in an arthropod host, which was likely due to the last blood meal of the tick contained SPyV1, rather than indicating actual infection of the tick.

5. Conclusions

In summary, our study investigated tick species in central and western China and analyzed the virome of ticks fed on different hosts. The viral diversity and evolution of full or novel viruses were thrown light on further. However, due to limited time and experimental conditions, we have not studied the pathogenic mechanism of potentially pathogenic viruses. With the gradual progress of the experiment, we will expand the collection range and sample size of tick to further enrich the data of the tick virus community in China. Furthermore, we hope to continue to deepen the mechanism of the potential disease-causing virus research.

Data availability

All genome sequences have been deposited into GenBank under accessions MZ244224–MZ244342. Quality-filtered sequence reads have been deposited in the sequence read archive (SRA) under BioProject ID PRJNA680460 and BioSample ID SAMN19003962.

Ethics statement

The sample collection and all the experiments in the present study were performed with an ethical approval given by Ethics Committee of Jiangsu University and the reference number is No. UJS2014017.

Author contributions

Zijun Yang: formal analysis; data curation; investigation; visualization; writing - original draft; writing - review & editing. Hao Wang: investigation; writing - review & editing. Shixing Yang: investigation; writing - review & editing. Xiaochun Wang: investigation; writing - review & editing. Quan Shen: investigation; writing - review & editing. Likai Ji: iInvestigation; writing - review & editing. Jian Zeng: investigation; writing - review & editing. Wen Zhang: conceptualization; funding acquisition; resources; investigation; methodology; validation; writing - review & editing. Haiyan Gong: conceptualization; funding acquisition; resources; investigation; methodology; writing - review & editing. Tongling Shan: conceptualization; funding acquisition; resources; investigation; methodology; writing – review & editing.

Conflict of interest

The authors have declared that no competing interests exist.

Appendix A. Supplementary data

The following are the Supplementary data to this article.