Yang et al. Virol J (2021) 18:163 https://doi.org/10.1186/s12985-021-01632-x RESEARCH Open Access Virome analysis of ticks in a forest region of Liaoning, China: characterization of a novel hepe-like virus sequence Zijun Yang1, Ju Zhang1, Shixing Yang1, Xiaochun Wang1, Quan Shen1, Guangming Sun3, Hao Wang2* and Wen Zhang1* Abstract Background: Ticks (class Arachnida, subclass Acari) are vectors of transmitting a broad range of pathogenic micro- organisms, protozoa, and viruses afecting humans and animals. Liaoning Province is rich in forests where diferent animals and, abundant Haemaphysalis longicornis ticks exist. Methods: Using viral metagenomics, we analyzed the virome in 300 Haemaphysalis longicornis ticks collected from June to August 2015 in the forested region of Liaoning Province, China. Results: From the 300 ticks, 1,218,388 high-quality reads were generated, of which 5643 (0.463%) reads showed sig- nifcant sequence identity to known viruses. Sequence and phylogenetic analysis revealed that viral sequences show- ing a close relationship with Dabieshan tick virus, Aleutian mink disease virus, adeno-associated virus, Gokushovirus, avian gyrovirus 2 were present in the virome of these ticks. However, the signifcance of these viruses to human and animal health requires further investigation. Notably, an hepe-like virus, named tick-borne hepe-like virus sequence, was obtained and was highly prevalent in these ticks with a rate of 50%. Nevertheless, one constraint of our study was the limited geographical distribution of the sampled ticks. Conclusion: Our study ofers an overview of the virome in ticks from a forest region of Liaoning Province and pro- vides further awareness of the viral diversity of ticks. Keywords: Virome of ticks, Metagenomic analysis, Dabieshan tick virus, Tick-borne hepe-like virus, Liaoning Province Background 158 cases of CCHFV (Crimean-Congo hemorrhagic fever Ticks (class Arachnida, subclass Acari) transmit a broad virus) infection were published from 1953 to 2016, with range of pathogenic microorganisms, protozoa, and an overall case fatality rate of 32.4% [3]. SFTS (Severe viruses and are the second most common vectors of dis- fever with thrombocytopenia syndrome), with a 5.3% eases afecting livestock, humans, and companion ani- national average mortality rate, was reported in 23 prov- mals [1, 2]. Te diseases caused by the tick-borne virus inces of China, with increased numbers yearly from 2010 are numerous and severe. In Africa, Asia, and Europe, to 2016 [4]. Moreover, the incidence of some tick-borne infections and transmissions in recent decades showed an increasing or fuctuating tendency due to various fac- *Correspondence: [email protected]; [email protected] 1 Department of Microbiology, School of Medicine, Jiangsu University, tors, mainly associated with increased tick-exposure, Zhenjiang 212013, Jiangsu, China especially with the enlargement of cities, taking the place 2 Department of Clinical Laboratory, Huai’an Hospital, Xuzhou Medical of forests, and exposing wild hosts to humans and live- University, Huai’an 223002, Jiangsu, China Full list of author information is available at the end of the article stock animals [5–7]. © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yang et al. Virol J (2021) 18:163 Page 2 of 12 Te rise of metagenomics analysis has transformed Tick sample pool preparation virus discovery and revealed a remarkable diversity of Te collected ticks were divided into six groups (Pool viruses sampled from ticks [8, 9]. Researchers found 01-06) based on their sampling month (early June, late a large monophyletic group of emerging viruses and June, early July, late July, early August, and late August) named this putative new virus family ’Chuviridae’ [9, (Additional fle 1). An additional table shows these pools 10]. Jingmen tick virus, a segmented RNA virus, was in detail (see Additional fle 1). Before homogenization, detected frstly from ticks in the Jingmen region of each tick pool was washed with 75% alcohol to remove Hubei Province in China [11]. Huaiyangshan virus, a contaminants on ticks and washed thrice with 1 mL of novel species of the genus Phlebovirus, was found in phosphate-bufered saline (PBS) to eliminate external human and tick samples with high prevalence [12, 13]. microbes. Te tick samples were homogenized, frozen, Together, these studies showed that using metagen- and thawed three times on dry ice, and the supernatants omics can express the diversity of viruses from ticks were then collected after centrifugation (5 min, 15,000g, massively. 4 °C). Liaoning Province, situated in the northeastern region of China, is owned by a temperate monsoon cli- Viral metagenomic analysis mate region with plenty of rainfall and sunshine. Fur- 500ul of each supernatant was fltered through a 0.45- thermore, it has 4.641 million hectares of forests and is μm flter (Millipore) to remove eukaryotic and bacterial rich in animal resources, with 827 species of animals, cell-sized particles. Te fltrate was treated for 60 min including amphibians, mammals, reptiles, and birds. at 37 °C with a DNases mixture (Turbo DNase from Researchers found that tick richness correlated with Ambion, Baseline-ZERO from Epicentre), benzonase forest size, even among locally common birds [14]. (Novagen), and RNase (Fermentas) to digest unprotected Ticks and tick-borne diseases have the evolution of nucleic acid [18, 19]. Nucleic acids (total DNA and RNA) cooperation with various wild animal hosts, and these were then extracted using a QIAamp Viral RNA Mini Kit hosts constitute reservoir hosts for ticks and tick-borne (QIAGEN) following the manufacturer’s instructions. pathogens[1]. Nevertheless, with urbanization and the Extractions were reverse-transcribed to cDNA using increasing range of human activity, a state of equilib- reverse transcriptase (Super-Script III, Invitrogen). Total rium between them may be of, and the risk of tick bites nucleic acids were subjected to RT reactions with Super- and infecting diseases increases. Haemaphysalis lon- Script III reverse transcriptase (Invitrogen), following gicornis (H. longicornis) is the predominant species in second-strand cDNA synthesis with Large (Klenow) frag- Liaoning Province [15], but the overview of viruses car- ment (NEB). Sixty-four libraries were then constructed ried by ticks has not yet been entirely elucidated. It is using Nextera XT DNA Sample Preparation Kit (Illu- necessary to investigate the viruses carried by ticks and mina) and sequenced using the HiSeq Illumina platform identify their natural habitats to prevent outbreaks of with 250 base pair-ends with dual barcoding for each tick-borne viral diseases [6]. pool [20]. Since the necessity and feasibility, this study aims to study the diversity and evolutionary origin of viruses Bioinformatics analysis in ticks from a forest of Liaoning Province by a viral metagenomic approach. Paired-end reads of 250 bp generated by HiSeq were debarcoded using vendor software from Illumina. Clonal reads were removed, and low-sequencing-quality tails Methods were trimmed using Phred. Adaptors were removed using Sample collection the default parameters of VecScreen[21]. Te cleaned According to climatic conditions of summer-autumn reads were assembled de novo within each barcode group favor tick proliferation, we collected 300 live adult ticks utilizing the ENSEMBLE assembler [22]. Te assembled from June to August during 2015 in a big forest park of contigs and singlets were compared to an in-house viral proteome database using BLASTx with an E-value cut- Dalian city in southern Liaoning (Fig. 1A) by the drag- 5 fag method. Six diferent batches of ffty ticks were sam- of of < 10¯ . Candidate viral hits were then contrasted to pled based on sampling time (early June, late June, early an in-house non-redundant (NVNR) protein database to July, late July, early August, and late August). Ticks were remove false-positive viral hits. Te NVNR database was placed in labeled vials and shipped on dry ice [16]. All the compiled using non-viral protein sequences extracted collected ticks were identifed using tick taxonomic keys from an NCBI nr fasta fle (based on annotation tax- by tick entomologists under the microscope at a magnif- onomy, excluding the virus kingdom). Contigs with- out signifcant BLASTx similarity to the viral proteome cation of × 56 [17]. Yang et al. Virol J (2021) 18:163 Page 3 of 12 A Liaoning Province China Dalian BC4000 8e+05 Adenoviridae Anelloviridae Bunyaviridae Flaviviridae 3000 Microviridae 6e+05 None−DNA virus total reads None−RNA virus viral reads None−unclassified virus Papillomaviridae s 2000 Parvoviridae 4e+05 reads Retroviridae read Tombusviridae Virgaviridae 2e+05 1000 0e+00 0 Pool01 Pool02 Pool03 Pool04 Pool05 Pool06 Pool01 Pool02 Pool03 Pool04 Pool05 Pool06 Pool Pool Fig. 1 The map of collection sites and the viral reads in each pool. A Map of tick collection sites. B The proportion of viral sequences in total reads.