METHODS
Sample collection
The questing ticks were collected by the flagging method, and
blood-sucking ticks were collected from cattle in
Shulan. These ticks were
identified to species as described elsewhere (Deng, 1978; Shao, Zhang,
Li, Huang, & Yan, 2020). Every 10
ticks were pooled into a 1.5 mL
Eppendorf tube according to the
collection sites and species and stored at -80°C until use.
RNA library construction and sequencing
After washing with 75% ethanol and
RNA/DNA-free water, pooled ticks
in tubes were added with 800 μL Dulbecco’s modified Eagle’s minimum
essential medium (DMEM) and two
stainless steel beads (3 mm
diameter), and crushed using the Tissuelyser (Jingxin, Shanghai, China)
at 70 Hz for 2 min. The lysates were centrifuged at 12000 rpm for 10 min
at 4 °C, and the supernatant was further pooled for library construction
according to the collection sites and species (Supplementary Table S1).
After digested with micrococcal nuclease (NEB, USA) in 37 °C for 2 h,
the pooled samples were used for viral RNA extraction with the TIANamp
Virus RNA kit (TIANGEN, Beijing, China).
The extracted RNA was subjected to metagenomic sequencing at Tanpu
Biological Technology Co., LTD (Shanghai, China). Briefly, the RNA from
each pool was used for library preparation with the
NEBNext® UltraTM RNA Library Prep Kit for
Illumina® (NEB, USA) according to the manufacturer’s
instructions. After adapter ligation, 10 cycles of PCR amplification
were performed for target enrichment. The libraries were pooled at equal
molar ratio, denatured and diluted to optimal concentration, and
sequenced with an Illumina NovaSeq 6000 System.
Transcriptome analysis
Transcriptome analysis was conducted as described elsewhere (Gordon et
al., 2020). Briefly, after trimming and removing low quality reads, the
paired-raw reads were purified by removing ribosomal RNA, host
contamination, and bacteria sequences using BBMap program
(https://github.com/BioInfoTools/bbmap), and assembled into contigs with
SPAdes v3.14.1 (https://github.com/abl ab/spades) and SOAPdenovo
v2.04 (https://github.com/aquaskyline/SOAPdenovo-Trans)
(Prjibelski, Antipov, Meleshko, Lapidus, & Korobeynikov, 2020; Xie et
al., 2014). After being compared with the nonredundant nucleotide (nt)
and protein (nr) database downloaded from GenBank using BLAST+ v2.10.0,
the assembled contigs were filtered to remove the host and bacterial
sequences. The relative abundance of the identified viruses was
determined by mapping the reads back to the assembled contigs using
Bowtie2 v2.3.3.1.
Viral genome confirmation and annotation
The assembled contigs were compared
with NCBI nucleotide and viral refseq database using BLAST (V2.10.0+),
and used as a reference for designing specific primers to confirm and
analyze the sequences of terminal ends, using the nested reverse
transcription-polymerase chain reaction (RT-PCR) and the rapid
amplification of cDNA ends (RACE) as described elsewhere (Ma et al.,
2021; Y. C. Wang et al., 2021). Detailed information on the primers for
the detection or whole genome amplification are shown in Supplementary
Table S2. Potential open reading frames (ORFs) in the viral sequences
were predicted using ORFfinder (https://www.ncbi.nlm.nih.gov/
orffinder/).
Virus classification
All the viruses identified in this study were classified according to
the latest International Committee on Taxonomy of Viruses (ICTV) report
of virus taxonomy
(https://talk.ictvonline.org/ictvreports/ictv_online_report/). A novel
viral species should be satisfied with one of the following conditions
as described before (Xu et al., 2021). namely, (i) <80%
nucleotide (nt) identity across the complete genome; or (ii)
<90% amino acid (aa) identity of the RNA-dependent RNA
polymerase (RdRp) domain with the known viruses. All novel viruses were
named as the collection sites that the virus was first identified,
followed by common viral names according to their taxonomy. All the
viral strains would be marked with ‘Northeatern (NE)’ to distinguish
them from the virus strains identified in other studies.
Phylogenetic analyses
To confirm the phylogenetic relationships of the viruses discovered in
this study, representative reference viral sequences were retrieved from
the GenBank database (Supplementary Table S3), and aligned using
ClustalW available within MEGA 7.0. Phylogenetic analyses were conducted
with the aligned sequences using the maximum-likelihood method in MEGA
version 7.0 with the best-fit
substitution model for each alignment (Kumar, Stecher, & Tamura, 2016).
A bootstrapping analysis of 1000 replicates were conducted in the
analysis, and the bootstrap values more than 70 were shown in the trees.