From 2015 to 2017, 7387 live adult ticks were plucked from infested cattle and buffalo in Nujiang Lisu Autonomous Prefecture (NJ), Dali Bai Autonomous Prefecture (DL) and Luxi County (LX) in Yunnan Province (Supplementary Figure S1 and Table 1), maintained alive in cases at room temperature before study. The ticks were identified morphologically as R. microplus by a trained expert and further confirmed by sequencing internal transcribed spacer 2 gene (ITS2). These ticks were washed three times using sterile phosphate-buffered saline (PBS, pH7.4), grouped (n = 11–20) depending on their body size and homogenized with 1 mL PBS for each group as described (Zhang et al. 2018). Homogenates were centrifuged at 5000 ×g for 10 min at 4 ℃ to remove debris, and supernatants were subpackaged and stored at - 80 ℃ until further processing.
Time Sampling location Number of groups Total ticks 2015 NJ 76 912 LX 45 540 2016 DL 351 4212 LX 47 564 2017 LX 79 410 Total 574 7387
Table 1. Summary of total tick sample collections in Yunnan Province from 2015 to 2017
Additionally, 100 serum samples of cattles, 100 serum samples from febrile patients and 100 healthy human serum samples from the same tick sampling location in LX were shipped to the laboratory in a cooler with ice pack or with dry ice, depending on the time required for transportation. Besides, 100 cattle serum samples were collected from a farm of Wuhan, Hubei Province.
According to our sampling location and time, our tick sample can be classified to four batches. And each 12 ticks from the same batch were divided into one group. For RNA-seq, three groups of ticks were mixed for one RNAseq library construction, and totally four libraries corresponding to each batches were constructed. Total RNA was purified using TRIzol LS reagent (Invitrogen, Calrsbad, CA) from 300 μL supernatant of each group. Then equivalent qualities of total RNA from three groups were mixed to generate one RNA pool (3–5 μg in total). RNA-seq library preparation was performed according to the protocol provided by Illumina as described (Li et al. 2015), and was further sequenced by the Hiseq 3000 platform (Illumina) to generate the 150 bp paired-end reads.
Raw sequencing reads with low quality were excluded, trimmed to remove adaptors using Trimmotic program and then subjected to a basic local alignment search tool (BLASTn) comparing against the tick genome to filter out the abundant host reads so as to simplify the following de novo assembly using Trinity program (Grabherr et al. 2011). Subsequently, the contigs were mapped to non-redundant nucleotide database (NT) and non-redundant protein database (NR) downloaded from NCBI using BLASTN and BLASTX respectively by using an E-value cutoff of 1e-5 to identify potential viral contigs. An overview of contig taxonomy was shown by MEGAN software with the BLASTN or BLASTX comparison results (Huson et al. 2007). Bowtie2 were used to obtain the raw reads of contig which mapping to a certain target viral genome and corresponding reads per kilobase million mapped reads (RPKM) were calculated (Langmead and Salzberg 2012).
Nested RT-PCR was performed to confirm the detection of RNA-seq with primer sets (Supplementary Table S1). That is cDNA from the tick pools which was originally subjected to RNA-seq were used as templates for PCR detection. Only positively detected potential virus would undergo further epidemiological survey.
The contigs which hit viral genome were used as templates for overlapping primers design. And the full-length genome of each virus was determined via genome walking, and 50- and 30- rapid amplification of cDNA ends (50-, 30 RACE) with commercial kit (Takara) according to the manufacturer's protocol.
All the PCR products were separated on 0.8% agarose gel and purified through agarose gel DNA extraction kit for Sanger sequencing.
Firstly, plasmid pET-28a-NP containing partial or complete nucleoprotein of each virus was constructed. Then recombinant plasmid pET-28-NP was electrotransformed into E.coli strain BL21 for protein expression induced with IPTG. And expressed NP was purified as described previously (Zou et al. 2016), which subsequently used for rabbit immunization to elicit polyclonal antibodies (pAbs) against NP. Western blots were performed to verify the effectiveness of the polyclonal antibody (anti-NP).
The same expression regions of pET-28a-NP were inserted into plasmid pCNDA3.1+ for eukaryotic expression. Then immunofluorescence assay was performed by using plasmid-transfected HEK293 cells. Cells were fixed with 4% paraformaldehyde for 15 min before being permeabilized with 0.5% Triton X-100 in PBS solution for 10 min. After rinsing with PBS solution, they were blocked with fish gelatin for 1 h, and then washed with PBS solution for another 3 min. The washing steps were repeated three times. Subsequently, bovine sera (diluted 1:20 in PBS solution) or polyclonal antibodies to NP (1:2000) as positive control group were incubated with cells for 2 h and washed as described earlier. Reactions among bovine sera were detected with FITC conjugated protein G (1:2000; Abcam) and the positive controls were detected with Alexa 488-labeled goat anti-rabbit (1:2000; Abcam). Cell transfected with empty pCDNA3.1+ was severed as negative control.
For each potential virus, phylogenetic trees were inferred based on the full-length RNA-dependent RNA polymerase (RdRp) protein which were aligned with their corresponding homologous references sequence. Alignment and phylogenetic analysis were conducted via ClustalW and maximum likelihood (ML) method which both implemented within MAGA version 6.0 (Tamura et al. 2013).
We use ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder) to predict potential protein, TMHMM v2.0 (https://www.cbs.dtu.dk/services/TMHMM/) to predict transmembrane domains, SignalP v4.0 (https://www.cbs.dtu.dk/services/SignalP/) to find likely signal sequence, NetOGlyc (https://www.cbs.dtu.dk/services/NetOGlyc/) to predict O-GalNAc glycosylation sites and NetNGlyc (https://www.cbs.dtu.dk/services/NetNGlyc/) to predict N-glycosylation sites.
Sample Collection and Preparation
Library Preparation and RNA-seq Sequencing
Viral Full-Length Genome Walking and PCRBased Epidemiological Survey
Preparation of Polyclonal Antibodies and Immunofluorescence Assay
Phylogenetic and Bioinformatics Analysis
Prediction of Protein Functional Domain
From 2015 to 2017, a total of 7387 ticks were collected from cattle and buffalo in the northwest (NJ and DL) and southeast (LX) areas of Yunnan Province and were divided into 574 groups (Supplementary Figure S1 and Table 1). Except that samples were collected continuously from LX for 3 years, ticks were collected from NJ in 2015 and from DL in 2016 (Table 1). These ticks were all half or full engorged R. microplus identified both by morphology and ITS2 sequencing (Fig. 1).
Figure 1. Molecular identification of tick samples. The phylogenetic analysis was performed via an ML method based on internal transcribed spacer 2 (ITS2) gene. The ticks collected in different year in this study were indicated by black dots.
Additionally, 100 serum samples of cattle and febrile patient and healthy human, respectively from Luxing County and 100 serum samples of cattle from Wuhan City in Hubei Province were acquired.
A total of four tick pools collecting from different regions of Yunnan during 2015 to 2017 generated ~ 28 Gb of data including 2.7 × 109 or so 150-base pair-end reads (Supplementary Table S2). After reads assembly and sequence comparison to the NT or NR database, 143 contigs from the four libraries were identified as viral sequences related to 9 viruses, concerning Phenuiviridae (n = 3), Chuviridae (n = 2), Rhabdoviridae (n = 1), Parvoriridae (n = 1) and Flaviviridae-like (n = 2) (Table 2).
No. Virus name Closest relative Identity with relative virus (aa) Classification of relative virus Coverage (%) RPKM 1 Yunnan mivirus 1 (YNMV1) strain YNMV1 Wuhan mivirus strain X78-1 86% Mivirus 94.2 4.8 2 Wuhan tick vrius 1 (WHTV1) strain YNrhabdoV1 Wuhan tick vrius 1 strain X78-2 97% Unassigned Rhabdovirdae 83.3 2.5 3 YN tick-associated phlebovirus 1 (YNPhelobV1) strain YN-PLV1 Lihan tick virus strain LH-1 S segment 89%
L segment 88%
Phlebovirus 86 1.57
4 Jingmen tick virus (JMTV) strain YNflaviV Jingmen tick virus isolate SY84 S1 segment 93%
S2 segment 99%
S3 segment 96%
S4 segment 99%
Unssigned (Jingmenvirus) S1:87.7
5 Bovine hokovirus 2 (BHKV) strain YNBHKV2 Bovine hokovirus 2 strain HK-B38 100% Tetraparvovirus 97 1.30 6 Bole mivirus (BLMV) strain YN-MV2 Bole tick virus 3 strain BL199 95% Mivirus 44.1 0.09 7 Bole tick virus 1 (BLTV1) strain YNPLV2 Bole tick virus 1 strain BL075 S/L segment 98% Phlebovirus 36.4 0.05 8 YN tick-associated flavi-like virus 1 (YN-FlaviV1) strain YN-FLV 1 Bole tick virus 4 80% Unassigned (Flavi-like) 30.2 0.03 9 YN tick-associated phlebovirus (YNPhelobV1) strain YN-PLV3 Blacklegged tick virus S segment 60%
L segment 65%
Phlebovirus 23.2 0.01
RPKM: Reads Per Kilobase per Million mapped reads
Table 2. Viruses identified in R. microplus in our study
RPKM values of the nine viruses suggested that the first five viruses (RPKM > 1.0) had much higher abundance than the last four viruses (RPKM < 1.0) (Table 2), in accordance with the genome coverage of RNA-seq that the former five viruses had several gaps along their genome sequences while dispersed short contigs flanked by large gaps were found along genome of the latter viruses. Genome walking was further performed with the first five viruses in order to acquire the full-length genome. Genome organization of these viruses was shown in Fig. 2, reflecting a great genomic diversity of tick-associated viruses.
Viral sequences share 86% and 95% identity at amino acid (aa) level with Wuhan mivirus (WHMV) strain X78-1 and Bole mivirus (BLMV) strain BL199, respectively were detected in our tick pools. WHMV and BLMV are identified in the R. microplus and Hyalomma asiaticum tick in China, respectively and assigned to the recently-proposed Mivirus genus of Chuviridae (Li et al. 2015). Based on the similarity, we considered virus related to WHMV as a novel chuvirus named Yunnan mivirus strain YN-MV1, and virus related to BLMV as a novel strain of BLTV named YN-MV2. Here we obtained the full-length genome of YN-MV1 (Fig. 2). YN-MV1 (GenBank MH814982) is a negative-strand circular RNA virus similar to WHMV, with genome length of 11, 395 nt which contains three open reading frames (ORFs) encoding RNA-dependent RNA polymerase (RdRp), glycoprotein (G) and nucleoprotein (NP) (Fig. 2). Phylogenetic tree showed that YN-MV1 is a novel member of Mivirus (Fig. 3A).
Figure 3. Phylogenetic relationship of A YNMV1 based on RdRp of members within Mononegavirales, B YN-rhabdov1 based on RdRp of rhabdovirus, C YN-PhleboV1 based on RdRp of bunyavirus, D YNflaviV1 based on NS3 and NS5 of flavivirus, E YN-BHKV2 virus based on RdRp of tetraparvovirus (indicated by red dot) with their closest viral family or genus. The trees shown here were inferred using an ML method with 1000 bootstraps.
Via blastn search, viral sequences demonstrated 97% identity to the Wuhan tick virus 1 (WHTV1) strain X78-2 were found in our tick pools. It is proposed to be a strain of WHTV1 termed YN-rhabdoV1 (GenBank MH814974). WHTV1, originally detected in same ticks in Hubei Province, currently remain unclassified in the family Rhabdoviridae and considered as a member of the dimarhabdovirus super group (Bourhy et al. 2005). The single negative-strand genome of YN-rhabdoV1 with a 10, 306 nt in length consists of four ORFs, two of which encodes RdRp and NP both being homologous with that of rhabdovirus (Fig. 2). The functions of the remaining two ORFs (ORF1 and ORF2) are unknown for no homologous known protein has been found. Phylogenetic analysis with other rhabdovirus shows that YN-rhabdoV1 is closer to Moussa virus, which is an unassigned rhabovirus, than to the other genus and forms a separated branch with a group of novel rhabodovirus-like virus identified mainly in ticks via NGS (Fig. 3B). This group of novel rhabodovirus-like virus may be considered as a new genus within Rhabdovirdae.
A number of tick-associated phlebovirus sequences were identified in our tick pools, and blastn or blastx search indicate these sequences mostly relate to viruses of Phlebovirus genus in Phenuiviridae. Viral sequences share 89% aa identity with Lihan tick virus (LHTV) which was originally identified in same tick species in central China, were considered to belong a novel phlebovirus named Yunnan tick-associated phlebovirus 1 strain YN-PheloboV1. Sequences share 98% aa identity with Bole tick virus 1 (BLTV1) strain BL075 are supposed to belong to a novel strain of BLTV1, tentatively named BLTV1 strain YNPheloboV2. And the remaining short contigs are considered as a novel phlebovirus named Yunnan tick-associated phlebovirus 3, sharing 60% aa identity with a blacklegged tick virus, which was originally identified in blacklegged tick from America. Like other phlebovirus, the genome of YN-PheloboV1 is supposed to consist three negative RNA segments termed S, M and L, respectively (Fig. 2). However, the M segment of both YN-PheloboV1 and LHTV haven't been identified yet. Phylogenetic tree shows that YN-PheloboV1 (Genbank No. MH814975 for S, Genbank No. MH814976 for M) is closest to Uukuniemi virus forming a separated branch with a group of novel putative phleboviruses which are characterized with unknown M segment (Fig. 3C).
Recently, a group of four segmented single-stranded positive RNA viruses related to flavivirus were identified in various arthropod and the prototype virus Jingmen tick virus (JMTV) strain SY84 was the firstly identified in R. microplus ticks collected in Hubei Province, China (Shi et al. 2016). Here, we also found the genomic sequences of a new JMTV strain YN-flaviV in the R. microplus ticks collected in 2017 in LX of Yunnan Province, which showed 93%–99% nt identity with strain SY84. Like the genome of JMTV, YN-flaviV comprises four segments (GenBank MH814977-MH814980) (Fig. 2), among which segment 1 and segment 3 respectively code protein related to nonstructural protein NS3 and NS5 of Flavivirus (family Flaviviridae). Proteins coded by segment 2 and segment 4 are quite unknown because no homologous proteins have been found. Via bioinformatics prediction, two N-glycosylation sites and three transmembrane domains were found on VP1 coded by segment 2, indicating VP1 may be a glycoprotein. Segment 4 has two ORFs coding VP2 and VP3. One signal peptide on the N terminal, one N- and O-glycosylation site was predicted on VP2. And VP3 is largely a membrane protein because 11 transmembrane domains were predicted on it. Phylogenetic tree with members of Flaviviridae shows that YN-flaviV clusters with a group of four segmented flavirus-like viruses named Jingmenvirus group, and were closest to members of Flavivirus (Fig. 3D). And this highly divergent lineage obviously separates into two sub-clades with one related to ticks and the other one related to insects or arthropods. We also found some short contigs related to NS3 and NS5 protein of flavivirus which share 80% aa identity with Bole tick virus 4 (BLTV4). BLTV4 was originally identified in Hyalomma asiaticum in Xinjiang Province of China and remained unclassified (Shi et al. 2016). We consider these flavi-like sequences detected in our study belong to a novel virus named Yunnan tick-associated flavi-like virus 1 (YNFLV1).
A new strain of Bovine hokovirus 2 (BHKV2) was found in our 2017-YNT RNA-seq library named YN-BHKV2 (GenBank MH814981), the genome of which shares 100% identity with BHKV2 strain BS-S13, which belongs to Tetraparvovirus genus of Parvoviridae. The genome of YN-BHKV2 consists of two ORFs, with ORF1 encoding the non-structural protein (NS1) and ORF2 encoding the vial capsid protein (VP1) as well as a putative transmembrane protein (VP2) encoded by an overlapping reading frame in ORF2 (Fig. 2). Phylogenetic analysis with other viruses of Tetraparvovirus genus shows YN-BHKV2 clustered with bovine hokovirus within ungulate tetraparvovirus group 1. This group of viruses was mainly detected in bovine from Hong Kong and domestic yaks in northwest China. However, different from the genotype epidemic in northwestern of China, another genotype is epidemic in Yunnan Province which was identical to that of Hong Kong (Fig. 3E).
PCR-based and immunofluorescence-based epidemiological survey was performed to investigate the prevalence of each virus in natural R. microplus tick during 2015–2017 (Table 3) and cattle and human sera (Table 4 and Fig. 4). IFA analysis of the positive control shows that the NP of each virus can be effectively expressed via transfected HEK293 (Fig. 4). YNMV1 virus has 20% or so positive rate among ticks from different landforms in different year. As for WHTV1, significantly high positive rate (42%) was detected in NJ in 2015, and relatively low positive rate (5%) or so was detected in LX in 2016 and 2017. Remarkably, serological assay shows high positive rate as 89% and 94% for YNMV1 and WHTV1, respectively among cattle serum. Comparing the above two viruses, prevalence of YN-PhleboV1 is much lower with 2.6% to 18.4%. Consistently, 8% cattle serum was positively detected with anti-YN-PhleboV1 antibody. And JMTV was only positively detected in one group of ticks from LX collected in 2015. Serological survey shows 18% cattle serum samples were detected positive for JMTV, meanwhile RNA was detected in four of these seropositive samples. Additionally, 63.1% tick samples from LX in 2017 and 40% cattle serum samples are positive for BHKV2. We also performed the same serological survey on 100 bovine serum samples from Hubei Province and healthy/febrile human serum from Yunnan LX, and no positive sample was detected with these five viruses. Collectively, these results suggest a stable and wild existence of YNMV1, WHTV1 and YN-PhelobV1 in R. microplus of Yunnan Province via circulation among ticks and cattle or may be other mammals.
2015 2016 2017 NJ
Yunnan mivirus 1 28.9%
Wuhan tick vrius 1 42%
YN tick-associated phlebovirus 1 2.6%
Jingmen tick virus 0 1/45
0 0 0 Bovine hokovirus 2 0 0 0 0 63.3%
Table 3. PCR-based epidemiological survey of each virus among tick samples
Cattle Serum sample Human serum sample Hubei
(n = 100)
(n = 100)
(n = 100)
(n = 100)
Yunnan mivirus 1 0 89% (89/100) 0 0 Wuhan tick vrius 1 0 94% (94/100) 0 0 YN tick-associated phlebovirus 1 0 4% (4/100) 0 0 Jingmen tick virus 0 18% (18/100) 0 0 Bovine hokovirus 2 0 40% (40/100) 0 0
Table 4. IFA-based epidemiological survey targeting NP protein of each virus