RESULTS
Tick collection and identification
From April 2020 to July 2021, a total of 2,031 ticks, including 204Haemaphysalis japonica , 393 H. concinna , 386Dermacentor silvarum and 1048 Ixodes persulcatus ticks,
were collected from NE China (Fig.1, Supplementary Table S1). The
collection sites were distributed in Ji’an (n=100), Dunhua (n=382), and
Shulan (n=300) in Jilin Province, Fangzheng (n=300), Mudanjiang (n=156),
Yichun (n=253), and Tahe (n=316) in Heilongjiang Province, and Songling
(n=224) in Inner Mongolia Autonomous Region (Fig.1, Supplementary Table
S1). Of these, five were located in Changbai Mountain (CBM), two in
Daxing’an Mountain (DXAM), and one in Xiaoxing’an Mountain (XXAM)
(Fig.1, Supplementary Table S1).
Identified RNA viruses
A total of 24 RNA libraries were constructed and sequenced, resulting in
245.8 GB clean data and ∼0.5 billion non-rRNA reads (Supplementary Table
S1). Totally, 2,059 viral contigs were obtained by de novo assembly from
∼0.93 million viral reads that accounted for 0.2% of the total non-rRNA
reads. Within each library, the viral reads ranged from 0.02% (library
SL3) to 0.53% (library JA1) of the total non-rRNA reads. After being
aligned by Blast and filtered by virus-host database, the viral contigs
were finally annotated to 22
viruses, belonging to the 8 viral
families, including Flaviviridae , Rhabdoviridae ,Nairoviridae , Phenuiviridae , Chuviridae ,Partitiviridae , Tombusviridae , and Solemoviridae ,
and one unclassified viral species (Table 1).
Viral genomic organization and taxonomy
For each of the 22 RNA viruses, the whole genomes of the representative
viral strains were further verified by Sanger sequencing (Table 1 and
Supplementary Fig. S1-S9). In total, 150 viral sequences from the 22
identified RNA viruses were determined (Supplementary Table S4). Of
them, 13 viruses were found in China
for the first time, and eight viruses were proposed as novel viral
species, as they were highly divergent to the previously identified
viruses (nt identity < 80% or RdRp aa identity
<90%), designated Tahe rhabdovirus 1-3 (THRV 1-3), Ji’an
nairovirus (JANV), Yichun nairovirus (YCNV), Mudanjiang phlebovirus
(MJPV), Yichun mivirus (YCMV), and Yichun tombus-like virus (YTLV),
respectively (Table 1). Another eight viruses, including Alongshan virus
(ALSV), Bole tick virus 4 (BLTV-4), Beiji nairovirus (BJNV), Jilin
luteo-like virus 2 (JLLV2), Xinjiang tick associated virus 1 (XTAV1),Ixodes scapularis associated virus 1 (ISAV1), Jilin partiti-like
virus 1 (JPLV1), and Ixodes scapularis associated virus 3
(ISAV3), showed the close relationships (nt identity > 80%
and RdRp aa identity > 90%) with previously described
tick-associated viruses that have not been approved by ICTV yet (Table
1). The remaining six viruses were all classified as known species, as
they showed close relationships and identical genome organizations with
ICTV-approved viruses, including tick-borne encephalitis virus (TBEV),
Songling virus (SGLV), Mukawa virus (MKWV), Sara tick phlebovirus
(STPV), Onega tick phlebovirus (OTPV), and Nuomin virus (NUMV) (Table
1).
Virome composition and abundance
Across the 24 libraries, 22 possessed 1–11 viral species, with the
exception of the libraries FZ1 and YC1, which had no virus detectable
(Fig. 2A). The ten libraries of I. persulcatus ticks had 4–11
viral species, with 11 in the library YC4, which included BJNV, YCNV,
MKWV, STPV, OTPV, NUMV, YCMV, JPLV1, JLLV2, ISAV1, and ISAV3. In
contrast, H. japonica , H. concinna , and D. silvarumlibraries only possessed 0–3 viral species. Of these 22 viral species,
17 species were identified in I. persulcatus , while only two,
three, and four species were detected in H. japonica , H.
concinna , and D. silvarum ticks, respectively (Fig. 2B).
Notably, most viruses were only identified in the specific tick species;
however, Tahe rhabdovirus 1 was found in the H. japonica ,H. concinna , and D. silvarum ticks, Ji’an nairovirus was
detected in the H. japonica and H. concinna ticks, and
Mukawa virus was identified in D. silvarum and I.
persulcatus ticks. It should also be noted that both ALSV and THRV3
were detected in only one I. persulcatus library. Interestingly,
some viral species, such as THRV2-3, SOLV, TBEV, and ALSV, were only
detected in ticks from DXAM, while other viruses, including JANV, MDPV,
YCNV, YCMV, BLTV4, YTLV, and XTAV1, were detected in ticks from XXAM and
CBM. There were also some viruses detectable in the libraries of three
regions, such as THRV1, BJNV, MKWV, NUMV, JPLV1, JLLV2, and ISAV1,
showing the wide distribution of these viruses (Fig. 2B). Of them, the
libraries of I. persulcatus ticks had relatively higher viral
reads than H. japonica , H. concinna , and D.
silvarum ticks, and the libraries of XXAM (Yichun) and DXAM (Tahe and
Songling) had relatively higher viral reads than that of CBM. Moreover,
NUMV and BJNV had relatively higher viral reads than other viruses (Fig.
2B).
Flaviviridae
According to the latest ICTV
report of virus taxonomy,Flavivirus ,Hepacivirus , Pegivirus , and Pestivirus are the
approved genera in family Flaviviridae . While in recent years, a
flavivirus-like group, the Jingmenvirus group, including a series of
genetically related viruses, such as Jingmen tick virus, ALSV, and
Yanggou tick virus, have been discovered (Fig. 3A). In this study, ALSV
was identified in the I. persulcatus library (TH4) from Tahe in
DXAM, and clustered together with ALSV strain H3 isolated from
tick-bitten patients in NE China (Fig. 3B) (Z. D. Wang et al., 2019),
but different from the isolates detected in I. persulcatus in
Russia and I. ricinus in Finland (Fig. 3B), with nt identities of
97.0–98.5% (Table S5, 6) (Kholodilov et al., 2020; Kuivanen et al.,
2019).
Three libraries (TH3, TH4, and SL4) of I. persulcatus ticks from
Tahe and Songling in DXAM were identified TBEV-positive, which formed a
different clade from the TBEV strains in XXAM and CBM, with nt
identities of 93.5–99.9% (Fig. 3C, Table S7).
Bole tick virus 4 (BLTV4)-NE
strains were phylogenetically grouped into the pestivirus-like group,
with nt identities of 77.5–83%
and RdRp aa identities of 94.6–96.9% to other BLTV4 isolates (Fig. 3A,
Table S8). BLTV4 was first identified in Hyalomma asiaticum ticks
in Xinjiang Uygur Autonomous
Region, China (M. Shi et al., 2016), and also discovered in Trinidad and
Tobago (Sameroff et al., 2019), Kenya (Y. Zhang et al., 2021), Romania
(Bratuleanu et al., 2021), and Thailand (Temmam, Chretien, et al.,
2019). Phylogenetic analysis showed that BLTV4-NE was different from
other strains identified in different tick species or regions (Fig. 3D).
In this study, the virus was only detected in two libraries of theD. silvarum ticks from CBM.
Nairoviridae
In the phylogenetic tree of the
family Nairoviridae , SGLV-NE, together with SGLV, formed a
separate clade from other viral members in the genusOrthonariovirus , including Ji’an nariovirus, Tacheng tick virus
1, and Tamdy virus (Fig. 4A). SGLV-NE was identified in two libraries
TH1 and TH2 of the H. concinna ticks from Tahe in DXAM,
and clustered together with SGLV
strains isolated from tick-bitten patients (Fig. 4B), with nt identities
of 92.4–99.2% (Table S9, 10) (Ma et al., 2021).
JANV, genetically related to SGLV with nt identities of 70.7–73.5%,
was a novel identified nairovirus (Table S9, 10). Four libraries were
detected JANV-positive, including DH1 and JA libraries of H.
japonica ticks in CBM, and MDJ1 and YC2 of H. concinna ticks in
XXAM (Fig. 4B).
BJNV and YCNV, belonging to an unclassified Norwavirus-like group,
showed close relationships to Norway nairovirus 1 and Grotenhout virus
(Fig. 4A), with nt identities of 75.4–79.6% (Segment L and S, Table
S11, 12). BJNV was identified in seven I. persulcatus tick
libraries in all the three regions, suggesting the wide distribution of
the virus in NE China. All the BJNV NE strains were clustered together
with BJNV strains identified in tick-bitten patients and I.
persulcatus tick in NE China and GSTV detected in Russia (Fig. 4C),
with nt identities of 96.2–100% (Segment L and S, Tables S11 and S12).
YCNV was only detected in three libraries (FZ3, YC3, and YC4) ofI. persulcatus ticks from Fangzheng and Yichun in CBM and XXAM.
Although YCNV showed nt identities of 82.3–83.9 (>80)
(Segment L and S, Table S11, 12) with BJNV, the virus formed a separate
clade, with aa (RdRp) identities of
87.7–88.5% (Fig. 4C, Table S11,
12), indicating that YCNV was a novel viral species that may be
different from BJNV.
Phenuiviridae
MKWV and MJPV, together with STPV and OTPV identified here, fell within
the genera Phlebovirus and Ixovirus of the familyPhenuiviridae , respectively (Fig. 5A).
MKWV NE strains were clustered
together with the strain MKW73 identified in I. persulcatus ticks
in Japan, with nt identities of
92.4–100% for L segment,
90.7–100% for M segment, and 93.5–99.9% for S segment (Fig. 5B,
Table S13, 14). Of the six MKWV NE strains, five were detected in theI. persulcatus ticks from Shuangzi, Dunhua, Yichun, and Tahe,
while one was identified in the D. silvarum ticks collected from
cattle in Shulan.
MJPV was only identified in two libraries in the I. persulcatusticks from Fangzheng and Mudanjiang in CBM, which was distantly related
to Kuriyama virus and
Mukawa virus strains, with low nt
identities of 75.8–77.4% for L segment,
67.6–68.7% for M segment M, and
69.8–70.8 % for S segment (Fig. 5B, Table S13, 14) (Matsuno et al.,
2018).
STPV and OTPV NE strains found in this study were clustered with STPV
and OTPV strains discovered in I. persulcatus ticks from Karelia
in Russia, with nt identities of 98.0–99.2% and 98.6–99.1%,
respectively (Fig. 5C, Table S15, 16). The two viruses formed separate
clades from each other, with nt identities of 56.4–57.3%, and were
identified in paired in I. persulcatus ticks in six libraries
from three collection sites, including Yichun in XXAM, and Tahe and
Songling in DXAM (Fig. 5C, Table S15, 16).
Rhabdoviridae
There were three novel rhabdoviruses identified in the study, namely,
Tahe
rhabdovirus 1 (THRV1), Tahe rhabdovirus 2 (THRV2), and Tahe rhabdovirus
3 (THRV3); they, together with Bole tick virus 2, Tacheng tick virus 3,
Huangpi tick virus 3, and Wuhan tick virus 1, were genetically grouped
into an unclassified Alphanemrhavirus-like group in the familyRhabdoviridae (Fig. 6A). A
total of eight libraries of H. japonica , H. concinna , and
D. silvarum ticks were detected THRV1, which formed close
relationship with Manly virus, with nt identities of 30.4–30.6% and
RdRp aa identities of 70.2–70.7%, respectively (Fig. 6B, Table S17).
However, THRV1 were divided into two different clades: one clade was
identified from libraries of Ji’an, Dunhua, Shulan, and Yichun in CBM
and XXAM, while the other clade found in libraries from Tahe and
Songling sited in DXAM, with nt identities of 81.4% and RdRp aa
identities of 93.6–93.7% (Fig. 6B, Table S17). Compared with THRV1,
THRV2 and THRV3 were only identified in the I. persulcatus tick
from Tahe in DXAM, with nt identities of 44.9–49.3% with THRV1 and
45.9–46.1% with each other (Fig. 6B, Table S17).
Chuviridae
Nuomin virus (NUMV) and Yichun mivirus (YCMV) identified here fell
within the Mivirus and Nigecruvirus genera in theChuviridae family, respectively (Fig. 7A). NUMV NE strains were
detected in all the ten I. persulcatus libraries in the three
regions in NE China, while no libraries from other tick species were
identified NUMV positive, indicating the wide distribution and
specificity of host tick species of the virus. The viral strains of NUMV
found in this study were clustered with other NUMV strains discovered in
humans from NE China and Lesnoe mivirus isolated from the I.
persulcatus ticks in Russia, showing nucleotide identities of
96.6–99.5% with each other (Table S18).
YCMV was a novel virus identified in this study, and clustered together
with Blacklegged tick chuvirus 2 found in I. scapularis ticks
from USA, with nt identities of 68.5%. YCMV was only detected in theI. persulcatus ticks from Dunhua and Yichun in CBM and XXAM (Fig.
7C), and shared high nt identity of 99.2%.
Partitiviridae
The Jilin partiti-like virus 1
(JPLV1) identified here fell within an unclassified clade, provisionally
designated as Deltapartitivirus-like group (Fig. 8A). JPLV1 NE strains
were clustered with JPLV1 strains JL/QG 1, JL/QG 2, and JL/QG 4 isolated
from I. persulcatus ticks in
Jilin Province, NE China, with the nt identity of more than 99%, and
JPLV1 was closely related to
Norway partiti-like virus 1
strains with high identities of nt 91.3–91.9% and aa 94.1–94.3%
(Table S19). Seven I. persulcatus libraries from all the three
regions were tested JPLV1 positive, with nt identities of more than 99%
to each other (Fig. 8B, Table S19).
Tombusviridae
The newly discovered Fangzheng
tombus-like virus (FTLV) fell within the Tombusviridae-like group in the
family Tombusviridae (Fig.
9A). FTLV NE strains were only found in I. persulcatus tick from
Yichun, Fangzheng, and Dunhua in XXAM and CBM (Fig. 9B), sharing a close
relationship (identity: nt 73.4–73.6% and aa 80.6–81.3%) to
Upmeje virus strain OTU9.IU20
that has been identified in Ixodes uriae in Sweden (Table S20)
(Pettersson et al., 2020).
Solemoviridae
Ixodes scapularisassociated virus 1 (ISAV1), Xinjiang
tick associated virus 1 (XTAV1), and
Jilin luteo-like virus 2 (JLLV2)
fell within the Sobemo-like virus group associated with the familySolemoviridae (Fig. 10A). In the aa RdRp phylogenetic tree,
ISAV1-NE was clustered with Norway
luteo-like virus 3 identified in Ixodes Ricinus ticks in Norway
and Ixodes scapularis associated virus 1 identified in I.
scapularis ticks in USA, with nt identities of 81.5–85.7% (Fig. 10B,
Table S21). All the six ISAV1 NE strains were found in the I.
persulcatus ticks from all the three regions in NE China, with nt
identities of 98.1–100% with each other (Table S21).
XTAV1 NE strains formed a different clade from ISAV1, with nt identities
of 61.9–62.8% (Fig. 10B, Table S21), and were detected in all the fourD. silvarum tick pools collected from Shulan and Dunhua sited in
CBM. The virus strains were clustered together with XJTAV1 strains 14-YG
and 16-T2 identified in Dermacentor nuttalli in Xinjiang Uygur
Autonomous Region, with nt identities of 95.9–96.2% (Table S21).
JLLV2 showed a close relationship with Norway luteo-like virus 2
identified in Ixodes ricinus ticks in Norway, with nt identities
of 87.7–88.2% and aa identities of 91.3–91.7% (Fig. 9C, Table S22).
JLLV2 NE strains were detected in the I. persulcatus ticks from
six libraries from all the three regions, and clustered with JLLV2
strains in I. persulcatus from Jilin and shared the closest
relationship with nt identity of more than 98% (Table S22).
Unclassified virus
Ixodes scapularis associated virus 3 (ISAV3) NE strains
identified in this study showed close relationship with ISAV3 and ISAV4
in Ixodes scapularis ticks from USA, but formed a different
clade, with nt identities of 86.7–88.2% (Fig. 11, Table S23) (Nakao et
al., 2017; Tokarz et al., 2018). Five libraries of I. persulcatusticks from Tahe, Songling, and Yichun in DXAM and XXAM were detected
ISAV3-positive, with nt identities of 93.7–99.8% (Fig. 11, Table S23).