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Diversity of RNA viruses of three dominant tick species in North
China
Tong Qin
Academy of Military Medical Sciences
Mingjie Shi
Academy of Military Medical Sciences
Meina Zhang
Academy of Military Medical Sciences
Zhitong Liu
Academy of Military Medical Sciences
Yi Sun ( sunyi@bmi.ac.cn )
Academy of Military Medical Sciences
Sun Yi
Academy of Military Medical Sciences
Article
Keywords: Virome, Diversity, high-throughput sequencing, Ixodidae, China
Posted Date: July 20th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1782867/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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Abstract
[Background] A wide range of bacterial pathogens have been identied in ticks, yet the diversity of viruses in ticks is largely unexplored.
[Methods] Here, we used metagenomics sequencing to characterize the diverse virome in these three species,
Haemaphysalis concinna
,
Dermacentor silvarum
, and
Ixodes persulcatus
, the principal tick species associated with pathogens transmitted in North China.
[Results] A total of 28 RNA viruses were identied and belonged to more than 12 viral families, including single strand positive sense RNA
virus (Flaviviridae, Picornaviridae, Luteoviridae, Solemoviridae and Tetravirus), negative sense RNA virus (MononegaviralesBunyavirales
and others) and double strand RNA virus (Totiviridae, Partitiviridae). Of them, Dermacentor pestivirus like virus, Chimay like rhabdovirus,
taiga tick Nigecruvirus, Mukava virus are presented as novel viral species, Nuomin virus, Scapularis ixovirus, Sara tick borne phlebovirus,
Tacheng uukuvirus, Beiji orthonariovirus had been established as human pathogens with undetermined natural circulation and
pathogenicity. Others virus including Norway mononegavirus 1, Jilin partitivirus, tick borne tetravirus, Pico-like virus, luteo-like virus 2, luteo-
like virus 3,Vovk virus, Levivirus and others Toti-like virus and Solemo-like virus involved with unknown pathogenicity to human and wild
animals.
[Conclusion] Extensive diverse of viruses frequently occur in two orders of Mononegavirales and Bunyavirales in the three tick species.
Comparatively,
I. persulcatus
ticks had signicant higher viral species than those in
H. concinna
and
D. silvarum
ticks. Our analysis
revealed that ticks are reservoirs for a wide range of viruses and suggests that discovery and characterization of tick borne viruses will
have implications for viral taxonomy and may provide insight into tick-transmitted viral zoonotic diseases.
Introduction
Ticks (Arachnida: Ixodida) are common hematophagous arthropods that have been implicated as vectors of human and animal diseases
world-wide (Guglielmone et al. 2014). Approximately 900 species of ticks have been described and taxonomically classied into four
families Argasidae (soft ticks), Ixodidae (hard ticks), Nuttallidae and newly proposed Deinocrtotonidae (Guglielmone et al. 2010, Peñalver
et al. 2017). Their propensity for feeding on multiple hosts, expansive range, and long life cycle underlines the importance of active
surveillance of ticks for the presence of potential pathogens threaten on human health (Jongejan and Uilenberg, 2004). Argasid and Ixodid
ticks combined transmit a greater diversity of viral, bacterial, and protozoan pathogens than any other arthropod vectors (Madison-
Antenucci et al. 2020). The increasing incidence of tick-borne disease worldwide is caused partly by the increased frequency of human
exposure to ticks or their endemic habitats, burgeoning tick populations and the discoveries of new tick-associated agents (Fang et al.,
2015, Madison-Antenucci et al. 2020). The signicance of tick-borne viral diseases (TBVDs) in human health has raised global health
concerns in recent decades due to the limited measures responding for suboptimal diagnostics, treatment options for emerging viruses,
and a scarcity of vaccines(Ortiz, et al. 2021). Attentions from clinicians and researchers has alerted the public according to the emergence
of novel viral pathogens causing febrile human diseases, such as Dabie bandaviru, also known as severe fever with thrombocytopenia
syndrome virus (SFTSV) (Yu et al. 2011, Liu et al. 2014)), Alongshan virus (Wang et al. 2019), Bourbon virus (Ejiri et al 2018), most recently
Jingmen tick virus (Jia et al. 2021), Yezo virus (Kodama et al. 2021), Toyo virus (Kobayashi et al. 2021) and Songlin virus (Ma et al. 2021),
which had been identied in China and neighbor countries. Additionally, the re-emergence and continuous spread of known tick-borne virial
diseases (TBVDs), such as Crimean–Congo hemorrhagic fever virus (CCHFV) (Monsalve-Arteagaid et al. 2020), Heartland virus (McMullan
et al., 2007), tick-borne encephalitis virus (Madison-Antenucci et al. 2020), Powassan virus (Grard et al., 2007) and African swine fever virus
(Gaudreault et al. 2020) positively correlate with the increasing incidence of TBVDs in humans and animals. The emergence of some
undened TBVDs pathogens as well as the dearth of data on tick virome highlights an urgent requirement for active viral surveillance and
discovery in ticks, which greatly promote our knowledge of the biodiversity and evolution of viruses vectored by ticks. Although it seems
plausible that traditional isolation via tissue culture has been proved a prerequisite gold standard to characterize a novel virus or insight a
known one. However, not all tick-borne viruses are amenable to be isolated due to technological limitations, extensive culture-independent
studies, such as, transcriptomics analysis following the high throughput sequences, have been attempted to examine tick virome (Li et al.
2015, Pettersson et al. 2017). Such studies might not only identify viruses associated with acute diseases but also could provide insights
into the pathogenesis of more controversial chronic illnesses associated with tick bites (Houldcroft et al. 2017). Therefore, proling viruses
in ticks and investigating the substantial contacts between ticks and human or animal hosts is urgent to better understand the viral sphere
vectored by ticks and identify the potential viral pathogens in China. In this study, virome of tick pools were proled via metagenomic
sequencing aimed at identifying the baseline viruses vectored by ticks and characterizing their biodiversity and evolution. And then, the
viral transmission patterns between ticks and human were discussed based on the active surveillance results of molecular prevalence and
potential exposure of human to these ticks. These ndings may shed lights on a more closely explore for the virome of ticks endemic to
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China and reveal the potential risks of TBVDs in human population, which helps to guide tick borne diseases prevention and control in
China.
Materials And Methods
Sample collection
Questing
Ixodes persulcatus
,
Haemaphysalis concinna
and
Dermacentor silvarum
were collected by sweeping ags on the vegetation in
Zhalantun county, Inner Mongolia autonomous region and Mudanjiang city, Heilongjiang province respectively. Morphological
identication of ticks was conducted using the standard key for Chinese ticks (Teng and Jiang, 1991) under stereomicroscopes.
Library preparation and sequencing.
Following species identication, total nucleic acid/RNA was extracted from three pools containing 30 females, males or nymphs of each
species. Before total RNA extraction, 300 µL of pooled homogenates was puried at room temperature by ltered (0.45 µM) and then
treated with RNase A (15 min), followed by Turbo DNase and Benzonase (MilliporeSigma, Burlington, MA, USA) (30 min) (Pettersson et al.
2017). The method is proved to enrich for viral particles due to the degradations of unprotected nucleic acids absence of a viral capsid
(Loens et al. 2007). The enriched total nucleic acid (11 µL) from each tick pool was then subjected to rst and second-strand cDNA
synthesis with Super Script IV reverse transcriptase (Invitrogen, Waltham, MA, USA) and exo-Klenow fragment (New England Biolabs,
Ipswich, MA, USA), respectively. For all libraries, the Ribo-Zero Gold Kit (Illumina) was used to removed ribosomal RNA under the
manufacturer’s guidance. Subsequently, all rRNA-depleted RNA-samples were resuspended to construct libraries using the KAPA Stranded
RNA-Seq Kit (KAPA biosystems, Roche) with barcode adapters (Bioo Scientic) following the manufacturer’s instructions. Qubit high
sensitive RNA assays (ThermoFisher Scientic) were performed to quantify cDNA-levels before, during and after library preparation and the
fragment sizes were simultaneously determined with a Agilent Bioanalzyer. Subsequently, equimolar amounts of nucleic acids were pooled
and submitted for sequencing in each library. All libraries were sequenced on a single lane (paired-end, 125 bp read-length) on an Illumina
HiSeq 2500 platform at the BGI Sequencing Centre (www.genomics.cn).
Quality checking, trimming and de novo assembly.
Sequencing raw reads were rstly subjected for adapter removal, quality trimmed using trimGalore
(www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (Krueger et al, 2021). Clean reads were de novo assembled using the Trinity
v2.8.5 program. (Grabherr et al. 2011, Haas et al. 2013).
Virus discovery and genome annotation.
Trinity assemblies with the length above 200 bp were subjected for BLASTN against all non-redundant nucleotide (nt) databases using a
local BLAST tool and BLASTX against all non-redundant protein (nr) databases (available as early as May 2022) of reference RNA viruses
as well as those recently published, with hits at an
e
-value of 1×10−5 or better collated. All potential virus assemblies were screened
against the Conserved Doman Database (www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi) with an expected value threshold of 1×10−3 to
identify viral gene segments.Putative viral contigs were further merged by high-identity overlaps a threshold value of 95% similarity with
using the SeqMan program of Lasergene package v7.1 (Burland 2000). To complete the remaining gaps, original reads were aligned to the
viral contigs again using Bowtie2 program (Langmead and Salzberg 2012), and the resultant assembly was veried in the Integrated
Genomics Viewer (Thorvaldsdóttir et al. 2013). A novel viral species should be satised with one of the following conditions described
before (Xu et al. 2021), namely, (i) <80 per cent nt identity across the complete genome; or (ii) <90 per cent amino acids identity of RNA-
dependent RNA polymerase (RdRp) domain with the known viruses. To eliminate possible endogenous viruses, all virial assemblies were
blasted against the
reference genomes of
I. persulcatus
(GCA_013339685.1),
D. silvarum
(GCA_013339745.1) and whole genome shotgun
database of Ixodida (Taxonomy ID: 6935, accession data:1/1/2022) respectively. If aligned bases of the query contigs covered more than
50% and the nucleotide similarity exhibited higher than 85% from any comparison with the above databases, they were discarded from the
downstream analysis. Transcripts abundance containing RNA-Seq fragment counts for each transcript (or gene) across each sample was
estimated using the alignment-based abundance estimation method RSEM (Xia
et al.
, 2011). The trimmed mean of M-values
normalization method (TMM) were employed to normalize the transcript abundance.
Multiple sequence alignments and evolutionary analysis.
To infer the evolutionary relationships of the viruses discovered, the protein translated RdRp open reading frame segments produced in this
study were combined with representative complete proteomes and (or) RdRp-segments of theBunyavirales,Mononegavirales, Flaviviridae,
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Luteoviridae,Partitiviridae, Totiviridae and Picoranviridae
were retrieved from NCBI Genbank (www.ncbi.nlm.nih.gov/genbank) and aligned
using Mafft v.7.266, employing the E-INS-i algorithm (Katoh and Standley, 2013). Ambiguous regions in the alignments were removed with
TrimAl v.1.2 (Capella-Gutierrez et al. 2009). Following sequence alignment, ProtTest v.3.4was employed to select the best-t model of
amino acid substitution (Darriba et al. 2011). Finally, maximum likelihood trees for all alignments were inferred using the best-t model of
amino acid substitution (LG+I+ Γ +F for all alignments) with 1000 bootstrap replicates with the PhyML v.3 program(Guindon et al. 2010).
Phylogenetic trees were edited and visualized with FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/gtree). All phylogenetic trees were
mid-point rooted for purposes of clarity only.
Data availability.
All sequence reads generated in this project are available under the NCBI Short Read Archive (SRA) under accessions SAMN26934333–
SAMN26934340 (BioProject ID: PRJNA819490) .
Results
Sampled from Mudanjiang city of Heilongjiang province
I. persulcatus
and
Hae. concinna
were assigned to 3 groups and 2 ones
respectively. Average 10.96Gb of data including 7.1-7.6 × 107or so 150-base pair-end reads were generated from male, female and nymph
group of
I. persulcatus
. While the male and female group of
Hae. concinna
generated ~11.18Gb of data including 6.8-8.0 × 107 or so 150-
base pair-end reads.
The third tick species,
D. silvarum
, collected from Zhalantun, Hulunbr, Inner Mongolia were also assigned to male,
female and nymph groups, each group generated ~ 12.30Gb of data including 7.5-8.6× 107or so 150-base pair-end reads. After quality
ltration and host subtraction, total of 614,213,814 reads remained which were assembled into 815,457 contigs. Assembled contigs were
compared to the NCBI Viral Genomes database, resulting of 1311, 1033 and 709 contigs from
I. persulcatus
,
D. silvarum
and
Hae. concinna
as viral origin through BLASTN and BLASTX, which were kept for a subsequent manual inspection (Supplemental Table 1). A prediction of
ORFs was also implemented to compared to the viral protein database through BLASTP. As a result, sequences of 28 putative viruses were
identied, with ve representing novel species. Seventeen viruses were identied within the
I. persulcatus
pool, eleven in
D. silvarum
while
only six were discovered in
H. concinna.
Of them, single-stranded positive-sense RNA viruses (Flaviviridae, Luteoviridae, Picornaviridae,
Solemoviridae and Tetraviridae), negative-stranded RNA viruses (Mononegavirales, Bunyavirales) and double stranded RNA viruses
(Totiviridae, Partitiviridae) were involved (Table 1).
Table 1. Virus identied in three dominant tick species in North China
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Voucher
species Genus No.
unique
contigs
contig
length Encoded
Fragments
mean
seq.
depth
relative
abundance
(RdRp%)
RdRp
Identity Top hit
(accession no)
Ixodes
persulcatus
Mivirus 3 10853 N, L, G,
VP4 1107-
3210 0.01–0.02 99.95 Nuomin virus
(UKS70436.1)
Nigecruvirus 3 11436 N, L, G,
VP4 2103-
3309 <0.01 83.70 Blacklegged tick
Chuvirus 2
(AUW34382.1)
Nucleorhabdovirus 3 2033-
5986 L, G 650–
1645 0.01–0.02 88.44 Chimay rhabdovirus
(AVM86063.1)
Alphanemrha
virus-like
4 5717-
6663 L, N 1905-
2221 0.07 49.46 Norway
mononegavirus 1
(ASY03261.1)
Ixovirus 2 6650-
6680 L, N 1105–
4732 0.25–0.47 99.67 Sara tick phlebovirus
1 (QPD01621.1)
Ixovirus 8 1380-
6630 L 277-
22219 0.02–0.07 74.1 Scapularis ixovirus
(YP_010086238.1)
Orthonairovirus 2 12840-
12869 L, S 105–
4732 0.01 99.04 Beiji nairovirus
(UFP37779.1)
Orthonairovirus 4 7842-
11076 L, N 1137-
4326 <0.01 93.25 South Bay virus
(ANT80542.1)
Orthobunyavirus 3 5780-
5800 L,G 1237-
2938 <0.01 38.25 Ixodes scapularis
bunyavirus
(BBD75425.1)
unclassied
Bunyavirales 6 4295-
9080 L 2231-
3091 <0.01 46.78 Ubmeje virus
(QKK82912.1)
Deltapartitivir
us-like
3 1510-
1628 L 1-521 0.01–0.02 98.1 Jilin partiti-like virus 1
(QTZ97684.1)
Luteoviridae 2 1419-
1628 L 427-
469 <0.01–0.07 93.8 Norway luteo-like
virus 2 (ASY03255.1)
Luteoviridae 3 1398–
1610 L 178–
506 <0.01–2.62 84.04 Norway luteo-like
virus 3 (ASY03257.1)
Riboviria 2 5389-
5427 L,N 1793-
1797 0.25–0.47 44.24 Vovk virus
(QKK82915.1)
Levivirus 2 620-
1899 L,H4bulk,
coat 206-
632 <0.01 42.27 Levivirus
sp
.
(QDH88856.1)
Totivirus 2 1100-
1330 L 509 0.01–0.02 38.17 Hubei toti-like virus
24 (YP_009336908.1)
Sobemovirus 2 2621 L 872 0.01–0.02 39.62 Hubei sobemo-like
virus 15
(YP_009330030.1)
Table 1(continued). Virus identied in three dominant tick species in North China
ssRNA(+) viruses
Sequences for a novel viral species belonging to family Flaviviridae, tentatively named
Dermacentor silvarum
pestivirus-like virus 1 (DSPV),
were identied in all the 3
D. silvarum
pools (Fig.1 panel A and B). DSPV comprises a single polyprotein that shares closest homology
within the NS3 and NS5 of viruses within genus Pestivirus (Sameroff et al. 2022), which clustered DSPV with other recently identied
pestivirus-like viruses, Bole tick virus 4 and Trinbago virus (Sameroff et al. 2019). Additional segmented avivirus, Jingmen tick virus were
either discovered in
I. persulcatus
with 97.16% and 96.90% amino acids similarities with RdRp (QFR36159.1) and VP3 protein
(QHW66956.1) respectively. These contigs were shown positioned on corresponding sites our phylogenetic tree of these Flavivirus
associated virus (Shi et al. 2016) constructed based on their RdRp genes (Fig.1 panel A and C).
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Voucher
species Genus No.
unique
contigs
contig
lengths Encoded
Fragments
mean
seq.
depth
relative
abundance
(RdRp%)
RdRp
Identity Top hit
(accession no)
Dermacenor
silvarum
pestivirus-like
viruses 4 14481-
15100 L. S 4827 0.01 90.81 Bole tick virus 4
(QUJ17979.1)
Alphanemrha
virus-like
3 6610-
6701 L, N 1241-
3305 <0.01 94.51 Rhipicephalus
associated
rhabdo-like virus
(QYW06859.1)
Alphanemrha
virus-like
2 6659-
6700 L 1893-
2215 0.01 48.42 America dog tick
rhabdovirus
(AUX13124.1)
Ixovirus 1 6650 L, S 4732 0.27 99.41 Sara tick
phlebovirus 1
(QPD01621.1)
Phlebovirus 2 2890-
3300 L, N,Ns 1079 0.04 93.53 Mukawa virus
(YP_009666332.1)
Uukuvirus 3 4537-
4550 L 1513 0.04 92.61 Tacheng
uukuvirus
(AWK68110.1)
Orthonairovirus 4 7842-
11076 L, N 1137-
4326 <0.01 93.25 South Bay virus
(ANT80542.1)
Solemoviridae 2 722-
1440 orf1, orf2 522-
1328 <0.01 100 Xinjiang tick
associated virus 1
(QBQ65134.1)
Picornavirus 2 648-
684 L 316-
328 0.01–0.07 33.79 Xiangshan
picorna-like virus 7
(UDL13977.1)
Totivirus 2 2008-
4566 L, orf1 774-
1052 0.01 45.10 Lonestar tick
totivirus
(AUX13136.1)
Totivirus 3 572-
881 L 235 <0.01 98.31 Xinjiang tick
totivirus
(QBQ65052)
Unclassied
Tetravirus 3 5310-
5514 L 1345-
1737 <0.01 71.53 tick borne
tetravirus
(QTE18640.1)
Haemaphyaslis
concinna
Alphanemrha
virus-like
1 7238 L 2170 0.01–0.02 84.21 Manly virus
(AYP67529.1)
Nucleorhabdovirus 4 1350-
1568 L ,N 537 0.01 73.53 blacklegged tick
rhabdovirus-1
(AUW34390.1)
Alphanemrha
virus-like
2 3489-
6500 L 1133–
2169 0.01–0.02 48.29 Tacheng Tick
Virus 3
(YP_009304331.1)
Alphanemrha
virus-like
2 2227-
2415 L,N 775-
804 0.01 51.40 Bole tick virus 2
(YP_009287864.1)
Sobemovirus 2 3700-
5092 L 1228-
1678 0.01 75.65 Hubei sobemo-like
virus 15
(YP_009330029.1)
Totivirus 2 2008-
4566 L, orf1 774-
1052 0.01 50.01 Lonestar tick
totivirus
(AUX13136.1)
Luteovirius, Picornavirus, Solemoviridae and Tetravirus
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Luteoviriales, Picornavirus Tetravirus and Solemoviridae members are also recognized as single-stranded positive-sense RNA viruses that
comprises many economically important pathogens parasite in human and animals (Ali et al. 2014)). In the present study, taiga tick luteo-
like virus were also identied with shared amino acid sequences of Norway luteo-virus 2 (88.44%, ASY03255.1) and Norway luteo-virus 3
(84.04%, ASY03257.1) respectively across 99% RdRp gene. The two virus grouped with
Ixodes scapularis
-associated viruses in the
phylogeny tree, although low abundant Norway luteo-virus 2 were present in one library (Supplemental Fig. 1 Panel A). Total 3
Solemoviridae members were discovered as well in the present study, of which, Sobemovirus like virus identied from
Hae. concinna
and
I.
persulcatus
were found identical to and Hubei sobemo-like virus 15 (YP_009330030.1) with 75.65% and 39.62% identities on the coverage
of RdRp over 2620 amino acids sequences. While another Solemoviridae member, Xinjiang tick associated virus 1 from
D. silvarum
were
shown 100% identical to those sequences on orf1 and orf2 respectively (Supplemental Fig. 1 Panel B). Additionally, another positive-sense
ssRNA viruses, tick borne tetravirus and picorna-like virus from
D. silvarum
exhibited 71.53% and 33.79% amino acids identities of RNA
polymerase genes with tick borne tetravirus-like virus (QTE18640.1) (Tokarz et al., 2018) and Xiangshan picorna-like virus 7 (UDL13977.1)
over 95% coverage spaced across L segments (Supplemental Fig. 1 Pane C and D). Further classication of the two ss(+)RNA viruses was
challenging due to high sequence divergence in combination with a limited number of available Tetravirus and Picornavirus sequences.
ssRNA(-) viruses
Mononegavirales
Chuviridae
Assembly of the contigs from
I. persulcatus
yield two virus belongs to Chuviridae. Among them, a mivirus, Nuomin virus, was discovered
from
Ixodes persulcatus
with shared viral RdRp amino acid (aa) sequences at99.95% on the 100% query coverage ofNuomin virus
(UKS70432.1) (Fig.2 Panel A and B). Nuomin virus was reported as a negative-strand circular RNA virus, whose genome at length of 10,863
nucleotides, coding open reading frames (ORFs) of RNA-dependent RNA polymerase (RdRp), glycoprotein (G), nucleoprotein (NP) and VP4
protein (Supplemental Fig. 2 panel A), which was shown high similarity to RdRp of Lesone mivirus (QPD01622.1) and Suffolk virus (YP
009177218.1) with distinct amino acids sequences on VP4. Nuomin virus had been recognized pathogenic to human by Liu et al. (2020) in
Tahe, Heilongjiang province, China, and then assigned to the recently-proposed Mivirus genus of Chuviridae (Li
et al.
2015). Fortunately, we
also achieved a novel Nigecruvirus, tentatively named Taiga tick Nigecruvirus (Fig.2 Panel A and B), which was shown at length of 11,436
nucleotides and identical to blacklegged tick Chuvirus 2 in L (AUW34382.1), G(AUW34383.1), NP(AUW34384.1) and VP4(AUW34385.1)
(Supplemental Fig. 2 panel B) based on the amino acids similarities at 83.07%, 79.94%, 66.22% and 67.31 respectively over the 100%
coverage. Homology searches revealed this virus to be very distant from genus
Mivirus
. The similarities of the four ORFs of the novel taiga
tick Nigecruvirus and Nuomin virus ensure that the two Chuviridae members prevalence in
I. persulcatus
in China (Shi et al. 2021).
Mononegaviridae-like virus
Except for Chuviridae, we also identied several viral contigs belonging to Mononegavirales (Afonso et al. 2016). Among which, one
nucleorhabdovirius and two alphanemrhavirus-like virus (Tacheng tick virus 3 and Norway mononegavirus 1) were discovered from
I.
persulcatus
with88.44%, 47.32% and49.46%identities to those aligned RdRp sequences of Chimay like rhabdovirus (62.52%
AVM86063.1),Tacheng tick virus 3, (YP_009304331.1) and Norway mononegavirus 1(ASY03261.1).Moreover, similar glycoprotein (G)
and nucleoprotein (NP)sequences were also obtainedfrom
I. persulcatus
and assigned as glycoprotein of Chimay
rhabdovirus(AVM86062.1) and nucleoprotein of Tacheng tick virus 3(62.17%, AJG39137.1) andNorway mononegavirus 1(57.43%,
KAG0427517.1) respectively. As a case of
H. concinna
,
assembly contigs obtainedwereshown with a high degree of diversity in order
Mononegavirales involved 3 alphanemrhavirus-like rhabdoviruses and one nucleorhabdovirus. Of alphanemrhavirus-like rhabdovirius, Bole
tick virus 2, Tacheng Tick Virus 3 and Manly virus were involved shared RdRp sequences with accession no. of reference strains as
YP_009287864.1(51.40%,804 aa), YP_009304331.1(48.29%,2179aa) andAYP67529.1 (84.21%, 2168 aa)in L segments respectively.
Bole tick virus 2 were also identied with nucleoprotein segment (73.02%, YP_009287860.1) over 100% coverage. The nucleorhabdovirus
(blacklegged tick rhabdovirus-1) shared similarities as 73.53% (AUW34390.1,1568aa) in RdRp genes of L segment. Whereas,
D. silvarum
from Zhaluntun, Inner Monogolia yield two alphanemrhavirus-like viruses identical to
Rhipicephalus
associated rhabdo-like virus
(QYW06859.1)and American dog tick rhabdovirus (AUX13124.1) with94.51% and48.42% similarities over the99.0% coverage. The details
of related protein fragments other than RdRp sequences in the viruses described above were shown in Table 1 (Fig.2 Panel A and C).
Bunyavirales
Bunyaviruses are segmented negative-stranded viruses that include at least 9 families and 13 genera, many of which were involved as
pathogens to human and animals (Kuhn et al. 2021). In our study, 11 species in 5 genera of 3 families in the order Bunyavirales were
Page 8/17
detected. Assembly of these contigs revealed the presence of 2 Orthonairovirus (Nairoviridae),3 Phleboviruses, 2 Ixovirus and one
uukuvirus (Phenuiviridae), one Orthobunyavirius (Peribunyaviridae) and 2 unclassied Bunyavirus from
I. persulcatus
and
D.
silvarum
,
whereas
no bunyavirales contigs detected in
Hae. concinna
(Table 1). The phylogenetic tree of Bunyavirales constructed based
on the RdRp genes indicated that these phleboviruses, orthonairovirus and unclassied bunyavirus clustered with referenced strains and
formed well-supported monophyletic groups as the “classic” schematic diagram involved with Orthohantanviridae, Narioviridae
Phenuiviridae, Peribunyaviridae and others (Fig. 3 Panel A).
Nairoviridae
Assembly
of
multiple contigs
from
I. persulcatus
and
D. silvarum
pools contained with coding sequences similar to viruses in the genus
Orthonairovirus by BLASTx. With CCHFV as a reference genome, assembly of these orthonairovirus-like contigs, provided 100% coverage of
L and S segments. The assembled sequences from
I. persulcatus
showed high identity (99.81%) to Beiji nairovirus (BJNV) (Fig. 3 Panel A
and B), a novel species initially discovered from
I. persulcatus
(Meng
et al. 2018) and human patients (Wang et al. 2021). BJNV reads were
the dominant viral reads obtained from all
I. persulcatus
pools and accounted for 5-7% of ltered reads. Moreover, South Bay virus were
also unrevealed from
D. silvarum
with 97.12% similarity in RdRp (AII01810.1) and 77.61% in nucleocapsid protein (AII01798.1) over the
100% coverage(Table 1, Fig. 3 Panel A and B). However, despite an exhaustive bioinformatics analysis, we were unable to identify any
contigs or reads with any similarity to M segments of Orthonairovirus.
Phenuiviridae
In the present study, total 6 species of three genera in the family Phenuiviridae presents in
Ixodes persulcatus
and
Dermacentor silvarum
.
The most diversity appeared in genus Phlebovirus, which currently comprised of over 70 phlebovirus isolated from ticks, mosquitoes, and
sand ies (Elliott and Brennan, 2014). We identied multiple contigs with homology to phleboviruses by BLASTx in pools of
I. persulcatus
and
D. silvarum.
The contigs derived from the pools of each tick species were assembled separately to a reference phlebovirus genome. We
obtained phlebovirus contigs from
D. silvarum
, which were identical to Mukawa virus with shared amino acids similarities inL segments
(98.60%, YP 009666332.1) and nonstructural protein (93.53%, YP_009666334.1) and nucleoprotein (99.60%, YP_009666333.1)over the
98% coverage. Moreover, an unclassied phlebovirus, Changping tick virus 2 (AJG39235.1), was harvested from
D. silvarum
with85.46%
similarityon the 100% sequence coverage of RdRp gene. Additionally, two members in genus
Ixovirus
was also achieved, one is Sara tick
phlebovirus from both
D. silvarum
and
I. persulcatus
with their RdRp identities up to 99.32% and 99.41% respectively across the 2210
amino acids sequences. Another
Ixovirus
in Phenuiviridae, Scapularis Ixovirus (previously cited as blacklegged tick phlebovirus 3,
YP_010086238.1), were also documented in
I. persulcatus
with74.1%identity in L segments. Fortunately, the third genus
Uukuvirus
in the
family Phenuiviridae were harvested from
D. silvarum
and identied as Tacheng uukuvirus (Tacheng tick virus 2,AWK68110.1)
with92.61% similarity on the amino acids sequences on polymerase gene(Table 1, Fig. 3 Panel A and C). All sequences assigned to the
Phenuiviridae family were presented with missed M segment coding for the viral glycoprotein.
Other Bunyavirales
Except Nairoviridae and Phenuiviridae, we also obtained another viral contigs belonging to Bunyavirales from
I. persulcatus
,
which
exhibited relative lower identity withvolzhskoetick virus 1 (72.41%, QPD01626.1) (Pettersson et al. 2020) and
Ixodes scapularis
bunyavirus (38.25%,BBD75425.1) (Orthobunyavirus, Peribunyaviridae) based on over 90% sequence coverage of RdRp gene. Finally,
Ubmeje virus (QKK82912.1) from
D. silvarum
were presented with lower identity as46.78%, which were also known as an unclassied
Bunyavirales (Table 1, Fig. 3 Panel A and D).
Double-stranded RNA viruses
Both Partitiviruses and Totiviridae are double-stranded RNA viruses mostly known abundant and diverse in arthropods, some of which are
pathogens to animal and human. Notably, several lineages of the partitiviruses appear to be, and the genomes of these hosts also
harbored related endogenous virus elements in host genomes (Nibert et al. 2014). We identied a highly divergent partiti-like virus, Jilin
partiti-like virus 1 from three
Ixodes persulcatus
pools. Interestingly, this virus formed a cluster with other arthropod associated partiti-like
viruses, which shared a close relationship with those found in host animals associated with arthropods. Whether Jilin partiti-like virus 1 is a
truly tick borne virus will need to be examined in details. Besides partiti-like virus, another dsRNA virus close to the family Totiviridae were
also discovered from
Hae. concinna
with relative lower amino acids identities withover 95% coverages of RdRp
genes(50.01%,AUX13136.1)and ORF1 (40.67.01%, AUX13135.1) ofLonestar tick totivirus. Meanwhile
I. persulcatus
yields contigs similar
to Hubei toti-like virus24based on over95%sequences L segment(38.17%, YP_009336908.1).As a case of
D. silvarum
,both Lonestar tick
totivirus and Hubei toti-like virus 24 were found with RdRp genes(Supplemental Fig. 3)
Page 9/17
Others
Other more distantly related viruses including unclassied Vovk virus (44.24% RdRp aa identity with QKK82915.1) (Wille
et al.
, 2020) and
Levivirus (42.27% RdRpaa identityQDH88856.131.75%H4bulk(QDH87337.1) and35.34%coat(UJQ85258.1)in the family of Leviviridae
were also detected from
I. persulcatus
.
Discussion
This study focused on the characterization of virome diversity of questing
D. silvarum
,
I. persulcatus
, and
H. concinna
ticks from North
China. The outcome from our sampling efforts supports the results throughout various Chinese regions, showing that
I. persulcatus
,
D.
silvarum
and
H. concinna
are three of the most abundant questing ticks within the regions (Jia et al. 2020). All three tick species have been
heavily implicated in the transmission of TBDs throughout the regions.
I. persulcatus
is the principal vector of the agents of Lyme
borrelioses, Rickettsioses and Babesioses along with
A. phagocytophilum
, and clinically relevant human pathogen Jingmen tick virus
(Alongshan virus) and far-eastern strain of TBEV (Li et al. 2022).
D. silvarum
and
H. concinna
had also been linked with several tick-borne
pathogen found throughout China, including
Francisella tularensis
,
Coxiella burnetii
,
Rickettsia
spp.,
Babesia
spp.,
Anaplasma
spp., TBEV
and SFTSV/Dabie bandavirus ((Meng et al 2019, Liu et al. 2022). Despite examining more pools of
D. silvarum
and
H. concinna
, we had
identied a greater number of viral contigs in
I. persulcatus
while no bunyavirales viral sequences was detected within
H. concinna
pools.
Combined, these studies support a hypothesis that different tick species can harbor diverse viruses at different levels. Interestingly, no viral
sequences for known TBEVs endemic to the region, besides low abundance Jingmen tick virus were identied from the sequencing data.
This most likely can be attributed to the low prevalence of these viruses within the tick populations along with sampling bias. For example,
data show that the prevalence of TBEV maintained within
I. persulcatus
populations in China is around 0.1% (Jiao et al., 2021). Since we
only examined 90
I. persulcatus
ticks, it is unlikely we would identify a positive tick.
Diverse Bunyavirus predominated in our HTS data.
More than 350 viruses is classied in the order Bunyavirales, which was assigned as 9 families and over 13 genera, with various species
only recently discovered and characterized (Kuhn et al. 2021). Our studies had demonstrated molecular evidences of the emergence of over
10 bunyaviruses present in ixodid ticks in China. Based on the phylogenetic analysis of the polymerase coding regions, Beiji
orthonairovirus and South Bay virus form a distinct phylogenetic lineage within currently described orthonairoviruses, which indicate that
the genus orthonairovirus is signicantly more diverse than previously appreciated. Based on a very short sequence fragment, the earlier
phylogenetic analysis of tick associated orthonairoviruses yielded two main phylogenetic clades, with which one clade being isolated
exclusively from ixodid ticks and the other from argasid ticks (Kuhn et al. 2016). The phylogeny has been proved complex and confounded,
as some previously unidentied genetically distinct or emerged nairovirus presents. A more accurate orthonairoviruses classication
scheme is urgent to be updated with oncoming contributions from genome sequencing of genetically unclassied nairoviruses, especially
those isolated from ticks of argasid or Ixodid. In addition to genus
Orthonariovirus
, we also identied several Phenuiviridae viruses from
I.
persulcatus
and
D. slivarum
. Family Phenuiviridae was latest updated representing the newly combined phleboviruses and Tenuivirus
along with newly erected genera
Goukovirus
and
Phasivirus
(Kuhn et al. 2021). Historically, phleboviruses were classied into at least three
phylogenetic clusters, each comprised of several potential species: the Uukuniemi group, the Bhanja group, and the STFS group based on
vector, genomic and serological relationships (Kuhn et al. 2021). Of them, Tacheng uukuvirus in
D. reticulatus
has been proved
phylogenetically close to a human clinical isolate with a history of tick bite in northwestern China (Dong et al., 2021),which raised widely
concerns about the health risk of
Dermacetor
ticks to transmit uukuvirus in China. As one of predominant human-bitten species, the
potential transmission ability of uukuvirus in
D. silvarum
remains to be determined in details. Furthermore, Mukawa virus, another novel
phleboviruses outsides the described three clades above, was rstly detected from
D. silvarum
. Together with the evidences of Mukawa
virus proliferations in Huh-7 cell line and the innate immune responses caused by the virial NSs during mice infection (Matsuno et al.
2018), the immense medical signicance of
D. silvarum
to human victims should be addressed and more detail surveys on the
epidemiologic characters of Mukawa virus should not be ignored. Similarly, although no clinical manifestations were documented to
elucidate the pathogenicity of some phleboviruses to human and animals, the discovery of Sara tick phlebovirus, Scapularis Ixovirus and
Changping tick virus 2 in our studies had deepen our knowledge about diverse Phenuiviridae viruses widely prevalence in China and
adjacent areas. However, the relative abundance of these bunyavirales virus were shown as quite lower among the virial transcripts
obtained. The expansive infectious ticks, for example, up to 3% infection rate of Beiji nairovirus in
I. persulcatus
(Liu et al. 2019), might
cause a series of human victims suffered from bunyavirus infections. Consideration of the unknown pathogenicity of Volzhskoe tick virus,
Ubmeje virus and
Ixodes scapularis
orthobunyavirus discovered in the present paper, the ndings of Bunyavirus-like contigs from
I.
persulcatus
and
D. silvarum
suggests that these viruses may have much medical relevance and broader geographical distributions,
perhaps throughout the range of the two ticks species, comparable to bacterial and protozoan pathogens vectored by them. Enhanced by
Page 10/17
the spreading ticks through the movements of viremic host reservoir, the real geographic ranges should be further extended. Actually, we do
not know whether these bunyaviruses are specic to their vector tick species. As a matter of fact, some tick-borne viruses can usually be
isolated from more than one tick species. CCHFV, for example, has been isolated from over 30 tick species and multiple genera throughout
Europe, Africa, and Asia, it is unlikely that all of the 30 species represent as competent vectors (Bente et al. 2013, Monsalve-Arteagaid et al.
2020). Although the HTS analysis suggests vector specicity for tick species, we cannot rule out the possibility that other tick species may
serve as vectors of these viruses.
One particularly notable characteristic of these bunyaviruses is the lack of recognizable glycoprotein-coding segment. Although we
recovered ~ 90% of the L and S segments for Sara tick phlebovirus, Mukava virus and other bunyavirus by HTS and dispelled the
possibility of viral integration, we were unable to identify any scenario with similarity to Bunyavirales M segments. We have considered
various explanations for this confounding result. Firstly, these viruses encode more complicated secondary structure of the M segments,
which may inhibit ecient cDNA synthesis, subsequentially results in the failure to detect by amplications (Houldcroft et al. 2017).
Alternatively, these viruses may employ special pathway in the process of cellular attachment and entry other than the well-known
glycoprotein precursor (GPC) depending manner (Welch et al. 2020). Finally, we also cannot exclude the possibility that an episome-like
form coded by S and L segments of these viruses. As such, they may not assembly an infectious virion but instead use other vehicles, for
instance, extracellular vesicles, for their disseminations to new hosts (Zhou et al. 2018).
High diversity Mononegavirales contigs predominated in our HTS data.
In our analysis, a novel Chuvirus, Taiga tick Nigecruvirus, were also unrevealed in
I. persulcatus
other than Nuomin virus. Phylogenic
analysis based on L, G, NP and VP4 fragments suggested that Taiga tick Nigecruvirus and blacklegged tick Chuvirus 2 formed a separate
monophyletic clade outside genus
Mivirus
under the Chuviridae branch of Jingchuvirales in the order Mononegavirales, although a
complete genetic characterization will be required to fully determine its taxonomy. The taiga tick Nigecruvirus were shown with high
identity in RdRp and relative lower identities in N and G genes with blacklegged tick Chuvirus 2, which no pathogenicity has been
documented involving human and other animals. However, the reported human pathogenicity of Nuomin virus in genus Mivirus from Tahe,
Heilongjiang province, suggested the potential threats of the taiga tick Nigecruvirus on human and animals via tick bites or other
transmission routes. Based on the phylogeny relationship, we considered the virus close related to blacklegged tick Chuvirus 2 as a novel
chuvirus named Taiga tick Nigecruvirus tentative, while Nuomin virus as a member of Mivirus, called Mivirus nuomin.
Rhabdoviridae are another diverse set of single-stranded negative sense RNA viruses with frequent host-switching features during their
evolution history (Longdon et al. 2015) and widely found to naturally infect both animals and plants. In the present study, almost all the
pools of the three tick species yield the positive contains of Rhabdovirus-like contigs, which demonstrates relatively high divergence of
RdRp sequences of the Rhabdoviruses. Among these rhabdoviruses, only two nucleorhabdoviruses Chimay rhabdoviruses and blacklegged
tick rhabdovirus-1 were harvested from
I. persulcatus
and
Hae. concinna
respectively. While, in the Alphanemrha virus-like group, extensive
diversity were observed with 3 species of viruses in
Hae. concinna
, 2 species in
D. silvarum
and one species in
I. persulcatus
. Together with
two nucleorhabdoviruses detected, diverse Rhabdoviruses in the three tick species might be associated with high frequencies of host
switches within their comparable broad host spectrums. It is worthy of noticed that more diversity of rhabdovirus is still unsampled due to
the limited tick samples or geographic sites.
Although our surveys has demonstrated wide array of high diverse viruses harbored in the three tick species sampled, however, the
limitations of the study were also prominent due to our sample bias. One constraint of our study was caused by the limited geographical
regions and aggregated distributions of these ticks, as our HTS analysis merely focused on specimens collected from a single location.
And also, we could not determine the pathogenicity and transmission capability of these viruses carried by these ticks. Necessary
investigations should be carried out in details to character their epidemiological features and determine their potential threat on public
health. Meanwhile, we still anticipate that many of the viruses uncovered by our HTS analysis along the distribution ranges of their
presumed natural cycle between vector ticks and host animals. we also speculate that analysis of ticks from diverse geographical areas
would reveal greater viral diversity with more emerged agents to be discovered in the future. And thus, accurate viral taxonomy would allow
more precise elucidation of tick associated virus evolutionary relationships (Shi et al. 2016).
Conclusions
In a sum, we metagenomically analyzed the virome of
I. persulcatus
,
D. silvarum
and
Hae. concinna
, species frequently associated with
pathogens potential transmitted in China. In addition to the exploration of viral diversity, our work also identied several tick borne viruses
with potential relevance for diseases of human and livestock, for example, Tacheng uukuvirus, Sara phlebovirus, Beiji nairovirus, Nuomin
virus and Jingmen tick virus and so on. The novel virus founded in the present study, Dermacentor pestivirus-like virus, taiga tick
Page 11/17
Nigecruvirus, Mukawa virus, South bay virus, and Scapularis Ixovirus do not allow reliable conclusions on the transmissibility issues
merely depending on available genetic similarities to human pathogens. Whatever, our discoveries can pave a corner stone for further
investigations and rational control strategy for tick borne viral diseases in China.
Declarations
Ethics approval and consent to participate
No human participants involving the present study, protocols for eld tick collections and samples processing were reviewed and
approved by the Institutional Ethics Review Board of Beijing Institute of Microbiology and Epidemiology (BIME-2020-019).
Consent for publication
All authors contributed to the manuscript and approved the submitted version.
Availability of data and materials
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession
number(s) can be found below: https://www.ncbi.nlm. nih.gov/PRJNA639641.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed
as a potential conict of interest.
Funding
This study was supported by State Key Program of Infectious Diseases of China (2017ZX10303404),.
Authors' contributions
Designed the study: Yi Sun, performed eld and lab-work: Tong Qin and Mingjie Shi, Performed bioinformatics work: Y. Sun, T. Qin. Wrote
the manuscript Y. Sun, T. Qin. Meina, Zhang and Zhitong Liu. All authors read and approved the manuscript.
Acknowledgements
We thank all the staff in the tick-borne disease department in Mudanjiang Forestry Central Hospital and Balin Clinics for their help with
sample collections.
References
1. Bolger AM, Lohse M & Usadel B. 2014. Trimmomatic: a exible trimmer for Illumina sequence data.
Bioinformatic
30, 2114-2120.
DOI:10.1093/bioinformatics/btu170
2. Burland TG. 2000. DNASTAR's Lasergene sequence analysis software. Methods Mol. Biol. 132, 71-91. DOI:10.1385/1-59259-192-2:71
3. Guglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Peña A and Horak IG. 2014. The hard ticks of the world (Acari:
Ixodida: Ixodidae). Springer, Heidelberg, xiii + 738 pp.
4. Peñalver E, Arillo A, Delclòs X, Peris D, Grimaldi D A, Anderson S R, Nascimbene P C And De La Fuente RP. 2017. Ticks parasitised
feathered dinosaurs as revealed by Cretaceous amber assemblages.
Nature Communications
, 8: 1924.
5. Guglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Peña A, Horak IG, Shao R, & Barker SC. (2010). The Argasidae,
Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species names.
Zootaxa
, 2528, 1-28. DOI:
10.11646/ZOOTAXA.2528.1.1
. Jongejan F And Uilenberg G. 2004. The global importance of ticks.
Parasitology
. 2004;129:S3–14.
7. Madison‐Antenucci, S., Kramer, L.D., Gebhardt, L.L., & Kauffman, E.B. 2020. Emerging tick-borne diseases.
Clinical Microbiology
Reviews
, 33 (2) e00083-18. DOI:10.1128/CMR.00083-18
. Fang LQ, Liu K., Li X-L, Liang S, Yang Y, Yao H-W, Sun R-X, Sun Y, Chen W-J, Zuo S-Q, Ma M-J, Li H, Jiang J-F, Yang X-F, Gray G C, Krause
P J And Cao W-C. 2015. Emerging tick-borne infections in mainland China: an increasing public health threat.
Lancet Infect Dis
.
Page 12/17
15(12): 1467–1479.
9. Ortiz DI, Piche-Ovares M, Romero-Vega LM, Wagman JM, & Troyo A. 2021. The impact of deforestation, urbanization, and changing
land use patterns on the ecology of mosquito and tick-borne diseases in central America.
Journal of Medical Entomology
, 58(4),
1546–1564, 13. DOI:10.1093/jme/tjaa209
10. Yu X, Liang M, Zhang S, Liu Y, Li J, Sun Y, Zhang L, Zhang Q, Popov VL, Li C, Qu J, Li Q, Zhang Y, Hai R, Wu W, Wang Q, Zhan F, Wang X,
Kan B, Wang S, Wan K, Jing H, Lu J, Yin W, Zhou H, Guan X, Liu J, Bi Z, Liu G, Ren J, Wang H, Zhao Z, Song J, He J, Wan T, Zhang J, Fu
X, Sun L, Dong X, Feng Z, Yang W, Hong T, Zhang Y, Walker DH, Wang Y, & Li D. (2011). Fever with thrombocytopenia associated with a
novel bunyavirus in China. T
he New England Journal of Medicine
, 364 16, 1523-32 . DOI:10.1056/NEJMoa1010095
11. Liu Q, He B, Huang S, Wei F, & Zhu X. (2014). Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis.
The
Lancet. Infectious diseases
, 14 8, 763-772 . DOI:10.1016/S1473-3099(14)70718-2
12. Wang, Z., Wang, B., Wei, F., Han, S., Zhang, L., Yang, Z., Yan, Y., Lv, X., Li, L., Wang, S., Song, M., Zhang, H., Huang, S., Chen, J., Huang, F.,
Li, S., Liu, H., Hong, J., Jin, Y., Wang, W., Zhou, J., & Liu, Q. 2019. A new segmented virus associated with human febrile illness in China.
The New England Journal of Medicine
, 380 22, 2116-2125. DOI: 10.1056/NEJMoa1805068
13. Ejiri, H., Lim, C., Isawa, H., Fujita, R., Murota, K., Sato, T., Kobayashi, D., Kan, M., Hattori, M., Kimura, T., Yamaguchi, Y., Takayama-Ito, M.,
Horiya, M., Posadas-Herrera, G., Minami, S., Kuwata, R., Shimoda, H., Maeda, K., Katayama, Y., Mizutani, T., Saijo, M., Kaku, K.,
Shinomiya, H., & Sawabe, K. 201). Characterization of a novel thogotovirus isolated from
Amblyomma testudinarium
ticks in Ehime,
Japan: A signicant phylogenetic relationship to Bourbon virus.
Virus Research
, 249, 57-65. DOI:10.1016/j.virusres.2018.03.004
14. Jia N, Liu H, Ni X, Bell-Sakyi L, Zheng Y, Song J, Li J, Jiang B, Wang Q, Sun Y, Wei R, Yuan T, Xia L, Chu Y, Wei W, Li L, Ye J, Lv Q, Cui X,
Guan Y, Tong Y, Jiang J, Lam TT, & Cao W. (2019). Emergence of human infection with Jingmen tick virus in China: A retrospective
study.
EBioMedicine,
43, 317 - 324. DOI:10.1016/j.ebiom.2019.04.004
15. Ma J, Lv X, Zhang X, Han S, Wang Z, Li L, Sun H, Ma L, Cheng Z, Shao J, Chen C, Zhao Y, Sui L, Liu L, Qian J, Wang W, & Liu Q. (2021).
Identication of a new orthonairovirus associated with human febrile illness in China.
Nature Medicine
. DOI:10.1038/s41591-020-
01228-y.
1. Monsalve-Arteagaid L, Alonso-Sard´on M., Bellido JLM, Santiago MBV, Lista MCV, Ab´an JL, Muro A, Belhassen-García M, 2020.
Seroprevalence of Crimean-Congo hemorrhagic fever in humans in the world health organization European region: a systematic
review.
PLoS Negl. Trop. Dis.
14, e0008094 DOI:10.1371/journal.pntd.0008094.
17. Kodama F, Yamaguchi H, Park E, Tatemoto K, Sashika M, Nakao R, Terauchi Y, Mizuma K, Orba Y, Kariwa H, Hagiwara K, Okazaki K,
Goto A, Komagome R, Miyoshi M, Ito T, Yamano K, Yoshii K, Funaki C, Ishizuka M, Shigeno A, Itakura Y, Bell-Sakyi L, Edagawa S,
Nagasaka A, Sakoda Y, Sawa H, Maeda K, Saijo M & Matsuno K. A novel nairovirus associated with acute febrile illness in Hokkaido,
Japan.
Nature Communications
, 2021, 12:5539.
1. Kobayashi D, Kuwata R, Kimura T, Faizah AN, Higa Y, Hayashi T, Sawabe K & Isawa H. Toyo virus, a novel member of the Kaisodi group
in the genus Uukuvirus (family Phenuiviridae) found in
Haemaphysalis formosensis
ticks in Japan.
Archives of Virology
. 2021, 166,
2751–2762.
19. Grard G, Moureau G, Charrel RN, Lemasson JJ, Gonzalez JP, Gallian P, Gritsun TS, Holmes EC, Gould EA, de Lamballerie X. 2007.
Genetic characterization of tick-borne aviviruses: new insights into evolution, pathogenetic determinants and taxonomy.
Virology
361,
80–92. DOI:10.1016/j.virol.2006.09.015
20. Gaudreault NN, Madden DW, Wilson WC, Trujillo JD and Richt JA. 2020. African swine fever virus: an emerging DNA arbovirus.
Frontiers in Veterinary Science, 7, 215. DOI: 10.3389/ fvets.2020.00215
21. Houldcroft, C.J., Beale, M.A., & Breuer, J. (2017). Clinical and biological insights from viral genome sequencing.
Nature Reviews.
Microbiology
, 15, 183 - 192. DOI:10.1038/nrmicro.2016.182
22. McMullan L, Folk SM, Kelly AJ, MacNeil A, Goldsmith CS, Metcalfe MG, Batten BC, Albariño, CG, Zaki SR, Rollin P. 2012. A New
Phlebovirus Associated with Severe Febrile Illness in Missouri.
N. Engl. J. Med.
367, 834–841.
23. Yu XJ, Tesh RB (2014) The role of mites in the transmission and maintenance of Hantaan virus (Hantavirus: Bunyaviridae).
Journal of
Infectious Diseases
, 210, 1693–1699. DOI: 10.1093/ infdis/jiu336
24. Marklewitz M, Zirkel F, Kurth A, Drosten C, Junglen S (2015) Evolutionary and phenotypic analysis of live virus isolates suggests
arthropod origin of a pathogenic RNA virus family.
Proceedings of the National Academy of Sciences
, USA, 112, 7536–7541.
DOI:10.1073/pnas.1502036112
25. Teng KF and Jiang ZJ. 1991. Economic insect fauna of China. Fasc 39. Acarina: Ixodidae. Beijing, Science Press, Academia Sinica. 1-
359.
Page 13/17
2. Pettersson, J.H., Shi, M., Bohlin, J., Eldholm, V., Brynildsrud, O.B., Paulsen, K.M., Andreassen, Å.K., & Holmes, E.C. (2017). Characterizing
the virome of Ixodes ricinus ticks from northern Europe.
Scientic Reports
, 7. DOI:10.1038/s41598-017-11439-y.
27. Loens, K.; Bergs, K.; Ursi, D.; Goossens, H.; Ieven, M. Evaluation of NucliSens easyMAG for Automated Nucleic Acid Extraction from
Various Clinical Specimens.
J. Clin. Microbiol.
2007, 45, 421–425.
2. Krueger F, James FO, Ewels PA, Afyounian E. and Schuster-Boeckler B. 2021. FelixKrueger /TrimGalore: v0.6.7.
DOI:10.5281/ZENODO.5127899.
29. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E,
Hacohen N, Gnirke A, Rhind N, Di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, & Regev A. 2011. Full-length
transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644-652.
30. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M.G., Blood, P.D., Bowden, J.C., Couger, M.B., Eccles, D.A., Li, B., Lieber, M.,
MacManes, M.D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N.T., Westerman, R., William, T., Dewey, C.N., Henschel, R., LeDuc, R.D.,
Friedman, N., & Regev, A. 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference
generation and analysis.
Nature Protocols
, 8, 1494-1512. DOI:10.1038/nprot.2013.084
31. Langmead B. and Salzberg S L 2012. Fast gapped-read alignment with Bowtie 2.
Nat. Methods
9, 357–359. DOI:10.1038/nmeth.1923.
32. Thorvaldsdóttir H, Robinson JT, & Mesirov JP. 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization
and exploration.
Briengs in Bioinformatics
, 14, 178 - 192. DOI:10.1093/bib/bbs017
33. Xia LC, Cram JA, Chen T, Fuhrman JA, & Sun F. 2011. Accurate Genome Relative Abundance Estimation Based on Shotgun
Metagenomic Reads.
PLoS ONE
, 6(12), e2799 DOI: 10.1371/journal.pone.0027992
34. Katoh, K. & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and
usability. Mol. Biol. Evol. 30, 772–780. DOI:10.1093/molbev/mst010
35. Capella-Gutierrez, S., Silla-Martinez, J. M. & Gabaldon, T. (2009). trimAl: a tool for automated alignment trimming in large-scale
phylogenetic analyses.
Bioinformatics
25, 1972-1973. DOI:10.1093/bioinformatics/btp348.
3. Darriba D, Taboada GL, Doallo R & Posada D. (2011). ProtTest 3: Fast selection of best-t models of protein evolution.
Bioinformatics
27, 1164-1165. DOI:10.1093/bioinformatics/ btr088.
37. Guindon, S., Dufayard, J., Lefort, V., Anisimova, M.O., Hordijk, W., & Gascuel, O. (2010). New algorithms and methods to estimate
maximum-likelihood phylogenies: assessing the performance of PhyML 3.0.
Systematic Biology
, 59(3), 307-21.
DOI:10.1093/sysbio/syq010
3. Sameroff S, Tokarz R, Charles RA, Jain K, Oleynik A, Che X, Georges K, Carrington CV, Lipkin WI, Oura C. Viral diversity of tick species
parasitizing cattle and dogs in Trinidad and Tobago.
Sci. Rep.
2019, 9, 10421. DOI:10.1038/s41598-019-46914-1
39. Shi M, Lin X, Vasilakis N, Tian J, Li, C, Chen L, Eastwood G, Diao X, Chen M, Chen X, Qin X, Widen SG, Wood TG, Tesh RB, Xu J, Holmes
EC, & Zhang Y. (2016). Divergent Viruses Discovered in Arthropods and Vertebrates Revise the Evolutionary History of the Flaviviridae
and Related Viruses.
Journal of Virology
, 90, 659 - 669. DOI: 10.1128/JVI.02036-15
40. Liu Q, Wang Z-D, Gao Y, Lv X, Han S, Zhang X, Shao J-W, Chen C, Li L, Hou Z-J, Sui L, Zhao Y, Wang B, Wang W, Song M. 2020.
Identication of a new chuvirus associated with febrile illness in China. https://doi.org/10.21203/rs.3.rs-104938/v1.
41. Li C, Shi M, Tian J, Lin X, Kang Y, Chen L, Qin X, Xu J, Holmes EC, & Zhang Y. (2015). Unprecedented genomic diversity of RNA viruses
in arthropods reveals the ancestry of negative-sense RNA viruses.
eLife, 4
. DOI:10.7554/eLife.05378
42. Shi J, Shen S, Wu H, Zhang Y, Deng F. (2021) Metagenomic proling of viruses associated with
Rhipicephalus microplus
ticks in
Yunnan province, China.
Virol. Sin.
36, 623–635. DOI:10.1007/s12250-020-00319-x.
43. Afonso CL, Amarasinghe GK, Bányai K, Bào Y, Basler CF, Bavari S, Bejerman NE, Blasdell KR, Briand F, Briese T, Bukreyev A, Calisher CH,
Chandran K, Cheng J, Clawson AN, Collins PL, Dietzgen RG, Dolnik O, Domier LL, Dürrwald R, Dye JM, Easton AJ, Ebihara H, Farkas SL,
Freitas-Astúa J, Formenty PB, Fouchier RA, Fu Y, Ghedin E, Goodin M, Hewson R, Horie M, Hyndman TH, Jiāng D, Kitajima EW, Kobinger
GP, Kondō H, Kurath G, Lamb RA, Lenardon S, Leroy EM, Li C, Lin X, Liu L, Longdon B, Marton S, Maisner A, Mühlberger E, Netesov SV,
Nowotny N, Patterson JL, Payne SL, Pawęska JT, Randall RE, Rima BK, Rota PA, Rubbenstroth D, Schwemmle M, Shi M, Smither S,
Stenglein MD, Stone DM, Takada A, Terregino C, Tesh RB, Tian J, Tomonaga K, Tordo N, Towner JS, Vasilakis N, Verbeek M, Volchkov
VE, Wahl-Jensen V, Walsh JA, Walker PJ, Wang D, Wang L, Wetzel T, Whiteld AE, Xiè JT, Yuen KY, Zhang Y, & Kuhn JH. (2016).
Taxonomy of the order Mononegavirales: update 2016.
Archives of Virology
, 161, 2351-2360. DOI:10.1007/s00705-016-2880-1
44. Wang Y, Wei Z, Lv X, Han S, Wang Z, Fan C, Zhang X, Shao J, Zhao Y, Sui L, Chen C, Liao M, Wang B, Jin N, Li C, Ma J, Hou Z, Yang Z,
Han Z, Zhang Y, Niu J, Wang W, Wang Y, & LIU Q. (2021). A new nairo-like virus associated with human febrile illness in China.
Emerging Microbes & Infections
, 10, 1200 - 1208. DOI: 10.1080/22221751.2021.1936197.
Page 14/17
45. Meng F, Ding M, Tan Z, Zhao Z, Xu L, Wu J, He B, & Tu C. (2019). Virome analysis of tick-borne viruses in Heilongjiang province, China.
Ticks and Tick-Borne Diseases
, 10 2, 412-420. DOI:10.1016/j.ttbdis.2018.12.002
4. Jiao J, Lu Z, Yu Y, Ou Y, Fu M, Zhao Y, Wu N, Zhao M, Liu Y, Sun Y, Wen B, Zhou D, Yuan Q, & Xiong X. 2021. Identication of tick-borne
pathogens by metagenomic next-generation sequencing in
Dermacentor nuttalli
and
Ixodes persulcatus
in Inner Mongolia, China.
Parasites & Vectors
, 14,287. DOI: 10.1186/s13071-021-04740-3
47. Kuhn JH, Adkins S, Agwanda BR, Al Kubrusli R, Alkhovsky Aльxoвcкий Cepгeй Bлaдимиpoвич SV, Amarasinghe GK, Avšič-Županc T,
Ayllón MA, Bahl J, Balkema-Buschmann, A, Ballinger, MJ, Basler, CF, Bavari, S, Beer, M, Bejerman, NE, Bennett, AJ, Bente DA, Bergeron É,
Bird BH, Blair CD, Blasdell KR Blystad, D Bojko J, Borth WB, Bradfut SB, Breyta R, Briese T, Brown PA, Brown J, Buchholz UJ, Buchmeier
MJ, Bukreyev A, Burt FJ, Büttner C, Calisher CH, Cao M, Casas I, Chandran K, Charrel RN, Cheng Q, Chiaki Y, Chiapello M, Choi I, Ciuffo
M, Clegg JC, Crozier I, Dal Bó E, de la Torre JC, de Lamballerie X, de Swart RL, Debat HJ, Dheilly NM, Di Cicco E, Di Paola N, Di Serio F,
Dietzgen RG, Digiaro M, Dolnik O, Drebot MA, Drexler JF, Dundon WG, Duprex WP, Dürrwald R, Dye JM, Easton AJ, Ebihara H, Elbeaino T,
Ergünay K, Ferguson HW, Fooks AR, Forgia M, Formenty PB, Fránová J, Freitas-Astúa J, Fu J, Fürl S, Gago-Zachert S, Gāo GF, García
ML García-Sastre A, Garrison AR, Gaskin T, Gonzalez J, Griths A, Goldberg TL, Groschup MH, Günther S, Hall RA, Hammond J, Han T,
Hepojoki JM, Hewson R, Hong J, Hong N, Hongo S, Horie M, H JS H T, Hughes HR, Hüttner Hyndman TH, Ilyas M, Jalkanen R, Jiāng D,
Jonson GB, Junglen S, Kadono F, Kaukinen KH, Kawate MK, Klempa B, Klingström J, Kobinger G, Koloniuk I, Kondō H, Koonin EV,
Krupovic M, Kubota K, Kurath G, Laenen L, Lambert AJ, Langevin SL, Lee B, Lefkowitz EJ, Leroy EM, Li S, Li L, Li J, Liu H, Lukashevich
IS, Maes P, de Souza WM, Marklewitz M, Marshall SH, Marzano SL, Massart S, McCauley JW Melzer M, Mielke-Ehret N, Miller KM, Ming
TJ, Mirazimi A, Mordecai GJ, Mühlbach HP, Mühlberger E, Naidu RA, Natsuaki T, Navarro JA, Netesov Heтёcoв Cepгeй Bиктopoвич SV,
Neumann G, Nowotny N, Nunes MR, Olmedo-Velarde A, Palacios GF, Pallás V, Pályi B, Papa Άννα Παπά A, Paraskevopoulou Σοφία
Παρασκευοπούλου S, Park AC, Parrish CR, Patterson DA, Pauvolid-Corrê, A, Pawęska JT, Payne SL, Peracchio C, Pérez DR, Postler TS,
Qi L, Radoshitzky SR, Resende RO, Reyes CA, Rima BK, Luna GR, Romanowski V, Rota PA, Rubbenstroth D, Rubino L, Runstadler JA,
Sabanadzovic S, Sall AA, Salvato MS, Sang R, Sasaya T, Schulze AD, Schwemmle M, Shi M, Shí X, Shí Z, Shimomoto Y, Shirako Y,
Siddell SG, Simmonds P, Sironi M, Smagghe G, Smither S, Song J, Spann KM, Spengler JR, Stenglein MD, Stone DM, Sugano J, Suttle
C, Tabata A, Takada A, Takeuchi S, Tchouassi DP, Teffer AK, Tesh RB, Thornburg NJ, Tomitaka Y, Tomonaga K, Tordo N, Torto B,
Towner JS, Tsuda S, Tu C, Turina M, Tzanetakis], IE, Uchida J, Usugi T, Vaira AM, Vallino M, van den Hoogen BG, Varsani A, Vasilakis
Νίκος Βασιλάκης N, Verbeek M, von Bargen S, Wada J, Wahl V, Walker PJ, Wang L, Wang G, Wang Y, Wang ]Y, Waqas MS, Wèi T, Wen
S, Whiteld AE, Williams JV, Wolf YI, Wu J, Xu L, Yanagisawa H, Yang C, Yang Z, Zerbini FM, Zhai L, Zhang Y, Zhang S, Zhang J, Zhang
Z, & Zhou X (2021). 2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders
Bunyavirales and Mononegavirales.
Archives of Virology
. (2021) 166:3513–3566. DOI:10.1007/s00705-021-05143-6
4. Elliott, R.M., & Brennan, B. (2014). Emerging phleboviruses.
Current Opinion in Virology
, 5, 50 - 57. DOI:10.1016/j.coviro.2014.01.011
49. Pettersson, J.H., Ellström, P., Ling, J., Nilsson, I., Bergström, S., GONZÁLEZ-ACUÑA, D.A., Olsen, B., & Holmes, E.C. (2020). Circumpolar
diversication of the
Ixodes uriae
tick virome.
PLoS Pathogens
, 16. DOI:10.1371/journal.ppat.1008759
50. Ali M, Hameed S, & Tahir M. (2014). Luteovirus: insights into pathogenicity.
Archives of Virology
, 159, 2853-2860.
DOI:10.1007/s00705-014-2172-6
51. Nibert, M.L., Ghabrial, S.A., Maiss, E., Lesker, T.R., Vainio, E.J., Jiāng, D., & Suzuki, N. (2014). Taxonomic reorganization of family
Partitiviridae and other recent progress in partitivirus research.
Virus Research
, 188, 128-41 . DOI:10.1016/j.virusres.2014.04.007
52. Tokarz R, Williams SH, Sameroff S, Sanchez Leon M, Jain K, & Lipkin WI. (2014). Virome Analysis of
Amblyomma americanum
,
Dermacentor variabilis
, and
Ixodes scapularis
Ticks Reveals Novel Highly Divergent Vertebrate and Invertebrate Viruses.
Journal of
Virology
, 88, 11480 - 11492. DOI: 10.1128/JVI.01858-14.
53. Li LJ, Ning NZ, Zheng YC, Chu YL, Cui XM, Zhang MZ, Guo WB, Wei R, Liu HB, Sun Y, Ye JL, Jiang BG, Yuan TT, Li J, Bian C, Bell-Sakyi
L, Wang H, Jiang JF, Song JL, Cao WC, Tsan-Yuk Lam T, Ni XB and Jia N (2022) Virome and blood meal-associated host responses in
Ixodes persulcatus
naturally fed on patients.
Front. Microbiol
. 12: 728996. doi: 10.3389/fmicb.2021.728996.
54. Liu, Z., Li, L., Xu, W., Yuan, Y., Liang, X., Zhang, L., Wei, Z., Sui, L., Zhao, Y., Cui, Y., Yin, Q., Li, D., Li, Q., Wei, F., Hou, Z., LIU, Q., & Wang, Z.
(2022). Extensive diversity of RNA viruses in ticks revealed by metagenomics in northeastern China.
bioRxiv
.
DOI:10.1101/2022.04.27.489762.
55. Wang Y-N, Jiang R-R, Ding H, Zhang X-L, Wang N, Zhang Y-F, Li Y, Chen J-J, Zhang P-H, Li H, Jiang J-F, Liu L-Z, Yu M-b, Wang G, Zhang
X-A and Liu W (2022) First Detection of Mukawa Virus in
Ixodes persulcatus
and
Haemaphysalis concinna
in China.
Front. Microbiol.
13:791563. doi: 10.3389/fmicb.2022.791563
5. Jia N; Wang JF; Shi WQ; Du LF; Sun Y; Zhan W.; Jiang JF; Wang Q; Zhang B; Ji PF; Zhao FQ; Cao WC. 2022. Large-scale comparative
analyses of tick genomes elucidate their genetic diversity and vector capacities.
Cell
,
2020,
182: 1-13.
Page 15/17
57. Paulsen, K.M., Pedersen, B.N., Soleng, A., Okbaldet, Y.B., Pettersson, J.H., Dudman, S.G., Ottesen, P., Vik, I.S., Vainio, K., & Andreassen,
Å.K. (2015). Prevalence of tick‐borne encephalitis virus in
Ixodes ricinus
ticks from three islands in north‐western Norway.
APMIS
, 123,
759 - 764. DOI:10.1111/apm.12412
5. Kuhn JH, Wiley MR, Rodriguez SE, Bào Y, Prieto K, Travassos da Rosa AP, Guzmán H, Savji N, Ladner JT, Tesh RB, Wada J, Jahrling PB,
Bente DA, & Palacios GF 2016. Genomic characterization of the genus Nairovirus (Family Bunyaviridae).
Viruses
, 8. 164;
DOI:10.3390/v8060164
59. Matsuno K, Kajihara M, Nakao R, Nao N, Mori-Kajihara A, Muramatsu M, et al. The unique phylogenetic position of a novel tick-borne
Phlebovirus ensures an ixodid origin of the genus Phlebovirus.
mSphere
. 2018 Jun 27;3(3) e00239-18. DOI:10.1128/mSphere.00239-
18
0. Bente, D.A., Forrester, N.L., Watts, D.M., McAuley, A.J., Whitehouse, C.A., & Bray, M. (2013). Crimean-Congo hemorrhagic fever: history,
epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral research, 100 1, 159-89. DOI:
10.1016/j.antiviral.2013.07.006
1. Welch SR, Scholte FE, Spengler JR, Ritter JM, Coleman-McCray, JD, Harmon JR, Nichol ST, Zaki SR, Spiropoulou CF and Bergeron É.
2020. The Crimean-Congo hemorrhagic fever virus nsm protein is dispensable for growth in vitro and disease in Ifnar-/- mice.
Microorganisms
, 8 , 775. DOI:10.3390/microorganisms8050775.
2. Zhou, W., Woodson, M., Neupane, B., Bai, F., Sherman, M.B., Choi, K.H., Neelakanta, G., Sultana, H., 2018. Exosomes serve as novel
modes of tick-borne avivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to
the vertebrate neuronal cells. PLoS Pathog. 14, e1006764.
3. Xu L, Guo MJ, Hu B, Zhou H, Yang W, Hui LX, Huang R, Zhan JB, Shi WF and Wu Y. 2021 Tick virome diversity in Hubei Province, China,
and the inuence of host ecology.
Virus Evolution
, 7(2), 1–12. DOI:10.1093/ve/veab089
4. Dong Z, Yang M, Wang Z, Zhao S, Xie S, Yang Y, Liu G, Zhao S, Xie J, Liu Q, & Wang Y. (2021). Human Tacheng tick virus 2 infection,
China, 2019.
Emerging Infectious Diseases
. DOI:10.3201/eid3201/191486.
5. Shi M, Lin X, Tian J, Chen L, Chen X, Li C, Qin X, Li J, Cao J, Eden J, Buchmann JP, Wang W, Xu J, Holmes EC, & Zhang Y. (2016).
Redening the invertebrate RNA virosphere.
Nature, 540
, 539-543. DOI:10.1038/nature20167
. Sameroff S, Tokarz R, Vucelja M, Jain K, Oleynik A, Boljfeti´c M, Bjedov L, Yates RA, Margaleti´c J, Oura CAL, Lipkin WI, Krajinovi´c LC,
and Markoti´c A. Virome of
Ixodes ricinus
,
Dermacentor reticulatus
, and
Haemaphysalis concinna
ticks from Croatia.
Viruses
2022, 14,
929. DOI:10.3390/v14050929
7. Longdon, B., Murray, G.G., Palmer, W.J., Day, J.P., Parker, D.J., Welch, J.J., Obbard, D.J., & Jiggins, F.M. (2015). The evolution, diversity,
and host associations of rhabdoviruses.
Virus Evolution
, 1. DOI:10.1093/ve/vev014.
Figures
Page 16/17
Figure 1
Phylogenetic analysis of representative branches in the family Flaviviridae based on the RdRp sequences (A). Enhanced region showing
the relationship of the unclassied pestivirus-like group in relation to pestivirus and Flavivirus (B). Enhanced region showing the
relationship of Jingmen tick virus in relation to family Flaviviridae (C)
Maximum likelihood tree inferred using the best-t model of amino acid substitution (LG + I + Γ + F for all alignments) with 1000 bootstrap
replicates.
Figure 2
Phylogenetic analysis of representative branches in order Mononegavirales. (A) Alignment of all branches belonging to order
Mononegavirales. (B) Enhanced region showing the relationship of Chuviridae group in relation to Rhabdoviridae. (C) Enhanced region
showing the relationships of Rhabdoviruses groups
Maximum likelihood tree inferred using the best-t model of amino acid substitution (LG + I + Γ + F for all alignments) with 1000 bootstrap
replicates
Page 17/17
Figure 3
Phylogenetic analysis of representative branches in order Bunyavirales
(A) Alignment of all branches belonging to order Bunyavirales; (B) Enhanced region showing Phenutenviridae members. (C) Enhanced
region showing Orthonarioviridae (D) Enhanced region showing Peribunyaviridae
Maximum likelihood tree inferred using the best-t model of amino acid substitution (LG + I + Γ + F for all alignments) with 1000 bootstrap
replicates.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
Qintongsupplementaltable.docx
LegendsforSupplementalFigures.docx
supplementalFigure1A.tif
supplementalFigure1B.tif
supplementalFigure1C.tif
supplementalFigure1D.tif
supplementalFigure2.tif
supplementalFigure3A.tif
supplementalFigure3B.tif
