Results
Sample collection from
rodents
In this study, between May 2017 and June 2021, we collected 588 throat
and anal swabs of 326 rodents belonging to the order Rodentiafrom Haikou City (CDC, Fucheng, Yanfeng, and Longtang), Baisha County
(Yinggeling, Huangjingjiaoling, and Bangxi) Dongfang City, Lingao
County, Tunchang County, Lingshui County, Ledong County, Chengmai
County, and Sanya City in the Hainan Province, which is the only
tropical island province in China (Figure 1 and Table S1). Rodent
species were morphologically identified and confirmed by mt-Cyt bgene sequences, including Rattus norvegicus ( 23.53%),Rattus tanezumi (38.24%), Rattus andamanensis(6.18%), Rattus losea (6.65%), Niviventer
fulvescens (19.41%), and Leopoldamys edwardsi (5.00%). The
species from Rattus under the Muridae are clustered on the
evolutionary tree and show a close distance to the Cricetulus of
the Circetidae and a far distance to the dipus andrhizomys . The 588 samples were combined into 28 pools according
to rodent species, swab types, and sample locations.
NGS sequencing
According to species, a total of
136G GB of nucleotide data (7,553,938 valid reads,150 bp long) were
obtained from 28 pools. Archaea,
bacteria, microbial eukaryotes, and invalid reads with no significant
similarity to any amino acid sequences in the viral NR protein database
were excluded. A comparison of the
total number of contigs and the proportion of viral reads from 6 rodent
species and 15 sampling locations is shown in Figure 2a-d. The viral
composition of each pooled sample is shown in Figure 2e.
The readings were filtered into
57,980 contigs, and the virus-associated contigs that best matched the
viral ORF proteins available in each pool were screened.
Metagenomic analysis and virome
overview
An overview of collection region,
rodent species, and virus-related contigs is shown in Table S3.
Mammalian virus-associated contigs were assigned to 15 families (Table
S4) and were composed of double-stranded (ds) DNA, dsRNA,
single-stranded (ss) DNA, ssRNA viruses and retro-transcribing. Viral
reads were assigned toFlaviviridae ,Coronaviridae , Arenaviridae , Astroviridae ,Adenoviridae , Parvoviridae , Papillomaviridae ,Herpesviridae ,Picornaviridae , Hepeviridae, and some arthropod viruses,
including Phenoviridae and Panviridae , as well as a large
group of unclassified RNA viruses. The heatmap shown in Figure 3
indicates that the viromes hosted by rodents might be related to their
geographic distribution. Flaviviruses and picornaviruses are widely
distributed in Chengmai, Lingao, and Baisha, with high abundance.
Coronaviruses, herpesviruses, and astroviruses are widely distributed
throughout the world. Pestivirus and Bocavirus are present only in the
mountainous forest area of Baisha.
The origins, host, structure
information and accession numbers of the viruses identified in this
study are shown in Table S5.
Characteristics of negative-stranded RNA
viruses
Arenaviridae
Arenaviruses (AreVs; order Bunyavirales , familyArenaviridae ) are a group of enveloped RNA viruses whose genome
contains at least two single negative-sense RNA segments totalling
approximately 10.5kb. AreVs are currently classified into four genera:Antennavirus, Hartmanivirus, Mammarenavirus, and Retarenavirus.Mammarenavirus cause central nervous system disease and
haemorrhagic fever in humans in Africa and Latin America [23-24].
Human pathogenic arenaviruses include the Tacaribe Complex (New-World)
and LCMV-LASV Complex (Old-World), which include the LASV and LCMV.
To identify partial genomes, representative reads of AreVs were obtained
from Rattus tanezumi collected from Lingshui (Yelin).
Primers
were designed for PCR amplification and using Sanger sequencing to cover
the gaps. We obtained a WENV
partial large segment (L) termed AreV-HMU-2 (Table S5). The preliminary
experiment in our study obtained a novel WENV complete sequence fromRattus norvegicus collected from Haikou (Daoke) in 2015, termed
the HMU virus. AreV-HMU-2 were detected in 9% (2/21) of the throat
swabs whereas the HMU virus was detected in 4% (1/22) of the anal
swabs. AreV-HMU-2 and HMU viruses showed 95.45% identity with each
other and 94.8% and 93.92% identity with the WENV 3, respectively.
Both viruses showed ≤ 79% identity with other mammalian pathogenic
arenaviruses (Table S6). The genome of the HMU virus comprises 7,147 bp
and shares high sequence similarity with the WENV isolate Rn-242
[19]. Additionally, AreV-HMU-2
and HMU virus phylogenetic analyses based on partial L showed that both
viruses were closely related to WENV and LORV and formed an independent
clade within the genus Mammarenavirus (Figure 4).
Characteristics of
positive-stranded RNA
viruses
Coronaviridae
Coronaviruses (CoVs; orderNidovirales , suborder Cornidovirineae , familyCoronaviridae ) are a group of enveloped RNA viruses with
unusually large positive ssRNA genomes of, 26-32kb [25,26]. The 2020
report of the International Committee on the Taxonomy of Viruses
(ICTV11https://talk.ictvonline.org/) states that CoVs can be
divided into four recognised genera: Alphacoronavirus ,Betacoronavirus , Gammacoronavirus , and
Deltacoronavirus . CoVs are etiological agents in animals and humans
that result in respiratory, hepatic, and enteric diseases such as cold,
severe acute respiratory syndrome (SARS) and (COVID-19)
[27-29].
To identify partial genomes, representative reads were obtained for CoVs
in Rattus tanezumi collected
from Haikou (Fucheng) and Lingao and Haikou (Yanfeng), andNiviventer fulvescenscollected from Baisha (Yinggeling).
We obtained a novel CoV strain
complete genome sequence, named CoV-HMU-1, and three
CoV RNA-dependent RNA polymerase
(RdRp) sequences and spike S segments, termed CoV-HMU-2, CoV-HMU-3, and
CoV-HMU-4, respectively. CoV-HMU-1 and CoV-HMU-2 were detected in 40%
(10/25) and 3% (1/32) of anal swabs, respectively. CoV-HMU-4 and
CoV-HMU-3 were both detected in 10% (2/20) of the throat swabs (Table
S5). Sequence similarity and phylogenetic analyses of RdRp and S
revealed that the identified CoVs could be classified asEmbecovirus under the genus Betacoronavirus , with RdRp and
S ORFs showing 96.1-99% and 95.2-97.9% nucleotide (nt)
identities, respectively (Table
S7). The tree topology showed that the RdRp of CoV-HMU-1 clustered withRattus norvegicus Betacoronavirus (KM349744), CoV-HMU-3 clustered
with Betacoronavirus 1 GCCDC4 (MW773844), and CoV-HMU-2 and CoV-HMU-4
clustered with Longquan Rl rat coronavirus (KF294371) (Figure 5a).
The two domains of the S protein of
CoVs are related to the invasive ability of the virus into the host
cell; the S1 domain is involved in cellular receptor binding and the S2
domain promotes the fusion between the viral capsid and the cell
membrane. The putative S1 region
was located at residues 493–873 for CoV-HMU-1, 463–775 for CoV-HMU-2
and CoV-HMU-4, and 202–442 for CoV-HMU-3. CoV-HMU-1 S1 shared 94.49%
amino acid (aa) identity with Betacoronavirus HKU24, and 59.62–59.26%
aa identity with lineages 2 and 3 CoVs (Figure 5b). CoV-HMU-2 and
CoV-HMU-4 shared high aa identities with lineage 2 CoVs in the S1 region
(88.46–96.31%) and showed 95.19% aa identities with each other.
CoV-HMU-3 shared 95.00% aa identity with Betacoronavirus 1 and
66.49-59.26% with CoV-HMU-2 and CoV-HMU-4. The putative S2 region was
located at residues 1081–1560 for CoV-HMU-1, 880–1408 for CoV1-HMU-2
and CoV-HMU-4, and 880–1419 for CoV-HMU-3.
Flaviviridae
Flaviviruses (orderAmarillovirales , familyFlaviviridae ) are a group of enveloped RNA viruses with
positive-sense ssRNA genomes of 9-13 kb.Flaviviridae can be
classified into four genera, Flavivirus , Hepacivirus ,Pegivirus , and Pestivirus , which include many important
arboviruses and mammalian viruses that are responsible for various mild
to severe infectious diseases in humans, primates, bats, horses, and
rodents [30-32]. Yellow fever, dengue, and Zika viruses are also
important human pathogens [33]. Pestiviruses (PestVs), including the
economically important bovine viral
diarrhoea virus and classical swine fever virus [34].
We obtained two novel complete PestV genome sequences, PestV-HMU-1 and
PestV-HMU-1, using representative reads for pestiviruses inLeopoldamys edwardsi collected from Baisha and Rattus
tanezumi collected from Chengmai County. The
Nest-PCR
was designed to screen for novel PestVs in the pool with read clues.
PestV-HMU-1 and PestV-HMU-2 were detected in 12% (2/17) and 6% (1/18)
of throat swabs, respectively (Table S5).
Additionally,
pairwise alignment revealed that the RdRp (non-structural protein 5,
NS5, or NS5B) of PestV-HMU-1 and
PestV-HMU-2 showed less than
62.39% and 93.33% aa identity with other PestV members (Table S8),
respectively. Phylogenetic analysis
based on RdRp aa sequences suggested that the two PestVs could be
assigned to two different lineages compared with known PestVs;
PestV-HMU-1 clustered with Kioloa
rodent PestV (OL452246) and PestV-HMU-2 clustered with Norway rat pestV
(NC025677) but appeared to represent a separate evolutionary lineage
(Figure 6). Sequence similarity and phylogenetic analysis revealed that
both novel sequences could be classified within the genus Pestivirus
under Flaviviridae .
Astroviridae
Astroviruses (AstroVs; order Nidovirales , familyAstroviridae ) are non-enveloped RNA viruses with positive-sense
ssRNA genomes of 6.8-7 kb. The AstroVs contained three overlapping ORF.Mamastrovirus es and astroviruses are members of the
Astroviridae family. AstroVs infect humans and animals, causing
diarrhoea, fever, and vomiting; the major clinical symptom is watery
diarrhoea. AstroVs may be associated with meningitis, aseptic
encephalitis, and meningoencephalomyelitis in humans and animals
[35].
We obtained a novel AstroV strain complete genome sequence termed
AstroV-HMU-4, and two AstroVs RdRp and capsid proteins, termed
AstroV-HMU-3 and AstroV-HMU-5, using representative reads for AstroVs inRattus tanezumi collected from Lingao and Chengmai. AstroV-HMU-3,
AstroV-HMU-4, and AstroV-HMU-5 were detected in 11% (2/18), 22%
(4/18), and 96% (24/25) of the anal swabs, respectively, and
AstroV-HMU-5 was detected in 68% (17/25) of throat swabs (Table S5).
The identity analysis of RdRp showed that the three identified AstroVs
had ≥97.59% aa identity with other known AstroVs (Table S9). Moreover,
evolutionary trees were constructed for complete protein sequences of
RdRp and CP. Phylogenetic analysis of RdRp and CP showed that the three
AstroVs clustered with Civet astrovirus (OM451116) and rodent astrovirus
(KT946730) within the genus Mamastrovirus ; however, AstroV-HMU-3
appeared to represent a separate evolutionary lineage (Figure 7).
Caliciviridae
Calicivirus virions are positive-sense, non-enveloped ssRNA viruses
belonging to the family Caliciviridae . Partial Calicivirus
RdRp (CaliV-HMU-1) was obtained
from Rattus norvegicus in Sanya (Table S5). However, the
sequence was difficult to compare at the nucleotide level and showed aa
identity with other known caliciviruses. Therefore, we suggest that
CaliV-HMU-1 belongs to a new species under Sapovirus , showing
68.06% aa identity with the human Sapovirus GI.
Hepeviridae
Hepeviruses include enterically transmitted small quasi-enveloped
viruses with positive-sense RNA genomes. Hepatitis E is an important
causative agent of acute sporadic viral hepatitis worldwide. We obtained
a partial RdRp sequence of
picornavirus from HaikouRattus norvegicus and named it HepeV-HMU-1 (Table S5). Sequence
similarity of partial RdRp indicated that HepeV-HMU-1 showed 92.16% nt
identity with Paslahepevirus isolated from Guangdong Rattus
norvegicus .
Picornaviridae
Picornaviruses are members of the
Picornaviridae family and are small, non-enveloped, and positive ssRNA
viruses. Picornavirus can cause skin, mucous membrane, intracranial,
heart, liver, nervous, and respiratory diseases in many vertebrate hosts
[36]. We obtained a partial RdRp sequence of the picornavirus from
Chengmai Rattus tanezumi and named it PicoV-HMU-1 (Table S5). Sequence
similarity of partial RdRp indicated that PicoV-HMU-1 shared 93.14% nt
identity with kobuvirus 1 isolated from Guangdong Rattus losea.
Characteristics of DNA
viruses
Parvoviridae
Parvoviruses (ParVs; order Piccovirales , familyParvoviridae ) are small, non-enveloped, non-segmented, ssDNA
viruses with an average genome size of 4-6 kb [37]. ParVs are
classified into three subfamilies: Densovirinae ,Hamaparvovirinae , and Parvovirinae . ParVs infect many
different animal hosts, including bovines, canines, bats, rodents, and
nonhuman primates. Cross-species transmission of Parvovirinae among
carnivores has been previously reported [38]. ParV B19 causes
hydrops fetalis in fetuses, erythema infectiosum in children, arthritis
in adults, and aplastic crisis in patients with hemoglobinopathies.
To identify the genomes, we used representative reads for ParVs inRattus tanezumi collected from Chengmai and Haikou (Fucheng),Rattus andamanensiscollected from Baisha (Bangxi), and Leopoldamys edwardsicollected from Baisha (Huangjingjiaoling) and obtained two novel ParV
complete genome sequences, ParV-HMU-1 and ParV-HMU-2. Moreover, three
ParV partial NS1 strains were obtained and named ParV-HMU-3, ParV-HMU-4,
and ParV-HMU-5, respectively. The analysis revealed that 14% (3/21),
11% (2/18), and 20% (4/20) of the anal swabs were positive for
ParV-HMU-1, ParV-HMU-4, and ParV-HMU-5, respectively. Moreover, 8%
(3/25) and 11% (2/18) of throat swabs were positive for ParV-HMU-2 and
ParV-HMU-3, respectively (Table S5). Genome identity analysis of NS1
revealed that ParV-HMU-4 and ParV-HMU-5 share ≥99.09% aa identity with
other known ParVs and with each other (Table S10). Pairwise alignment of
NS1 indicated that ParV-HMU-1, ParV-HMU-2, and ParV-HMU-3 showed high
diversity (66.36 %, 68.79 %, and 75.58% aa identity with other known
ParVs, respectively). Evolutionary trees constructed for NS showed that
the five ParVs were clustered with rat bocavirus (KT454512) underbocavirus ; however, ParV-HMU-1, ParV-HMU-2, and ParV-HMU-3
appeared to represent a separate evolutionary cluster (Figure 8a).
Papillomaviridae
Papillomaviruses (PVs; orderZurhausenvirale s, family Papillomaviridae , subfamilyFirstpapillomavirinae , and Secondpapillomavirinae ) are a
diverse group of small, non-enveloped viruses with dsDNA genomes of 5-8
kb. However, ancestral PVs are composed of four major ORFs that encode
early (E1, E2) and late (L1 and L2) proteins. PVs can cause persistent
infections in the skin and mucosal membranes in humans and mammals and
may also cause epidermal proliferative lesions.
Representative reads for PVs in Rattus norvegicus collected from
Tunchang (Nandian), Rattus tanezumi collected from Haikou
(Yanfeng), and Niviventer collected from Baisha (Yinggeling) were
used to obtain two novel PV strains with complete genome sequences,
named PV-HMU-5 and PV-HMU-6, and two PVs L1 sequences, named PV-HMU-3
and PV-HMU-4. PV-HMU-3 was detected in 45% (9/20) of throat swabs
whereas PV-HMU-5 was detected in 61% (11/18) of throat swabs and 55%
(10/18) of anal swabs.
PV-HMU-6
was detected in 50% (9/18) of throat swabs and 38% (7/18) of anal
swabs whereas PV-HMU-4 was detected in 10% (2/20) of throat swabs
(Table S5). Pairwise alignment revealed that the L segments of PV-HMU-3
and PV-HMU-4 showed 98.98% and 100.00% aa identity with known PVs
(Table S11), respectively. Moreover, genome identity analysis of L
revealed that
PV-HMU-5
and PV-HMU-6 showed 89.96% and 89.63% nt identities with all other
known
PVs,
respectively. Phylogenetic analysis based on the L nucleotide sequences
revealed that four PVs were assigned to the genusFirstpapillomavirinae ; however, PV-HMU-3, PV-HMU-4, and PV-HMU-5
clustered with each other; PV-HMU-6 clustered with Mastomys natalensis
papillomavirus 1 (MRU01834) and separated from the members of the PVs
(Figure 8b).
Characteristics of unclassified RNA
viruses
In the present study, we identified
seven unclassified RNA viruses in swabs of different rodent species from
different sites on Hainan Island (Table
S4). The seven unclassified virus
families mainly belonged to the unclassified Picornavirales underPisuvricota . After annotating the complete or partial ORF or RdRp
coding regions of these fully or partially sequenced viruses,
many unclassifiedPicobirnaviridae and Picornaviridae reads belonged to
pools from the hosts ofRattus and Niviventer .
Moreover, reads ofRhabdoviridae ,Dicistroviridae , and Chuviridae appeared in pools from
Rattus hosts.Data analysis showed that rodents
carry a larger proportion of viral RNA and a wider range of phylogenetic
diversity. Therefore, further research on rodent-borne RNA will have
important implications for expanding our understanding of the viral
spectrum and pathogenicity of rodent-borne viruses.