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\begin{document}

\title{Diversity and independent evolutionary profiling of rodent-borne
pathogens in tropical island, China}



\author[1]{Youyou Li}%
\author[1]{Chuanning Tang}%
\author[1]{Yun Zhang}%
\author[1]{Zihan Li}%
\author[1]{Gaoyu Wang}%
\author[1]{Ruoyan Peng}%
\author[1]{Yi Huang}%
\author[1]{Xiaoyuan Hu}%
\author[2]{Henan Xin}%
\author[2]{Boxuan Feng}%
\author[2]{Xuefang Cao}%
\author[2]{Yongpeng He}%
\author[2]{Tonglei Guo}%
\author[2]{Yijun He}%
\author[2]{Haoxiang Su}%
\author[1]{Xiuji Cui}%
\author[1]{Lina Niu}%
\author[2]{Zhiqiang Wu}%
\author[2]{Jian Yang}%
\author[3]{Fan Yang}%
\author[1]{Gang Lu}%
\author[2]{Lei Gao}%
\author[2]{Qi Jin}%
\author[4]{Meifang Xiao}%
\author[1]{Feifei Yin}%
\author[5]{Jiang Du}%
\affil[1]{Hainan Medical University}%
\affil[2]{Chinese Academy of Medical Sciences \& Peking Union Medical College}%
\affil[3]{Chinese Academy of Medical Sciences \& Peking Union Medical College Institute of Pathogen Biology}%
\affil[4]{Hainan Women and Children's Medical Center}%
\affil[5]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 03, 2024}


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\selectlanguage{english}
\begin{abstract}
The risk of emerging infectious diseases (EID) is increasing globally.
More than 60\% of EIDs worldwide are caused by animal-borne pathogens,
and most viral pathogens are rodent-borne. This study aimed to
characterise the virome and analyse the phylogenetic evolution and
diversity of rodent-borne viruses in Hainan Province, China. We
collected 588 anal and throat samples from rodents, combined them into
28 pools according to their species and location, and processed them for
next-generation sequencing and bioinformatics analysis. The diverse
viral reads closely related to mammals were assigned to 15 viral
families. Molecular clues of the important rodent-borne viruses were
further identified by polymerase chain reaction for phylogenetic
analysis and annotation of genetic characteristics such as coronavirus,
arenavirus, picornavirus. We identified a pestivirus in Leopoldoms
edwardsi and two bocaviruses in Rattus andamanensis and Leopoldoms
edwardsi from the national nature reserves of Jianfengling and Bangxi
with low amino acid identity to known pathogens are proposed as the
novel species, and their rodent hosts have not been previously reported
to carry~these viruses. These results expand our knowledge of viral
classification and host range and suggest that there are highly diverse,
undiscovered viruses that have evolved independently in their unique
wildlife hosts in inaccessible areas, which may cause zoonosis if they
cross their host barrier. Our virome and phylogenetic analyses of
rodent-borne viruses provide basic data for the prevention and control
of human infectious diseases caused by rodent-borne viruses in the
subtropical area of China.%
\end{abstract}%



\sloppy


\section*{Introduction}

{\label{introduction}}

Owing to economic globalisation and environmental changes, the risk of
emerging infectious diseases (EID) is increasing globally, posing
threats to human health, the economy, and society. Approximately 60.3\%
of human EIDs are caused by zoonotic pathogens, originating from
rodents, bats, birds, and other wildlife {[}1-3{]}, with viral or prion
pathogens accounting for 25.4\% of all EID events {[}1{]}.Coronavirus
Disease 2019 (COVID-19) and Ebola hemorrhagic fever (EBHF) bleong to EID
events. Rodential is the largest mammalian group on Earth, with 35
families, 389 genera, and approximately 2700 species
(\textasciitilde{}43\% of all mammal species) worldwide {[}4{]}. Rodents
are widely distributed, diverse in species, have a strong reproductive
ability, migrate in groups, and are mostly concentrated in densely
populated and humid and warm places such as Central and Southern China,
providing them with many opportunities to interact with humans.

Rodents carry Yersinia pestis, Hantaviruses (HanVs), and Leptospira,
which cause plague, haemorrhagic fever, and leptospirosis, respectively
{[}5{]}. Moreover, several important zoonotic viruses carried by rodents
are members of the \emph{Hantaviridae} ,\emph{Flaviviridae}
,\emph{Arenaviridae, Reoviridae} ,\emph{Piconaviridae} , \emph{and
Togaviridae} families {[}4-9{]}. Many of these family viruses include
multiple agents associated with acute gastroenteritis or febrile
illnesses that can cause severe diseases in humans. Lassa virus (LASV),
lymphocytic choriomeningitis virus (LCMV), Tacaribe, Machupo, Junin,
Guanarinto, Sabia, and the recently discovered White-water Arroyo and
Lujo viruses are high-impact pathogens belonging to
the\emph{Arenaviridae} family {[}10-13{]}. HanVs infect a wide range of
mammalian hosts; Hanta, Seoul, Puumala, and Sin Nombre viruses are
etiological agents of haemorrhagic fever with renal syndrome and
hantavirus cardiopulmonary syndrome in humans {[}7{]}. Virus-host
co-divergence and cross-species transmission have played an important
role in the evolution and taxonomy of HanVs {[}7{]}. Additionally, rat
hepatitis E is the causative agent of hepatitis in humans {[}14{]} and
tick-borne encephalitis virus {[}15{]} causes human meningitis,
encephalitis, and meningoencephalitis in the northern European
subcontinent. Severe fever with thrombocytopenia syndrome {[}16{]},
Crimean Congo haemorrhagic fever, and Omsk haemorrhagic fever viruses
are transmitted by rodents to humans as vectors {[}5{]}.

Next-generation sequencing (NGS), viral metagenomic analysis is rapidly
evolving in the study of natural environments {[}17{]}, humans, and
animals {[}18{]}. Recently, metagenomic and meta-transcriptomic analyses
have been increasingly used to investigate the composition of
r\emph{odent} virome. For example, the Aichi virus and novel circovirus
have been detected in wild rodent faecal samples in the analysis of
mouse enterovirus diversity {[}4{]}. The detection of multiple
infectious viruses in rodent lungs in mainland Southeast Asia {[}18{]}.
Hainan Island is a tropical island province that has been geographically
isolated from mainland China for a long time; therefore, its ecological
environment and microorganisms are independent to some degree. Owing to
the unique climate and geographical environment of Hainan Island, it is
rich in diverse mammalian species, especially rodents. The Hainan
Province is an important free trade port in China; therefore, the spread
of viral pathogens from this province is expected to increase through
economic globalisation. In our previous studies, the Wenzhou virus
(WENV) belonging to\emph{Arenaviridae} from \emph{Rattus norvegicus} was
detected in Daoke Village, Hainan, and a complete genome sequence was
obtained {[}19{]}. The present study aimed to . We collected 588 throat
and anal swabs from 326 rodents belonging to 6 species and grouped the
swabs into 28 pools according to the rodent species, swab types, and
sample locations. We sequenced the virome, revealed that coronaviruses,
flaviviruses, parvoviruses, astroviruses, and papillomaviruses are
carried by rodents, and revealed the viral abundance of each pool. The
annotation and phylogenetic description of rodent-borne viruses will
help us to explore their origin, spread mode, and evolutionary patterns.

\section*{Materials and methods}

{\label{materials-and-methods}}

\subsection*{Ethics statement}

{\label{ethics-statement}}

This study was conducted in sampling procedures were approved by the
Ethics Committee of Hainan Medical University (approval number:
HMUEC20180059).

\subsection*{Swabs samples of rodents}

{\label{swabs-samples-of-rodents}}

We collected 588 throat and anal swab samples from 326 rodents from
multiple locations in 9 counties or cities in the Hainan Province, the
tropical island province in China, from May 2017 to June 2021. Samples
(15-20) were grouped into pools according to their sampling locations
and species. Morphology was combined with mitochondrial
cytochrome\emph{b} (mt-\emph{cyt b} ) to identify murine species and
analyse the congruence between viruses and their hosts (Table S1). The
collected samples were quickly immersed in the maintenance medium in the
virus-sampling tube (Yocon Biology, Beijing, China) to ensure the sample
quality, and transported to the laboratory within 24 h using the
low-temperature cold chain {[}6{]}. The samples were divided into three
parts evenly upon arrival at the laboratory and were stored at -80 \selectlanguage{ngerman}°C
storage before subsequent experiments, which is consistent with a
previous study {[}6{]}.

\subsection*{Viral nucleic acid library construction and
NGS}

{\label{viral-nucleic-acid-library-construction-and-ngs}}

The 588 samples were combined into 28 pools based on swab type and
sample location. Swab samples were passed through 0.45\selectlanguage{greek}µ\selectlanguage{english}m filters
(MilliporeSigma, Burlington, MA, USA) to remove eukaryotic and
bacterium-sized particles. The filtrate was ultracentrifuged at 100,000
\selectlanguage{ngerman}×\emph{g} and 4 \selectlanguage{english} for 3 h. The precipitate collected from 28 pool
samples was resuspended in Hank's balanced salt solution and digested
with DNase (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA,
USA) to decompose and remove unprotected nucleic acids. Viral RNA was
extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany).
Viral cDNA was generated using primer K-8N and Superscript III Reverse
Transcriptase (Invitrogen, Thermo Fisher Scientific), as previously
described {[}20{]}. Sequence-independent polymerase chain reaction (PCR)
amplification products were purified and subjected to magnetic bead
sorting. Viral nucleic acid libraries constructed by pre-processing,
such as sonication, were analysed using an Illumina HiSeq2500 sequencer
(Illumina Inc., San Diego, CA, USA) for a single read of 150 bp in
length. Sequence data were deposited in the National Center for
Biotechnology Information (NCBI) sequence reads archive under the
accession number PRJNA892773.

\subsection*{Metagenomic sequencing}

{\label{metagenomic-sequencing}}

Quality-controlled reads of each sample were assembled using Trinity
V2.5.1 {[}21{]}. DIAMOND was used to compare the contigs against the
non-redundant protein database from NCBI. All blastx results of contigs
were annotated with taxonomy id by an in-house database, which combined
accession files
(\emph{ftp://ftp.ncbi.nih.gov/pub/taxonomy/accession2taxid/} ), and
other information obtained from the NCBI Entrez server. Non-redundant
viral contigs larger than 500 nucleotides (nt) were selected. Contigs
related to bacteriophages, plant viruses, and insect viruses were
excluded. Read mapping was performed using BWA-MEM {[}22{]}. Low-quality
mappings were removed using Converm (v0.6.1,
\emph{https://github.com/wwood/CoverM} ) with two preset parameters:
min-read-percent-identity 0.90 --min-read-aligned-percent 0.75. The
relative viral abundance was also generated from Coverm using the
transcripts per million (tpm) method. A heatmap for visualising the
mouse-related virome profile was plotted using the pheatmap package in
R.

\subsection*{Viral genome sequencing}

{\label{viral-genome-sequencing}}

Molecular clues from metagenomic analyses were used to classify sequence
reads into viral families or genera using MEGAN. To identify the partial
or complete genome, representative viral open reading frames
(ORF)-related reads were selected for read-based PCR and sequencing.
Reads with accurate genomic locations were used to design specific
nested PCR primers and to identify partial genomes, which is consistent
with a previous study {[}6{]}. cDNA was generated using random primers
and Superscript III Reverse Transcriptase (Invitrogen). The remaining
genomic sequences were analysed using genome walking and 5\selectlanguage{english}'- and 3\selectlanguage{english}'-
rapid amplification of cDNA ends (Invitrogen; and Takara Bio, Kyoto
Japan). The primers used to amplify the sequence obtained in this study
are shown in (Table S2).

\subsection*{Genome annotation}

{\label{genome-annotation}}

The viral basic nucleotide sequences structure of the genomes and the
amino acid encoded by effective ORFs and its location were deduced by
comparing with the sequence of other virus families. Prediction of
conservative protein families and domains using Pfam
(http://www.ebi.ac.uk/services/proteins), BLASTP
(\emph{https://blast.ncbi.nlm.nih.gov} ), and InterProScan 5
(\emph{http://www.ebi.ac.uk/services/proteins} ). Routine sequence
alignment was performed using Clustal Omega
(\emph{http://www.ebi.ac.uk/Tools/} ). The evolution tree beautification
was performed using ITOL. (\emph{https://itol.embl.de/} ).

\subsection*{Viral prevalence}

{\label{viral-prevalence}}

Nearly complete or partial genomic sequences of the viruses obtained by
NGS sequencing were used as templates to design specific, semi-nested
primers for the non-structural gene for PCR and screening for viruses in
each filtrate sediment and individual sample (Table S2). PCR was
performed using Go Taq Colorless Master Mix (Promega). Nest PCR using
two microliters of the first-round PCR product as the template of the
second round of PCR has high specificity and sensitivity. The thermal
cycling conditions for both PCRs were 94 degC for 5 min, followed by 35
cycles at 94 degC for 30 s, 57 degC for 35 s, 72 degC for 30 s, and a final
elongation step at 72 degC for 10 min. The PCR products were analysed
using 1.5\% agarose gel electrophoresis and ultraviolet imaging.

\subsection*{Phylogenetic and data
analyses}

{\label{phylogenetic-and-data-analyses}}

We used the ClusterW package to align the nucleotide sequences and
deduce the amino acid sequences
(https://rdrr.io/bioc/muscle/man/muscle-package.html) and default
parameters in MEGAX. Relatively conserved viruses were selected to
construct a phylogenetic tree using the maximum likelihood method.
According to the operation rules, the best substitution model was
evaluated using the model selection package function of MEGAX with 1000
bootstrap replicates. The NCBI basic local alignment search tool was
used to perform pairwise amino acid alignment between the reference
sequences and the new virus.

\section*{Results}

{\label{results}}

\subsection*{Sample collection from
rodents}

{\label{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 \emph{Rodentia}from
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-\emph{Cyt
b}gene sequences, including \emph{Rattus norvegicus (}
23.53\%),\emph{Rattus tanezumi} (38.24\%), \emph{Rattus
andamanensis}(6.18\%)\emph{,} \emph{Rattus losea} (6.65\%),
\emph{Niviventer fulvescens} (19.41\%), \emph{and Leopoldamys edwardsi}
(5.00\%). The species from \emph{Rattus} under the \emph{Muridae} are
clustered on the evolutionary tree and show a close distance to the
\emph{Cricetulus} of the \emph{Circetidae} and a far distance to the
\emph{dipus} and\emph{rhizomys} . The 588 samples were combined into 28
pools according to rodent species, swab types, and sample locations.

\subsection*{NGS sequencing}

{\label{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.

\subsection*{Metagenomic analysis and virome
overview}

{\label{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 to\emph{Flaviviridae}
,\emph{Coronaviridae} , \emph{Arenaviridae} , \emph{Astroviridae}
,\emph{Adenoviridae} , \emph{Parvoviridae} , \emph{Papillomaviridae}
,\emph{Herpesviridae} ,\emph{Picornaviridae} , \emph{Hepeviridae,} and
some arthropod viruses, including \emph{Phenoviridae} and
\emph{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.

\subsection*{Characteristics of negative-stranded RNA
viruses}

{\label{characteristics-of-negative-stranded-rna-viruses}}

\subsubsection*{Arenaviridae}

{\label{arenaviridae}}

Arenaviruses (AreVs; order \emph{Bunyavirales} ,
family\emph{Arenaviridae} ) 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:\emph{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 \emph{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 from\emph{Rattus 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 \selectlanguage{english}[?] 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).

\subsection*{Characteristics of positive-stranded RNA
viruses}

{\label{characteristics-of-positive-stranded-rna-viruses}}

\subsubsection*{\texorpdfstring{Coron\emph{aviridae}}{Coronaviridae}}

{\label{coronaviridae}}

Coronaviruses (CoVs; order\emph{Nidovirales} , suborder
\emph{Cornidovirineae} , family\emph{Coronaviridae} ) 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: \emph{Alphacoronavirus}
,\emph{Betacoronavirus} , \emph{Gammacoronavirus} , \emph{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 \emph{Rattus tanezumi} collected from Haikou (Fucheng) and Lingao and
Haikou (Yanfeng), and\emph{Niviventer fulvescens}collected 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
as\emph{Embecovirus} under the genus \emph{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 with\emph{Rattus 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.

\subsubsection*{Flaviviridae}

{\label{flaviviridae}}

Flaviviruses (order\emph{Amarillovirales} , family\emph{Flaviviridae} )
are a group of enveloped RNA viruses with positive-sense ssRNA genomes
of 9-13 kb.\emph{Flaviviridae} can be classified into four genera,
\emph{Flavivirus} , \emph{Hepacivirus} ,\emph{Pegivirus} , and
\emph{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
in\emph{Leopoldamys edwardsi} collected from Baisha and \emph{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 \emph{Flaviviridae} .

\subsubsection*{\texorpdfstring{Astr\emph{oviridae}}{Astroviridae}}

{\label{astroviridae}}

Astroviruses (AstroVs; order \emph{Nidovirales} ,
family\emph{Astroviridae} ) are non-enveloped RNA viruses with
positive-sense ssRNA genomes of 6.8-7 kb. The AstroVs contained three
overlapping ORF.\emph{Mamastrovirus} es and \emph{astroviruses} are
members of \emph{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
in\emph{Rattus 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 \selectlanguage{english}[?]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 \emph{Mamastrovirus} ;
however, AstroV-HMU-3 appeared to represent a separate evolutionary
lineage (Figure 7).

\subsubsection*{\texorpdfstring{Calici\emph{viridae}}{Caliciviridae}}

{\label{caliciviridae}}

Calicivirus virions are positive-sense, non-enveloped ssRNA viruses
belonging to the family \emph{Caliciviridae} . Partial Calicivirus RdRp
(CaliV-HMU-1) was obtained from \emph{Rattus norvegicus} in Sanya (Table
S5)\emph{.} 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
\emph{Sapovirus} , showing 68.06\% aa identity with the human Sapovirus
GI.

\subsubsection*{Hepeviridae}

{\label{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 Haikou\emph{Rattus
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 \emph{Rattus norvegicus} .

\subsubsection*{\texorpdfstring{\emph{Picornavir}idae}{Picornaviridae}}

{\label{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 \emph{Guangdong Rattus losea.}

\subsection*{Characteristics of DNA
viruses}

{\label{characteristics-of-dna-viruses}}

\subsubsection*{Parvoviridae}

{\label{parvoviridae}}

Parvoviruses (ParVs; order \emph{Piccovirales} ,
family\emph{Parvoviridae} ) are small, non-enveloped, non-segmented,
ssDNA viruses with an average genome size of 4-6 kb {[}37{]}. ParVs are
classified into three subfamilies: \emph{Densovirinae}
,\emph{Hamaparvovirinae} , \emph{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
in\emph{Rattus tanezumi} collected from Chengmai and Haikou
(Fucheng),\emph{Rattus andamanensis}collected from Baisha (Bangxi), and
\emph{Leopoldamys edwardsi}collected 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
\selectlanguage{english}[?]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) under\emph{bocavirus} ; however, ParV-HMU-1,
ParV-HMU-2, and ParV-HMU-3 appeared to represent a separate evolutionary
cluster (Figure 8a).

\subsubsection*{Papillomaviridae}

{\label{papillomaviridae}}

Papillomaviruses (PVs; order\emph{Zurhausenvirale} s, family
\emph{Papillomaviridae} , subfamily\emph{Firstpapillomavirinae} , and
\emph{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 \emph{Rattus norvegicus} collected from
Tunchang (Nandian), \emph{Rattus tanezumi} collected from Haikou
(Yanfeng), and \emph{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
genus\emph{Firstpapillomavirinae} ; 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).

\subsection*{Characteristics of unclassified RNA
viruses}

{\label{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 \emph{Picornavirales} under\emph{Pisuvricota} . After
annotating the complete or partial ORF or RdRp coding regions of these
fully or partially sequenced viruses, many
unclassified\emph{Picobirnaviridae} and \emph{Picornaviridae} reads
belonged to pools from the hosts of\emph{Rattus} and \emph{Niviventer} .
Moreover, reads of\emph{Rhabdoviridae} ,\emph{Dicistroviridae} , and
\emph{Chuviridae} appeared in pools from Rattus hosts\emph{.}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.

\section*{Discussion}

{\label{discussion}}

With economic globalisation and environmental changes, the scope of
human activities continues to expand, which increases the possibility of
pathogens jumping from rodent hosts to humans and domestic animals. The
risk of zoonotic EID is elevated in tropical forest areas experiencing
land-use change and wildlife biodiversity (mammal species richness),
based on the prediction of demographic, environmental, and biological
correlates associated with EID events {[}39{]}. Traditional virus
detection methods rely on cell culture based on visible cytopathic
effects and molecular detection of known viral genome sequences.
However, the application of NGS technology enables the sequencing of
pathogenic genomes in one sample, including unknown pathogens. Hainan
Island is an important trade hub and tropical monsoon area with large
animal species diversity. Therefore, virome and phylogenetic analyses of
rodent-borne viruses in Hainan Island can provide insights into the
prevention and control of rodent-borne diseases in China.

The virome data obtained in this study revealed that the risk of
rodent-borne pathogens on Hainan Island should not be underestimated. In
this study, we collected swab samples from six species and three genera
of Muridae, covering nine districts of Hainan Province, to analyse the
viral diversity carried by rodents living next to humans and living in
natural reserves. The novel CoVs, PestVs, AstVs, ParVs, and PVs
identified in this study increase our knowledge of the viral
classification of each viral family. Many rodent-borne viruses detected
in this study were not only found on Hainan Island but also in the
southwestern (Yunnan, Hunan) and northern (Jilin, Ningxia, Inner
Mongolia, Xinjiang) regions of China {[}20{]}, Thailand, Lao PDR,
Cambodia, and other parts of the world {[}18{]}. Moreover, we also
identified widely distributed respiratory tract or enteric RNA viruses,
such as AstroV, pestivirus, papillomavirus, picornaviruses, rotavirus,
and picobirnavirus, which is consistent with a previous study {[}20{]}.
However, the abundance of these viruses in the metagenomic analysis in
the present study was different from that previously reported in rodent
lung samples {[}18{]}. Our data was not abundant in viruses that are
transmitted through blood or bodily fluids, such as phleboviruses,
hepaciviruses, Hantaan viruses, and arteriviruses. This phenomenon may
be due to the different tissue tropisms of these viruses. The results of
the viral positivity rate suggest that these viruses infect hosts
primarily through the respiratory tract, such as CoVs and PestVs.
Moreover, some viruses were positive in anal swabs but negative in
throat swabs. These results may be attributed to insufficient collection
of virus-infected exfoliated host cells during the collection of swabs
or to the different replication characteristics of faecal/oral- or
respiratory-transmitted viruses, which may be detected at different
locations in different cycles {[}19{]}. Some viruses, such as
AstroV-HMU-5, were detected in both throat and anal swabs, indicating
that the host may carry the virus for a long time and may transmit it
via the faecal-oral route (food- or water-borne routes) {[}40{]}.
Additionally, PV-HMU-5 was detected with PV-HMU-6 in the same host, and
throat and anal swabs showed PV positivity in multiple hosts, indicating
that this host group carried the PVs for a long time, which increases
the probability of the virus becoming a potential pathogen.

The murine hepatitis virus in murine coronaviruses was first isolated in
1949 {[}41{]}, and a variant named sialodacryoadenitis coronavirus was
detected in rats in 1970 {[}42{]}. Coronavirus recombination events are
highly common, such as feline CoV type I and canine coronavirus
recombination events {[}43{]}. Human CoVs recombine to produce three
genotypes {[}44{]} and SARS-CoV constantly undergoes evolutionary
recombination {[}45{]}. The results of the phylogenetic analysis of the
RdRp and S protein strains of CoVs were consistent. The four CoVs
identified in this study shared high RdRp and S protein identities with
known murine CoVs and clustered with murine CoVs under the subgenus
Embecovirus in the evolutionary tree. CoV-HMU-4, CoV-HMU-2, CoV-HMU-3,
and mouse hepatitis virus showed a relatively close distance in the
evolutionary tree, especially CoV-HMU-3 and mouse hepatitis virus, under
the same branch. Based on the rodent hosts of known and newly discovered
CoVs, our results suggest that the same virus can be detected in
different regions and rodents, indicating that rodents carrying CoVs may
not exhibir geographic and host specificity. The high similarity of CoVs
observed in our study suggests that each lineage shares a common
ancestor. Additionally, beta-CoVs were detected in the throat and anal
swabs of rodents and only a few clues were found in rodent lungs,
suggesting that these viruses primarily infect the upper respiratory
tract whereas the absence of alpha-CoVs in the two types of samples
indicates that alpha-CoVs may not be the main cause of lower respiratory
tract infections in rodents.

Pestiviruses may cause clinical illnesses, such as acute diarrhoea,
acute haemorrhagic syndrome, wasting disease, and transplacental
infection, leading to fetal death {[}46{]}, and posing serious health
and economic burden. PestV genomic sequences have been detected in bat
and rat samples using NGS {[}8, 47{]}. In the present study, pairwise
alignment revealed that the RdRp of PestV-HMU-1 and PestV-HMU-2 showed
low aa identities with known pathogens, suggesting that they may be
novel species, and rodents have not been previously reported to
carry~these viruses. PestV-HMU-1 was carried by the \emph{Leopoldamys
edwardsi} collected from the Huanjinjiaoling reserve and PestV-HMU-2 was
carried by the \emph{Rattus tanezumi} collected from the Chengmai.
Moreover, PestV-HMU-1 was also present with ParV-HMU-2 in \emph{Leop}
oldamys edwardsi from the Huanjinjiaoling reserve of Hainan. The
abundance and diversity of viruses are high in the central mountainous
areas of Hainan.

Multiple infectious ParVs have been detected in animals; porcine ParV
causes reproductive failure in pigs {[}48{]} whereas canine and feline
panleukopenia ParVs are pathogenic to dogs and cats, respectively
{[}49{]}. ParV infection can also cause serious harm to humans; toad
virus, tusavirus, and cutavirus are mainly detected in children with
diarrhoea {[}50{]}. ParV-HMU-1 and ParV-HMU-2, carried by \emph{Rattus
andamanensi} and \emph{Leopoldamys edwardsi}collected from the two
nature reserves in Baisha, showed low identities with known pathogens.
Overall, the virome reported in this study is highly diverse. These
results extend our knowledge of viral taxonomy and host range and show
that in inaccessible areas, there are still highly diverse viruses that
have evolved independently in their unique wildlife hosts. If these
viruses cross the host barrier, they are highly likely to cause
zoonosis.

Our results provide a profile of the composition of the rodent-borne
virome and a baseline for rodent-borne viruses on Hainan Island, China.
We detected PestVs and bocaviruses with low identities that were not
previously reported to be carried by rodents. The associated viral
genome sequences obtained in our study were corroborated, allowing us to
further understand the phylogenetic relationship of the viruses on
Hainan Island to other regions and obtain data on the diversity and
independent evolutionary profiling of rodent-borne pathogens. This study
suggests that mammals still harbour a large number of uncharacterised
viruses, which require further investigation. This study provides basic
data for the prevention and control of rodent-borne infectious diseases,
evaluates the potential pathogenicity of newly discovered
microorganisms, and provides a baseline for future virome studies and
identification of potential pathogens in EIDs.

\section*{Conclusions}

{\label{conclusions}}

In this study, we report multiple novel viruses from different viral
families in nasal and anal swabs collected from rodents in the Hainan
Province. These findings greatly increase our knowledge of the
rodent-borne virus community on Hainan Island and provide basic data for
analysing the evolutionary independence of viruses. The pathogenicity
and the associated impact of these novel viruses on humans and animals
should be evaluated in further studies.

\textbf{Author Contributions}

FY, LG and QJ designed the study. YL, CT, YZ, ZL, GW, PR, YH, XH, XC,
LN, HS, MX and GL collected the specimens and performed the experiments.
HX, BF, XFC, ZW, JY, YPH, TG, YJH and FY analysed the data. YL and JD
wrote the manuscript. All authors read and approved the final
manuscript.

\textbf{Disclosure statement}

No potential conflict of interest was reported by the author(s).

\textbf{Funding}

This work was supported by Key Research and Development Plan of Hainan
Province (No. ZDYF2020150, and ZDYF2022SHFZ085), the National Natural
Science Foundation of China (Nos. 32060015, 81860367 and 82060378), CAMS
Innovation Fund for Medical Sciences (CIFMS)'' (grant 2021-I2M-1-037),
the Major Science and Technology Program of Hainan Province (No.
ZDKJ202003), the Hainan Provincial Natural Science Foundation of China
(Nos. 820RC628 and 2019RC218), the Scientific Research Foundation of the
Higher Education Institutions of Hainan Province, China (No.
Hnky2021-40), the Research Project of the Hainan Academician Innovation
Platform (No. YSPTZX202004), the Hainan Talent Development Project (No.
SRC200003), Graduate student innovation grant of\selectlanguage{english} Hainan\selectlanguage{english} Medical
University (No. Hys2020-33, and Qhys2021-356).

\section*{Data availability statement}

{\label{data-availability-statement}}

Sequence data were deposited in the National Center for Biotechnology
Information (NCBI) sequence reads archive under the accession number
PRJNA892773.

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\textbf{Figure 1.} Sample collection in the Hainan Island and
metagenomics of rodents according to species and locations. The maps of
Haikou City, Baisha County, Dongfang City, Lingao County, Tunchang
County, Lingshui County, Ledong County, Chengmai County, and Sanya City
in Hainan Island, showing the locations of the samples. Rodent numbers
and species are also shown on the map with different shapes, colours,
and sizes.

\textbf{Figure 2.} (a-d\textbf{).} Comparison of the number of total
contigs and proportions of viral reads from the six rodent species.
Comparison of the numbers of total reads and proportions of viral reads
from the fifteen sampling locations. (e\textbf{).} The proportions of
the number of reads assigned to each viral family in each of the 28
pools are shown in the bar graph.

\textbf{Figure 3.} Rodent-related virome profile in the transcriptomic
data from the 28 pools. Expression is reported in log2(TPM), where TPM
refers to transcripts per million.

\textbf{Figure 4.} Phylogenetic tree of arenaviruses (AreVs). The
phylogenetic tree is based on the partial L protein (RdRp) acid
sequences of AreVs. Phylogenetic trees were constructed by the maximum
likelihood method using the best-fit models (LG+G+I). AreVs identified
in this study are labelled in red. Host genus and location of each virus
are labelled by the 5-point stars and dots of different colours. The
outer colour rings represent additional taxonomic information about
these viruses.

\textbf{Figure 5} . Phylogenetic tree of coronaviruses (CoVs). (a) The
phylogenetic tree is based on the amino acid sequences of complete
RNA-dependent RNA polymerase (RdRp) genes and S (spike) genes of CoVs.
(b)Phylogenetic trees were constructed by the maximum likelihood method
using the best-fit models (WAG+G). All CoVs identified in this study are
labelled in red. Host genus and location of each virus are labelled by
the 5-point stars and dots of different colours. The outer colour rings
represent additional taxonomic information about these viruses.

\textbf{Figure 6} . Phylogenetic tree of pestiviruses (PestVs). The
phylogenetic tree is based on the amino acid sequences of complete
RNA-dependent RNA polymerase (RdRp) (NS5) genes of PestVs. Phylogenetic
trees were constructed by the maximum likelihood method using the
best-fit models (rtREV+G). All PestVs identified in this study are
labelled in red. Host genus and location of each virus are labelled by
the 5-point stars and dots of different colours. The outer colour rings
represent additional taxonomic information about these viruses.

\textbf{Figure 7} . Phylogenetic tree of astroviruses (AstroVs). (a-b)
The phylogenetic tree is based on the amino acid sequences of complete
RNA-dependent RNA polymerase (RdRp) genes and CP genes. Phylogenetic
trees were constructed by the maximum likelihood method using the
best-fit models (mtREV+G+I). All AstroVs identified in this study are
labelled in red. Host genus and location of each virus are labelled by
the 5-point stars and dots of different colours. The outer colour rings
represent additional taxonomic information about these viruses.

\textbf{Figure 8} . Phylogenetic tree of parvoviruses (ParVs) and
papillomaviruses (PVs). (a) Phylogenetic tree based on the amino acid
sequences of complete NS5 genes of ParVs. Phylogenetic trees were
constructed by the maximum likelihood method using the best-fit models
(rtREV+G+I). (b) Phylogenetic trees are based on the amino acid
sequences of complete L genes of PVs and constructed by the maximum
likelihood method using the best-fit models (LG+G). All ParVs and PVs
identified in this study are labelled in red. Host genus and location of
each virus are labelled by the 5-point stars and dots of different
colours. The outer colour rings represent additional taxonomic
information about these viruses.\selectlanguage{english}
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