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\begin{document}

\title{Epidemiology and genetic characteristics of murine kobuvirus from fecal
samples of Rattus losea, Rattus tanezumi and Rattus norvegicus in
Southern China}



\author[1]{Minyi Zhang}%
\author[1]{Fang-Fei  You}%
\author[1]{Fei Wu}%
\author[1]{Huan He}%
\author[1]{Qiushuang Li}%
\author[1]{Qing Chen}%
\affil[1]{Southern Medical University}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Most recently, murine kobuvirus (MuKV), a novel recognized
member of the family Picornaviridae, has been initially identified from
fecal samples of Rattus norvegicus in China. The circulation of MuKV
from other murine rodent species is not fully understood, prompted us to
investigate the prevalence and genetic characterization of MuKV in
Rattus losea, Rattus tanezumi and Rattus norvegicus in Southern China.
Results: Between 2015 and 2017, a total of 243 fecal samples from three
murine rodent species were screened for the presence of murine kobuvirus
(MuKV) from three cities in Southern China, with an overall prevalence
of 23.0\% (56/243). Phylogenetic analysis based on the complete VP1 gene
suggested our sequences were genetically closely related to each other
and the Chinese strains. Three complete polyprotein nucleotide sequences
of MuKV and the genome organization were acquired in the present study.
Phylogenetic analyses also showed our sequences belong to the members of
genus Kobuvirus within the genotype of Achivirus A. Conclusion: The
present study indicated that MuKV is very common in murine rodent
populations.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Picornaviruses (family \emph{Picornaviridae} ) are small, non-enveloped
RNA viruses with single-stranded and positive-sense polarity. As of
March 2020, 147 species divided into 63 genera have been known currently
in the family
\emph{Picornaviridae}(\emph{https://www.picornaviridae.com/}), many of
which had been found to result in significant diseases in humans and a
wide variety of animals. The genus \emph{Kobuvirus} is a novel
recognized member of the family \emph{Picornaviridae} , which contains a
genomic organization similar to that of other picornaviruses and has a
linear genome ranging from 8.2 to 8.4 kb in length. The genome structure
of \emph{Kobuvirus} is composed of VPg, 5' UTR\textsuperscript{IRES-V},
a large open reading frame (ORF) encoding a leader protein (L) and other
three functional regions (P1-P2-P3), 3' UTR and poly (A) tail. The P1
gene region encodes the structural proteins (VP0-VP3-VP1), whereas P2
(2A\textsuperscript{H-Box/NC}-2B-2C) and P3
(3A-3B\textsuperscript{VPg}-3C\textsuperscript{pro}-3D\textsuperscript{pol})
gene regions encode non-structural proteins (Khamrin, Maneekarn, Okitsu,
\& Ushijima, 2014). Of these, the 3D region as the most conserved region
plays an essential role in viral replication (Lescar \& Canard, 2009),
whereas the VP1 gene is the highly variable motif encoding the most
important viral capsid protein that determines the pathogenicity and
antigenicity for kobuvirus (Reuter, Boros, \& Pankovics, 2011).

Since the determination of the first human kobuvirus (Achi virus, AiV)
in a patient with nonbacterial diarrhea~in March 1989, Japan (Yamashita,
Sakae, Ishihara, Isomura, \& Utagawa, 1993), the novel kobuvirus strains
have been determined as members of the genus \emph{Kobuvirus} from a
variety of animal species. In 2003, bovine kobuvirus (BKV) as a
cytopathic agent was first recognized in the culture medium of HeLa
cells that had been used over 30 years in a laboratory in Japan
(Yamashita et al., 2003). Porcine kobuvirus (PKoV) was initially
discovered in 2008 from the fecal samples of domestic pigs in Hungary
(Reuter, Boldizs\selectlanguage{ngerman}ár, Kiss, \& Pankovics, 2008). Subsequently, ovine
kobuvirus (OKV) that was found to be related to BKV was initially
isolated from young, healthy and domestic sheep in Hungary in 2009
(Reuter, Boros, Pankovics, \& Egyed, 2010). In the last ten
years,\emph{Kobuviruses} have been described worldwide from a wide range
of hosts, including black goats in South Korean (Lee et al., 2012),
rabbits in Hungary (Pankovics et al., 2016), dogs in South Korean and
Africa (Oem, Choi, Lee, Lee, \& Choi, 2014; Olarte-Castillo et al.,
2015), cats in South Korean and China (Cho et al., 2014; Niu et al.,
2019), ferrets in the Netherlands and Sweden (Smits et al., 2013), rats
(You et al., 2020) and bats (Wu et al., 2016) in China, wolves (Melegari
et al., 2018), red foxes (Di Martino et al., 2014) and roe deer (Di
Martino et al., 2015) in Italy.

According to the International Committee on Taxonomy of Viruses
(ICTV),\emph{Kobuviruses} comprise six officially recognized species to
date. The species \emph{Aichivirus A} contains Achi virus (found in
humans), canine kobuvirus, feline kobuvirus, murine kobuvirus and a
kobuvirus found in sewage (Ng et al., 2012), whereas \emph{Aichivirus B}
consists of bovine kobuvirus (Yamashita et al., 2003), ferret kobuvirus
(Smits et al., 2013) and ovine kobuvirus (Reuter et al., 2010).
Regarding\emph{Aichivirus C} , two distinct types have been described,
including porcine kobuvirus and caprine kobuvirus (Reuter, Boldizsár, \&
Pankovics, 2009), while cattle kobuvirus (Otomaru et al., 2016), rabbit
(Pankovics et al., 2016) and bat kobu-like viruses
represent\emph{Aichivirus D, E} and \emph{F} , respectively.

Murine kobuvirus (MuKV) belongs to a member of the
species\emph{Aichivirus A} that initially detected from the stools of a
canyon mouse (\emph{Peromyscus crinitus} ) in the USA in 2010 (Phan et
al., 2011). Subsequently, MuKVs were identified in Hungary (Reuter et
al., 2011), Vietnam (Lu et al., 2018) and USA (Williams et al., 2018)
from fecal samples of several rodent species, including striped field
mouse (\emph{Apodemus agrarius} ), Norway rat (\emph{Rattus norvegicus}
),\emph{Rattus losea} , \emph{Rattus argentiventer} and \emph{Mus
musculus} . We have recently initially conducted an epidemiological
study for rat kobuvirus in \emph{Rattus norvegicus} from Guangdong,
China (You et al., 2020). However, lack of information on kobuvirus in
other murine rodent species in China; here, we investigate the
prevalence and genetic characterization of MuKV in \emph{Rattus losea} ,
\emph{Rattus tanezumi}and \emph{Rattus norvegicus} among several regions
in Southern China.

\textbf{Materials and methods}

\textbf{Sample collection}

A total of 243 fresh fecal samples were collected between October 2015
and September 2017 from the rats with unknown health status captured
close to human residences in three different regions in China, including
Xiamen city in Fujian province, Yiyang city in Hunan province and Malipo
county in Yunnan province. Individual fresh stool samples were
immediately placed in RNase-free tubes with 700\selectlanguage{greek}μ\selectlanguage{english}l phosphate-buffered
saline (PBS) (0.3\% homogenate) and stored at -80 until further use.
These stool specimens have also been examined for rat bocavirus (Xiong,
Zhou, et al., 2018), porcine bocaviruses (Xiong et al., 2019) and torque
teno virus (Xiong, Mo, et al., 2018).

\textbf{Nucleic acid extraction and cDNA synthesis}

Viral nucleic acid was extracted from 200\selectlanguage{greek}μ\selectlanguage{english}l of each stool supernatant
using the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Japan), and the
obtained nucleic acid was then reverse-transcribed to synthesize cDNA
using Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland)
in accordance with the manufacturer's instructions. The cDNA was used as
the template directly for polymerase chain reaction (PCR) or kept frozen
at -20.

\textbf{Detection for kobuvirus and other emerging diarrhea viruses}

The presence of MuKV was detected by PCR using universal primers
according to the previous description, UNIV-kobu-F/UNIV-kobu-R,
targeting a 217-bp fragment of partial 3D region for all kobuvirus
strains (Reuter et al., 2009). Two sets of primers for a longer 3D gene
(461bp) and complete VP1 gene (831bp) of MuKV were also used in order to
examine the nucleotide sequences among the 217-bp 3D gene-positive
samples, in line with the previous research (You et al., 2020). Each PCR
mixture was conducted in a total volume of 25\selectlanguage{greek}μ\selectlanguage{english}l, including 12.5\selectlanguage{greek}μ\selectlanguage{english}l Green
Master Mix (Promega, USA), 8.5\selectlanguage{greek}μ\selectlanguage{english}l sterilized H\textsubscript{2}O, 2\selectlanguage{greek}μ\selectlanguage{english}l
cDNA template and 1.0\selectlanguage{greek}μ\selectlanguage{english}M each of the primers. The mixtures were amplified
by 95 for 2min, 40 cycles of 95 for 30s, 56 for 1min, 72 for 1min,
with a final elongation step at 72 for 5min. The amplified products
were analyzed by 1.0\% agarose gels electrophoresis and further
sequenced using ABI Prism 3730xl DNA Analyzer (Applied Biosystems,
Foster City, CA, USA). Moreover, kobuviruses have found to be related to
gastroenteritis and considered as one of the emerging diarrhea viruses
in recent years, containing \emph{Bocavirus, Salivirus, Cosavirus,
Bufavirus, Parechovirus} and \emph{Tusavirus} (Finkbeiner et al., 2008);
we thereby screened for other emerging diarrhea viruses among these
samples according to PCR assays previously reported (Kapoor et al.,
2010; Kapoor et al., 2008; Phan et al., 2014; Shan et al., 2010; Yang et
al., 2016).

\textbf{Genome acquisition of complete polyprotein gene}

Three MuKV-positive samples were randomly selected to amplify the
full-length polyprotein gene sequences using ten pairs of primers
designed in this study on the Benchling website
(\emph{https://benchling.com}), referred to the unpublished sequences in
the Genbank database (accession number: MF352432.1). The primer
sequences were shown in Table 1. After sequencing, there full-length
polyprotein genomes were assembled using Lasergene SeqMan software
(DNASTAR, Inc. USA, Wisconsin, Madison), and available in Genbank under
the accession numbers MW292480-MW292482.

\textbf{Genetic and phylogenetic analysis}

The nucleotide sequences of MuKV identified in the present study were
compared to the corresponding sequences of other kobuvirus strains
previously reported in the Genbank database using Basic Local Alignment
Search Tool (BLAST;\emph{https://www.ncbi.nlm.nih.gov}). Multiple
sequences were aligned with the corresponding regions of kobuvirus
reference strains retrieved from Genbank database using the CLUSTAL W
program in Molecular Evolutionary Genetics Analysis (MEGA version 7.0,
Oxford Molecular Ltd., UK). The pairwise nucleotide (nt) and amino acid
(aa) identities among all sequences were calculated using MegAlign
program (DNASTAR, Inc. USA, Wisconsin, Madison). The open reading frame
(ORF) was predicted for the obtained genome sequences by ORF finder
(\emph{https://www.ncbi.nlm.nih.gov/orffinder}). Similarity plot
analysis of the full-length polyprotein genome was performed using
SimPlot 3.5.1 software. The RNA secondary structure prediction for 5'
and 3' UTR was conducted by Mfold program (Zuker, 2003). Phylogenetic
analyses were generated by maximum-likelihood method with 1000 bootstrap
replicates using MEGA v7.0 and further visualized in FigTree v1.4.0
software. The model selections were based on the results of 'Find best
DNA/Protein models' in MEGA v7.0.

\textbf{Results}

\textbf{Detection of MuKV in stool samples}

Two hundred and forty three fecal samples of murine rodents from Xiamen
(n=96), Malipo (n=48) and Yiyang (n=99) in Southern China were screened
for the presence of MuKV by PCR utilizing a universal primer pair
targeted to their corresponding 3D regions, with an overall prevalence
of 23.0\% (56/243). The MuKV was examined at the highest positive rate
of 28.1\% (41/146) in \emph{Rattus norvegicus} , following by 23.7\%
(9/38) in \emph{Rattus tanezumi} and 10.2\% (6/59) in \emph{Rattus
losea} . Regarding other emerging diarrhea viruses,
human\emph{Bocavirus} , \emph{Parechovirus A} and \emph{Parechovirus B}
were found in the same stool samples, while the co-infection of MuKV
with other emerging diarrhea viruses was not observed in the present
study.

\textbf{Phylogenetic analysis of 3D gene and VP1 gene sequences}

Twenty-two representative sequences of partial 3D gene (461nt) were
selected randomly for further PCR amplification and sequencing within
these positive samples. Sequences isolated from different areas and
different species were highly conserved with each other, at the
nucleotide level of 92.7\%-99.5\% and deduced amino acid level of
95\%-100\%. Maximum-likelihood phylogenetic analysis based on partial 3D
regions was generated with representative kobuvirus strains from
multiple species, indicating that our sequences belonged
to\emph{Achivirus A} and displayed the closet relatedness with MuKVs.
Interestingly, only one strain (YY101) determined during this study
showed the closest relationship to the Chinese MuKV strain, GZ85,
isolated from \emph{Rattus norvegicus} (You et al., 2020), while the
other 21 sequences formed a major group with MuKV reference strains
identified from USA, China and Hungary (Figure 1).

Meanwhile, seven complete VP1 regions (917nt) were successfully
determined in the present study, which share 88.7\%-99.3\% nucleotide
identities and 95.1\%-100\% amino acid identities with one another. In
contrast to other references kobuvirus previously reported in Genbank
database, they have the highest sequence similarity with the Chinese
MuKV strain, MM33 (accession number MN648600.1), at the nucleotide level
of 91.3\%-93.9\%. A phylogenetic tree has been conducted based on the
seven VP1 sequences identified in the current study and the available
complete nucleotide sequences for MuKV VP1 from three different
countries, indicating that our VP1 sequences clustered along with two
MuKV strains, GZ80 and GZ84, previously described in Guangdong, China,
while other kobuvirus reference strains from China, Hungary and Vietnam
formed another groups (Figure 2). The partial 3D and complete VP1
nucleotide sequences of MuKV determined in the present study have been
lodged within the Genbank database under the accession numbers
MW292451-MW292472 and MW292473-MW292479 , respectively.

\textbf{Genomic and phylogenetic analyses of complete polyprotein
genome}

Three complete MuKV polyprotein genome sequences (except the 5' and
3'UTR) were successfully obtained from three MuKV-positive fecal samples
using ten primer pairs in the current study, designating MuKV/XM34/CHN
(accession no. MW292482), MuKV/XM86/CHN (accession no. MW292481) and
MuKV/YN27/CHN (accession no. MW292480). The obtained genome sequence of
MuKV/XM34/CHN was 7714nt long and possessed one complete ORF (7293nt)
encoding a potential polyprotein of 2430 amino acids, whereas the
acquired 7714-nt-long genome strain of MuKV/XM86/CHN and 7711-nt-long
genome strain of MuKV/YN27/CHN contained the complete ORF of 7293nt
(2430aa) and 7290nt (2429aa) in length, respectively. Interestingly, the
lack of one-amino-acid was found in 3A region of the strain
MuKV/YN27/CHN when multiple aligned with the genome of MuKV/XM34/CHN and
MuKV/XM86/CHN, and they shared 92.5-97.1\% nucleotide identities and
97.1-98.1\% amino acid identities with each other. The predicted genomes
of our study strains have the same genome organization as identified for
other kobuviruses, and the detailed information was shown in Figure 3A.

The genomes identified in the present study showed the highest
similarity to the currently published MuKV strain GZ85 (MN648601.1) as
the closest match determined by BLASTn search, with 93.2-94.5\%
nucleotide identities. Moreover, the comparison results indicated our
study strains were also closely related to the previously described
MuKVs. Consequently, we conducted a comparative sequence analysis for
each functional region between our study strains and representative
kobuviruses available in the Genbank database. The kobuviruses
identified in the present study showed amino acid identities with those
of rat (96.0-98.3\%), human (80.1-80.7\%), canine (80.1-80.5\%), and
feline kobuvirus reference strains (80.1-80.3\%) based upon the complete
polyprotein gene. For MuKV/XM34/CHN and MuKV/XM86/CHN, the highest
nucleotide identities were presented in P2 region of rat kobuvirus GZ85
strain, with values of 94.6\% and 94.7\%, respectively, while they also
shared the highest amino acid identities (98.7\%) to the P2 region of
rat08/rAiA/HUN. Regarding MuKV/YN27/CHN, the highest nucleotide (95.4\%)
and amino acid identities (99.8\%) were found in P2 and P1 regions of
GZ85, respectively (Table 2). Besides, the similarity plot analysis was
generated to further analyze the genetic characteristics, contrasting
the complete polyprotein nucleotide gene of our study strains and one
feline kobuvirus strain WHJ-1/MF598159.1 (used as an out-group sequence)
to the rat kobuvirus reference strain GZ85/MN648601.1 (used as a query
sequence). The findings suggested the sequences displayed relatively
high similarities to the query sequence in the VP0, VP3, 2A-2C, 3C and
3D regions, whereas different similarities were exhibited in the L, VP1,
3A and 3B regions. Additionally, MuKV/YN27/CHN presented considerably
higher similarities in the L, 3A, 3B regions and the sequence connecting
2A and 2B than MuKV/XM34/CHN and MuKV/XM86/CHN (Figure 3B).

Table 3 shows the predicted protease-cleavage sites of our study strains
and other reference kobuviruses, including Q/G, P/Q, Q/T, Q/S, Y/V and
Q/A. The predicted protease-cleavage sites between L and VP0 (Q/G), 2A
and 2B (Q/G), 2B and 2C (Q/G), 2C and 3A (Q/G), and 3C and 3D (Q/S) are
conserved among kobuviruses from different species. Besides, the
protease-cleavage sites between 3A and 3B is Q/A for the kobuvirus
strains, except for porcine kobuvirus (Q/G).

Maximum-likelihood phylogenetic analysis was conducted based on the
complete polyprotein nucleotide sequences between our study strains and
other representative members of family \emph{Picornaviridae} ,
suggesting the strains identified in the present study were located at
the same branch as the members of genus \emph{Kobuvirus} , where the
sequences of MuKV are the closest relatives (Figure 4).

\textbf{Discussion}

The circulation of kobuvirus in rats has been reported in Guangdong,
China, but only from \emph{Rattus norvegicus} . To our best knowledge,
the present study represents the first study to investigate the
prevalence of murine kobuvirus isolated from three murine rodent species
in southern China, including \emph{Rattus losea} , \emph{Rattus
tanezumi}and \emph{Rattus norvegicus} .

The detection results indicated that fifty-six strains of MuKV were
identified in fecal samples from these murine rodents, with an
approximate detection rate of 23.0\% (56/243). Of these, the prevalence
of MuKV in \emph{Rattus losea} , \emph{Rattus tanezumi} and \emph{Rattus
norvegicus} was 10.2\% (6/59), 23.7\% (9/38) and 28.1\% (41/146),
respectively, confirming these three murine rodent species in China are
infected with MuKV, as many studies reported that the kobuvirus
infection is widely distributed in the world (Mili\selectlanguage{polish}ć\selectlanguage{english}evi\selectlanguage{polish}ć \selectlanguage{english}et al., 2020).
The MuKVs were examined in 50\% of\emph{Rattus norvegicus} fecal samples
in USA in 2014 and later the MuKV strains were presented in
approximately 50\% of \emph{Rattus norvegicus}in Hungary (Boros et al.,
2019; Firth et al., 2014), while the detection rate of MuKV was 17\% in
Vietnam (Lu et al., 2018). Thus, our present finding confirmed murine
rodent species are hosts of kuboviruses. The detection of kobuvirus is
common in murine rodents, even if the different prevalence.

Several previous studies have detected kobuviruses isolated from
different animals with diarrhea, suggesting kobuviruses were the
causative agent for gastroenteritis (Niu et al., 2019; Wang,
Fredrickson, Duncan, Samuelson, \& Hsiao, 2020). Simultaneously, it has
been described that kobuviruses have a serious impact on systemic
infections (Ribeiro et al., 2017). Although the health status of the
analyzed live-trapped murine rodents in the present study is currently
unknown due to none of observable clinical manifestation, the frequent
presence of kobuviruses identified in fecal samples implied a viral
affinity to the gastrointestinal tract (Mili\selectlanguage{polish}ć\selectlanguage{english}evi\selectlanguage{polish}ć \selectlanguage{english}et al., 2020).
Furthermore, a previous study suggested that kobuviruses are frequently
mixed infected with other pathogens (Jackova et al., 2017). Hence, we
simultaneously screened for those as emerging diarrhea viruses,
including \emph{Bocavirus, Salivirus, Cosavirus, Bufavirus,
Parechovirus} and \emph{Tusavirus,} while no co-infection was found in
this investigation. Considering that murine rodents are in close contact
with human life, there is still a high risk of human transmissibility of
MuKVs increasing human health concerns. Taken together, these give
valuable insight into future experimental studies focused on its
pathogenesis and the real associations with infectious diseases,
especially for gastroenteritis.

Phylogenetic analysis based upon partial 3D gene confirmed our sequences
to be members of genus \emph{Kobuvirus} of species \emph{Achivirus A} ,
clearly branched together with murine, canine and feline kobuviruses and
human Achi viruses. The 21 MuKV sequences identified in the current
study formed a large group with the American, Chinese and Hungarian MuKV
strains, indicating no geographic clustering based on 3D region in line
with previously published results (Mili\selectlanguage{polish}ć\selectlanguage{english}evi\selectlanguage{polish}ć \selectlanguage{english}et al., 2020), whereas only
one strain (YY101) showed the closest relationship to the Chinese MuKV
strain, GZ85. Moreover, our sequences were more likely to
phylogenetically cluster at the same branch according to their host
species.

The VP1 capsid protein of picornaviruses is the most immunodominant
portion of kobuvirus determining the antigenicity and pathogenicity; it
is the most variable structural protein for kobuviruses as well (Chen et
al., 2013; Reuter et al., 2011). The phylogenetic analysis based on the
complete VP1 nucleotide sequences of the study strains with the species
of murine rodent (in italics) shows our study strains clustered closely
together with the Chinese strains and separated from other published VP1
sequences of MuKVs from Hungary and Vietnam, indicating the MuKV strains
from China may possess a similar evolution background and could be
circulating among the different murine rodent populations in China (Y.
Wang et al., 2020). This finding presents the geographical
location-specific clustering of the selected VP1 strains, while
geographic clustering has not been observed in the 3D region. Whether
these MuKV sequences from different geography impact protein function
still warrants research attention in the future.

The acquirement of complete polyprotein genomes in the present study
allowed us to obtain information on the genetic characteristics of MuKV
strains circulating in murine rodents in Southern China, which was named
MuKV/XM34/CHN (7296nt), MuKV/XM86/CHN (7296nt) and MuKV/YN27/CHN
(7293nt). Genomic analysis revealed that the polyprotein of our study
strains have a similar genome architecture to other kobuviruses,
including L , VP0, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D.
Interestingly, one-amino-acid deletion was presented in the deduced 3A
viral protein region of MuKV/YN27/CHN, similar to the canine strain,
CH-1, and feline strain, WHJ-1. According to the previous studies,
one-amino-acid deletion was also found in VP0 region of feline kobuvirus
from a diarrhoeic cat (Niu et al., 2019). In contrast, thirty-amino-acid
deletion was presented in 2B region of porcine kobuvirus from health
piglets that might be associated with the pathogenicity of porcine
kobuvirus (Jin et al., 2015). Nevertheless, natural mutation and
recombination exist in viruses of the family \emph{Picornaviridae} ,
playing an important role in genetic diversities (Lukashev, 2010).
Whether this amino acid deletion has an impact on the pathogenicity of
kobuviruese from different species requires further investigation via
structure prediction and genomic analysis. A phylogenetic analysis
of\emph{Kobuvirus} and other members of the family
\emph{Picornaviridae}demonstrated that our study strains belong to the
members of genus\emph{Kobuvirus} .

Furthermore, a phylogenetic analysis based on the complete polyprotein
sequences of our study strains and different kobuvirus species
(\emph{Achivirus A-F} ) revealed our MuKV strains were more closely
related to canine kobuvirus and human Achi virus than to bovine
kobuvirus. This result suggests that cross-species transmission of
kobuviruses can occur due to frequent contact between rats, dogs, and
humans in the natural environment, in accordance with a prior study
donmenstrating multiple cross-species transmissions have a possibility
to exist within and among mammalian species (Lu et al., 2018). These
findings increase further understanding of evolution and genomic
characteristics for \emph{Kobuvirus} in murine rodent populations.

Besides, the 49-nt-long partial 3'UTR of MuKVs identified in our study
showed the highest sequence identities (98.0\%-100\%) to the Chinese
strain Wencheng-Rt386-2 (accession no. MF352432.1). The predicted RNA
secondary structure of the partial 3'UTR also contain the characteristic
''barbell-like'' structure with conserved motifs of AGGGAAC (Figure 5),
which is identical in other partial 3'UTR of MuKV references currently
available in the Genbank database. The presence of barbell-like
structures was recognizable in the genus of \emph{Kobuvirus} among
different nucleotide positions of the 3'UTR gene (Boros et al., 2019;
Choi, Lee, Lee, \& Oem, 2015). It is important to note that intense
studies should figure out the question about the functions of this
structure.

In conclusion, we first identify kobuviruses in \emph{Rattus losea}
and\emph{Rattus tanezumi} , expanding the host range of kobuviruses. The
combined findings of this study provide molecular characteristics of
MuKV and show widespread circulation in different murine rodent species.
The limitation of the present study is small sample size. Therefore,
more detailed analyses including epidemiological and experimental
investigations are needed to emphasize the pathogenicity, genetic
diversity and potential risk to the public health of murine kobuvirus.

\textbf{Ethics Statement}

The research protocol has been approved by the Animal Ethics and Welfare
Committee of the School of Public Health, Southern Medical University
and met the guidelines for the Rules for the Implementation of
Laboratory Animal Medicine (1998) from the Ministry of Health, China.

\textbf{Data available statement}

All data generated or analyzed during this study are included in this
published article. Access to raw data can be acquired by connecting to
the corresponding author via email.

\textbf{Acknowledgments}

We are grateful for the generous support of our teammates in the sample
collection and technical assistance. This work was supported by the
National Natural Science Foundation of China (Grant No.81973107).

\textbf{Conflict of interest statement}

No conflict of interest exits in the submission of this manuscript.

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