Discussion
The circulation of kobuvirus in rats has been reported in Guangdong,
China, but only from 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 Rattus losea , Rattus tanezumiand 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 Rattus losea , Rattus tanezumi and 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 (Milićević et al., 2020). The MuKVs were examined in 50% ofRattus norvegicus fecal samples in USA in 2014 and later the MuKV
strains were presented in approximately 50% of Rattus norvegicusin 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 (Milićević 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 Bocavirus, Salivirus, Cosavirus, Bufavirus,
Parechovirus and 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 Kobuvirus of species 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 (Milićević 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 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 ofKobuvirus and other members of the family Picornaviridaedemonstrated that our study strains belong to the members of genusKobuvirus .
Furthermore, a phylogenetic analysis based on the complete polyprotein
sequences of our study strains and different kobuvirus species
(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 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 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 Rattus losea andRattus 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.