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.