Kobuviruses belong to the genus Kobuvirus, within the family Picornaviridae, and they are small (27–30 nm diameter), spherical, non-enveloped, positive-sense, single-stranded RNA viruses (1). They all share a common genome organization, with a length of 8.2–8.4 kb. The genome consists of a 5′ untranslated region (UTR), a single open reading frame (ORF) that encodes a large polyprotein of 2,400–2,500 amino acids which is divided into a leader (L) protein, three structural proteins (VP0, VP3, and VP1), seven non-structural proteins (2A−2C and 3A−3D), a 3′UTR, and a poly(A) tail (2). Of these, the VP1 protein is the most variable, whereas the 3D gene is highly conserved (3).

Kobuvirus was first isolated from a fecal sample from a patient with acute gastroenteritis in Japan in 1989 (4). Kobuviruses are widespread worldwide, and are associated with gastroenteritis, respiratory infections, and other clinical symptoms (5). They have been found in a wide range of hosts including humans (4), cattle (6), sheep (7), pigs (8), dogs (9), cats (10), foxes (11), ferrets (12), goats (13), rabbits (14), bats (15), mice (16), and rats (17). They can also be detected in the environment, such as in sewage (18). The International Committee on Taxonomy of Viruses has classified the genus Kobuvirus into six species; Aichivirus A (AiV A; formerly Aichi virus), Aichivirus B (AiV B; formerly bovine Kobuvirus), Aichivirus C (AiV C; formerly porcine Kobuvirus), Aichivirus D (AiV D; formerly Kagovirus 1), Aichivirus E (AiV E; formerly rabbit Kobuvirus), and Aichivirus F (AiV F; formerly bat Kobuvirus), and 20 genetic types (19). Human Kobuvirus, canine Kobuvirus, murine Kobuvirus (MuKV), feline Kobuvirus, and roller Kobuvirus belong to Aichivirus A (20).

Rodents are considered potential reservoirs of several novel pathogens including Kobuvirus. MuKV was first detected in Peromyscus crinitus and Peromyscus maniculatus feces in the USA in 2011 (16). MuKVs were subsequently found in Vietnam (17), Hungary (21, 22), and the USA (23, 24) in stool samples from various murine species including Rattus norvegicus, Rattus tanezumi, Rattus argentiventer, Rattus losea, Rattus exulans, Mus musculus, and Bandicota indica. We recently conducted studies using fecal samples to determine the circulation of MuKV from Rattus norvegicus in Guangdong, China (25), and investigated the prevalence and genetic characterization of MuKV in Rattus losea, Rattus tanezumi, and Rattus norvegicus in several regions in southern China (26). Related research was limited to fecal samples, however, and detailed data pertaining to Kobuvirus in different samples of several rodent species was lacking.

Multiple studies have shown that kobuviruses can cause gastroenteritis in humans and other animal species, but there is limited information on Kobuvirus transmission routes. In humans Kobuvirus has been detected in feces and serum samples (4, 27). Porcine Kobuvirus viraemia has been reported (28), and a seroprevalence study in dogs and cats suggests that high percentages of these animals contain IgG antibodies against Aichivirus A (29). MuKV has been found in lung, brain, heart, and liver samples from rodents (23, 24), and canine Kobuvirus was identified in the brain, lungs, tonsils, and liver of the same puppy (30), indicating that kobuviruses might have a broad tissue tropism. To date the transmission route of MuKV in murine rodents remains unknown. To expand knowledge of the transmission route of kobuviruses, the current study investigated detection rates of MuKV in fecal, serum, throat, and lung tissue samples from murine rodents.

To the best of our knowledge, this is the first study to investigate the prevalence and genomes of kobuviruses in multiple samples (fecal, lung, throat swab, and serum) from different murine rodent species (Rattus norvegicus, Rattus tanezumi, and Rattus rattus). We investigated 413 fecal samples, of which 143 (34.6%) were positive for Kobuvirus, with detection rates of 34.9% in Rattus norvegicus, 29.4% in Rattus tanezumi, and 0% in Rattus rattus. In our previous study, detection rates were 23.7% in Rattus tanezumi, 10.2% in Rattus losea, and 28.1% in Rattus norvegicus in southern China (26), and the prevalence in Rattus norvegicus was 12.6% in Guangzhou (25). MuKVs were detected in 50% of Rattus norvegicus fecal samples in New York in 2014 (24), and were present in 55.5% of free-living and laboratory rats (Rattus norvegicus) in Hungary (22). In New York (Manhattan and Queens sites) in 2018, MuKV was detected in 17.5% of Mus musculus samples (23), and in Vietnam the prevalence rate of MuKV in rats was 17% (17). These findings reveal that kobuviruses are prevalent among different species of murine rodents. In this report, although the prevalence of kobuviruses in fecal samples did not differ significantly between the species, the difference among seasons was significant, with the highest prevalence in winter, suggesting that murine Kobuvirus is prevalent throughout the year, but more attention should be paid to disease prevention and control in winter. Furthermore, both female and male rats can carry Kobuvirus, and there was no statistical difference between the sexes.

Previous studies suggest that kobuviruses may play a role as causative agents in gastroenteritis diseases (5), and are often found in gastrointestinal tract samples from humans and other animals (5, 9–20), suggesting that Kobuvirus is transmitted via the fecal-oral route or consumption of contaminated food or water. The detection of kobuviruses in sewage samples in South Africa (18) and Tunisia (35), in surface waters in Venezuela (36), in sewage and surface waters in the Netherlands (37), and in sewage and river waters in Japan (38), and a new Kobuvirus closely related to canine Kobuvirus detected in sewage samples in Japan (34) supports this contention. In the current study, only MuKV was found in rodents feces, and in phylogenetic analyses all sequences obtained clustered together with the sewage-derived Kobuvirus found in Nepal (33), and shared more than 86% nucleotide and 87% amino acid identities, confirming that MuKV is mainly transmitted via the fecal-oral route through contaminated water and raw and treated sewage, which is concordant with previous Tunisian, Japanese, and Dutch studies (35, 37, 38). Furthermore, we recently conducted two initial epidemiological studies investigating MuKV in murine rodent stool samples (25, 26) which also provide evidence that is congruent with this finding.

Seroepidemiologic studies in Japan, Germany, France, Spain, and Tunisia (27, 39–42) indicate general circulation of kobuviruses in human populations, suggesting an important role of Kobuvirus infection in gastroenteritis. However, a report of porcine viremia suggests that Kobuvirus can escape the gastrointestinal tract into the circulatory system in immunocompetent virus-infected hosts (28), and its high prevalence in dog and cat serum samples indicates the importance of Kobuvirus detection in animal serum specimens (29). Further investigations are required to confirm whether the digestive tract is the main location of Kobuvirus replication. Notably, kobuviruses have also been identified in other sample types. MuKV was found in feces and liver samples from Mus musculus (23), and in fecal, lung, brain, and heart specimens from an infected Rattus norvegicus (24). Interestingly, canine Kobuvirus was detected in brain, lung, tonsil, and liver samples from a puppy, but feces and gastrointestinal tract samples from that same puppy were negative (30). These findings confirm widespread viral dissemination in multiple tissues of animals, but also expand the potential tissue tropisms and transmission routes of kobuviruses.

In neighbor-joining phylogenetic analysis based on partial 3D or VP1 regions, the MuKV sequences isolated from Rattus norvegicus and Rattus tanezumi in the current study clustered with other rat-derived Kobuvirus sequences identified in China, Hungary, and the USA. In phylogenetic tree analysis based on partial 3D regions, the sequences obtained clustered tightly together in a clade according to the species, confirming that the 3D gene is conserved. Notably, however, phylogenetic analysis based on the VP1 regions indicated that such origin-specific lineages of species were not clear. Although the above results suggest that MuKV has distinctive characteristics that differ from other Aichivirus A viruses, the possibility of cross-species transmission in Kobuvirus cannot be ignored. Historical transmissions between rabbits and bats, between rodents and bats, and between cows and pigs have been reported (17, 43, 44), raising the possibility of cross-species transmission in the future; which could result in a sustained threat to public health due to increased contact between humans and animals. More evidence is required to prove cross-species transmission of Kobuvirus, and assess the pathogenicity posed to human health.

Amino acid mutations cause changes in protein phenotype, and are considered most deleterious. Both the 3D and VP1 regions investigated in the current study are important in the stability of the Kobuvirus genome, and its replication. In amino acid heterogeneity analysis, the sequences identified in the study were highly similar, and the 3D region was relatively conserved whereas the VP1 region had more amino acid deletions and substitutions. These results are concordant with a previous review indicating that VP1 is the most variable structural protein and the 3D gene is highly conserved among kobuviruses (3). Estimation of these changes in genomic regions would be helpful with respect to the characteristics of kobuviruses.

In total, 28 complete gene sequences of MuKV were successfully acquired in this study. The polyproteins of the sequences were all cleaved into 11 viral proteins, including L, P1 (VP0, VP3, and VP1), P2 (2A−2C), and P3 (3A−3D), and the predicted cleavage sites were Q/G, P/Q, Q/H, Q/S, Q/T, Q/A, Q/P, Y/V, Q/C, and A/T, according to alignment with known Kobuvirus cleavage sites. Comparisons of the protease cleavage sites in Kobuvirus sequences isolated from humans and animals indicated that the protease cleavage site sequences are highly conserved, supporting a previous review suggesting that the predicted cleavage sites are similar to those in other picornaviruses (3). As representatives of two species, SZ59 shared relatively high nucleotide and amino acid identity with SZ171 (94.0 and 99.3%, respectively), and with other full reference sequences. SZ59 and SZ171 had the highest similarity to the MuKV sequences (93.3–93.6% nucleotide and 98.1–98.3% amino acid identities), with identity values <60% similar to ovine, porcine, and bovine sequences. In similarity plot analysis, SZ59 and SZ171 had lower similarities at the L, VP0, VP1, and 3A regions, and the region at the start of the 3D gene, indicating possible changes in these regions.

In the current study, all the lung, throat swab, and serum samples were negative for Kobuvirus. This may be related to an insufficient sample size, lack of representation in rat species, and geographic distribution. We also screened Kobuvirus-positive samples for Rosavirus, Bocavirus, and Hunnivirus, and 52, 43, and 6 samples, respectively, were co-infected with these viruses. These results are consistent with previous studies demonstrating that kobuviruses are frequently present in mixed infections with other pathogens (45, 46).

The present study provides evidence of fecal-oral transmission of MuKV, and increases the knowledge of molecular characteristics and evolution of MuKV. Further epidemiological and laboratory studies are needed to verify fecal-oral transmission of MuKV.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The animal study was reviewed and approved by Animal Ethics and Welfare Committee of the School of Public Health, Southern Medical University.

YG and QC conceived of the project. QC obtained the funding. YG contributed to the writing of the article and analyzed the data. YG, WH, and JF performed the experiment. YG, WH, JF, YL, and HH collected the samples. All authors have read and approved the manuscript for publication. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (Grant No. 81973107). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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