Coronaviruses (CoVs), the largest positive-sense RNA viruses, have caused infections in both humans and animals. The cross-species transmission of CoVs poses a serious threat to public health. Rodents and bats, the two largest orders of mammals, serve as significant natural reservoirs for CoVs. It is important to monitor the CoVs carried by bats and rodents. In this study, we collected 410 fecal samples from bats and 74 intestinal samples from rats in Yunnan Province, China. Using RT-PCR, we identified one positive sample for alphacoronavirus (TC-14) from Rhinolophus sinicus (Chinese rufous horseshoe bat) and two positive samples for betacoronavirus (GS-53, GS-56) from Apodemus ilex (Rodentia: Muridae). We successfully characterized the complete genomes of TC-14 and GS-56. Phylogenetic analysis revealed that TC-14 clustered with bat CoV HKU2 and SADS-CoV, while GS-56 was closely related to rat CoV HKU24. The identification of positive selection sites and estimation of divergence dates further helped characterize the genetic evolution of TC-14 and GS-56. In summary, this research reveals the genetic evolution characteristics of TC-14 and GS-56, providing valuable references for the study of CoVs carried by bats and rodents in Yunnan Province.
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