Pangolin HKU4-related coronaviruses found in greater bamboo bats from southern China

Coronavirus (CoV) spillover originating from game animals, particularly pangolins, is currently a significant concern. Meanwhile, vigilance is urgently needed for coronaviruses carried by bats, which are known as natural reservoirs of many coronaviruses. In this study, we collected 729 anal swabs of 20 different bat species from nine locations in Yunnan and Guangdong provinces, southern China, in 2016 and 2017, and described the molecular characteristics and genetic diversity of alphacoronaviruses (αCoVs) and betacoronaviruses (βCoVs) found in these bats. Using RT-PCR, we identified 58 (8.0%) bat CoVs in nine bat species from six locations. Furthermore, using the Illumina platform, we obtained two representative full-length genomes of the bat CoVs, namely TyRo-CoV-162275 and TyRo-CoV-162269. Sequence analysis showed that TyRo-CoV-162275 shared the highest identity with Malayan pangolin (Manis javanica) HKU4-related coronaviruses (MjHKU4r-CoVs) from Guangxi Province, whereas TyRo-CoV-162269 was closely related to HKU33-CoV discovered in a greater bamboo bat (Tylonycteris robustula) from Guizhou Province. Notably, TyRo-CoV-162275 has a putative furin protease cleavage site in its S protein and is likely to utilize human dipeptidyl peptidase-4 (hDPP4) as a cell-entry receptor, similar to MERS-CoV. To the best of our knowledge, this is the first report of a bat HKU4r-CoV strain containing a furin protease cleavage site. These findings expand our understanding of coronavirus geographic and host distributions.