Isolation and Characterization of an HKU4-related coronavirus circulating in Malayan pangolin

As the mostly trafficked mammal, pangolins may play important role in coronaviruses zoonotic transmission cycle. Here, we report the circulation of a Tylonycteris bat coronavirus HKU4-related coronavirus from smuggled Malayan pangolins ( Manis javanica ), including 4 viral nucleotide positive and another 7 viral seropositives from 86 animals. Four almost identical (99%) genomes were identified and a MjHKU4r-CoV- 1 virus was isolated, which uses human dipeptidyl peptidase-4 (hDPP4) as receptor and human proteases for cell infection, and this infection is enhanced by a furin cleavage site that is absent in the bat HKU4r-CoVs. In addition, the MjHKU4r-CoV-1 spike showed higher binding affinity to the hDPP4 or wider host range than the bat HKU4-CoV. Finally, MjHKU4r-CoV-1 is productively infectious and pathogenic in human airway, intestinal organs or in hDPP4 transgenic mice. Our work highlighted the importance of pangolins as susceptible hosts of bat-related coronaviruses that poised for human emergence.