Natural transmission of bat-like severe acute respiratory syndrome Coronavirus 2 without proline-arginine-arginine-alanine variants in Coronavirus disease 2019 patients

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains the furin cleavage Proline-Arginine- Arginine-Alanine (PRRA) motif in the S1/S2 region, which enhances viral pathogenicity but is absent in closely related bat and pangolin coronaviruses. Whether bat-like coronaviral variants without PRRA (ΔPRRA) can establish natural infections in humans is unknown. Methods. Here, we developed a duplex digital polymerase chain reaction assay to examine ΔPRRA variants in Vero-E6- propagated isolates, human organoids, experimentally infected hamsters, and coronavirus disease 2019 (COVID-19) patients. Results. We found that SARS-CoV-2, as currently transmitting in humans, contained a quasispecies of wild-type, ΔPRRA variants and variants that have mutations upstream of the PRRA motif. Moreover, the ΔPRRA variants were readily detected despite being at a low intra-host frequency in transmitted founder viruses in hamsters and in COVID-19 patients, including in acute cases and a family cluster, with a prevalence rate of 52.9%. Conclusions. Our findings demonstrate that bat-like SARS-CoV-2ΔPRRA not only naturally exists but remains transmissible in COVID-19 patients, which has significant implications regarding the zoonotic origin and natural evolution of SARS-CoV-2.