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-ArginineArginine-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 (APRRA) can establish natural infections in humans is unknown.Methods. Here, we developed a duplex digital polymerase chain reaction assay to examine APRRA 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, Delta PRRA variants and variants that have mutations upstream of the Delta PRRA motif. Moreover, the Delta PRRA 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-2(Delta PRRA) 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.