Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS‐CoV‐2 variants

Increasing evidence supports inter‐species transmission of SARS‐CoV‐2 variants from humans to domestic or wild animals during the ongoing COVID‐19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS‐CoV‐2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS‐CoV‐2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, to evaluate the impact of S mutations, we tested 27 pseudoviruses of SARS‐CoV‐2 carrying different spike mutants by infecting Hela cells expressing different angiotensin‐converting enzyme 2 (ACE2) orthologs from 20 animals. Of these 27 pseudoviruses, 20 bear single mutation and the other 7 were cloned from emerging SARS‐CoV‐2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (B.1.429), and Mu (B.1.621). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS‐CoV‐2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS‐CoV‐2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammal ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS‐CoV‐2, potentially contributing to the spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS‐CoV‐2 variants with these two mutations.