In vitro and in vivo characterization of a bat merbecovirus with ACE2- and DPP4-independent cell entry.

Betacoronaviruses, which have caused three human outbreaks within the last two decades, are thought to originate from bats, raising the concern that bat coronaviruses could cause a novel human outbreak in the future. To determine whether the bat merbecovirus EjCoV-3 strain, previously detected in Eptesicus japonensis in Japan, has the potential to infect humans, we analyzed its cellular entry mechanism. Cellular entry of EjCoV-3 via the spike protein requires protease treatment and is mediated by an unknown receptor, other than DPP4 or ACE2. We generated cultivable recombinant EjCoV-3 using bacterial artificial chromosome-based reverse genetics and found that it efficiently replicated in human respiratory and intestinal cell cultures as well as nasal ciliated epithelium in hamsters. These findings suggest that bat merbecovirus with ACE2- and DPP4-independent cell entry has the potential to cause human infections, highlighting the importance of extensive bat surveillance for pandemic preparedness. IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused three significant outbreaks in the past two decades and are believed to have originated from bats. To investigate the potential for future outbreaks, we generated a Japanese bat-derived MERS-related coronavirus, designated EjCoV-3, using reverse genetics. Our results showed that EjCoV-3 does not utilize ACE2 and DPP4, cell entry receptors for SARS-CoV and MERS-CoV, as a means of infection. However, we found that EjCoV-3 is the first bat merbecovirus capable of efficiently replicating in human respiratory cells and the respiratory tract of hamsters. These findings provide new insight into the potential for MERS-related coronaviruses that do not use ACE2 and DPP4 to infect the human respiratory tract, highlighting the importance of preparedness for outbreaks caused by these viruses.