Difference in the ACE2 utilization by SARS-CoV and SARS-CoV-2
We transfected the HeLa cells with the 20 plasmids expressing different ACE2s individually or empty vector as a control. At 48 h post transfection, the cells were infected with SARS-CoV-BJ01, SARS-CoV-2 or Pangolin CoV pseudovirus. After 48 h of infection, the cells were lysed and subjected to luciferase assay to evaluate the cell-entry efficiency of the pseudoviruses mediated by different ACE2s. As shown inFigure 2 , little luminescence signals could be observed in samples from HeLa cells transfected with empty vector and infected by any of the three pseudoviruses, indicating that native HeLa without ACE2 could not mediate the pseudovirus entry. Luminescence signals from cells expressing crucian, crocodile or viper snake ACE2 were also low, indicating that fish and reptilian ACE2s could barely mediate the pseudovirus entry. Cells expressing chicken or mouse ACE2 showed a strong luminescence signal when infected by the SARS-CoV-BJ01 pseudovirus but not by the SARS-CoV-2 pseudovirus, indicating that SARS-CoV-BJ01 could use chicken or mouse ACE2 for cell entry but SARS-CoV-2 could not. Pangolin CoV was capable of utilizing both chicken and mouse ACE2s, but its utilizing efficiency of chicken ACE2 was much lower than SARS-CoV-BJ01. On the contrary, SARS-CoV-2 pseudovirus ignited a stronger luminescence than SARS-CoV-BJ01 or Pangolin CoV pseudovirus in cells expressing bat ACE2, indicating the highest utilizing capability of bat ACE2 by SARS-CoV-2. For the other ACE2s, infection of all the three pseudoviruses led to strong luminescence signals, implying that all the three SARSr-CoV were capable of utilizing a broad range of ACE2s.
A broad host range is supposed to lead to effective interspecies transmission of virus and more potential to cause a pandemic. The COVID-19 pandemic caused by SARS-CoV-2 is the most severe worldwide pandemic in the recent years, surpassing the SARS pandemic in 2003, so it is likely to speculate that SARS-CoV-2 has a broader host range. Surprisingly, our cell-entry result showed that SARS-CoV-2 had a smaller range of ACE2 utilization than SARS-CoV. SARS-CoV-2 could not utilize mouse or chicken ACE2 which could be used by SARS-CoV, indicating a narrower host range of SARS-CoV-2, especially in murine and birds. The reason is probably that the host range of SARS-CoV-2 is broad enough to support its transmission from bats to humans, and lack of infection to some kinds of animals does not affect such transmission due to redundant routes. Thus, it is not suggested to over-interpret the determination of the host range on the possibility of a virus to cause pandemics, especially for the viruses with broad host ranges.
Notably, SARS-CoV-2 has a better utilization of bat ACE2 than SARS-CoV. Though SARS-CoV originate from bat-SARSr-CoV, the utilization of bat ACE2 by SARS-CoV is quite limited which is supported by the previous reports (Ren, Qu et al. 2008, Ge, Li et al. 2013). According our current results, SARS-CoV-2 utilizes Chinese horseshoe bat ACE2 much better than SARS-CoV, indicating a higher homology between SARS-CoV-2 and its ancestor. This speculation is supported by the phylogenetic analysis of viral genomes in the previous study (Zhou, Yang et al. 2020).
Our cell-entry result showed that SARS-CoV and SARS-CoV-2 could use a wide variety of mammalian ACE2s, which is further supported by the reports about the susceptibility of various mammals to SARS-CoV-2 infection (Shi, Wen et al. 2020). However, the utilization of fish and reptilian ACE2s was quite poor for both SARS-CoV and SARS-CoV-2. This can be explained by the remote phylogenetic relationship between fish/reptilian ACE2s and mammalian ACE2s. By comparison, bird ACE2s have closer phylogenetic relationship with mammalian ACE2s, and thus, bird ACE2s could be used by some but not all SARSr-CoVs, such as SARS-CoV. This indicates that SARSr-CoVs are more likely to be transmitted by mammals and birds but not fish and reptiles, and more attention should be paid to domestic mammals and birds to prevent CoV pandemic.