The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, poses a significant threat to global public health and the economy. While multiple vaccine candidates and therapeutic antibodies are under development, understanding SARS-CoV-2's origins and transmission dynamics is crucial for effective control. SARS-CoV-2 is believed to have originated from bats, but its intermediate host remains unknown. Coronaviruses (CoVs) are enveloped viruses with single-stranded RNA genomes, categorized into four genera: alpha, beta, gamma, and deltaCoVs. Several betaCoVs, including SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused significant outbreaks. Bats are known natural reservoirs for various viruses, including CoVs. Phylogenetic analyses suggest SARS-CoV-2's close relationship to bat CoVs, such as RaTG13 and RmYN02. However, the intermediate host remains elusive, with potential candidates including minks, pangolins, and cats. SARS-CoV-2, like other CoVs, requires binding to host cell receptors to initiate infection. The angiotensin-converting enzyme 2 (ACE2) protein serves as the receptor for both SARS-CoV and SARS-CoV-2. The SARS-CoV-2 spike (S) protein's receptor-binding domain (RBD) specifically interacts with ACE2. Therefore, analyzing the binding of SARS-CoV-2 RBD to ACE2 orthologs from various species could help identify potential intermediate hosts.

The literature extensively documents the zoonotic origins of many coronaviruses. Studies have established the link between bats and several human coronaviruses, including HCoV-NL63 and HCoV-229E. SARS-CoV's origin in bats and its potential intermediate host, civets, have been well-documented. Similarly, MERS-CoV's association with bats and dromedary camels has been investigated through serological studies. The discovery of pangolin coronaviruses with high sequence similarity to SARS-CoV-2 has fueled the hypothesis of pangolins as potential intermediate hosts. Reports on SARS-CoV-2 infection in minks and experimental transmission in cats further highlight the broad host range of the virus. Previous research characterizing the interaction between SARS-CoV RBD and ACE2 has laid the groundwork for understanding the virus's receptor binding. This study builds upon this existing knowledge by investigating the interaction between SARS-CoV-2 RBD and ACE2 orthologs from a wider range of animal species.

This study used a multi-faceted approach to investigate the binding of SARS-CoV-2 RBD to ACE2 orthologs from 26 different animal species, encompassing domestic animals, pets, and wild animals. Phylogenetic analysis of ACE2 sequences was conducted to establish evolutionary relationships among the studied species. Key residues in hACE2 responsible for SARS-CoV-2 RBD interaction were identified and compared across species to predict susceptibility. Flow cytometry (FACS) was employed to assess the binding of SARS-CoV-2 RBD and SARS-CoV RBD to cell-surface expressed ACE2 orthologs. Surface plasmon resonance (SPR) assays provided quantitative measurements of binding affinities. Finally, pseudovirus transduction assays were used to evaluate the functionality of ACE2-RBD interactions in facilitating viral entry.

Phylogenetic analysis revealed varying degrees of evolutionary distance between the ACE2 orthologs of the studied species and human ACE2. Comparison of key RBD-binding residues showed that monkey ACE2 is identical to human ACE2, while European hedgehog, lesser hedgehog tenrec, and chicken ACE2 showed the most substitutions. Flow cytometry demonstrated that SARS-CoV-2 RBD interacted with ACE2 orthologs from primates, lagomorphs, pholidota, perissodactyla, most carnivores, and most artiodactyls, but not from rodentia, insectivora, afrotheria, or galliformes. Five bat species showed varied interactions. SARS-CoV RBD exhibited similar binding patterns, with exceptions for civet and alpaca ACE2. SPR analysis quantified binding affinities, revealing that monkey ACE2 bound SARS-CoV-2 RBD with similar strength to human ACE2. Other species displayed varying degrees of weaker binding, with some showing significantly reduced affinity. Notably, civet ACE2 showed no binding to SARS-CoV-2 RBD, while alpaca ACE2 showed weak binding despite lacking detectable interaction in FACS assays. Pseudovirus transduction assays corroborated the binding data, showing that ACE2 orthologs from susceptible species facilitated viral entry.

The findings demonstrate a broad host range for SARS-CoV-2, indicating potential susceptibility in several animal species. The similar binding mode of cat ACE2 to SARS-CoV-2 RBD, as revealed by cryo-EM, further supports this conclusion. The variations in binding affinity among different species might reflect differences in susceptibility and transmission efficiency. The lack of binding to civet ACE2 by SARS-CoV-2 RBD, in contrast to SARS-CoV RBD, may suggest distinct receptor-binding specificities between the two viruses. This study's results provide insights into potential intermediate hosts for SARS-CoV-2 and underscore the importance of monitoring animal populations for the virus to prevent future outbreaks. Further research is needed to investigate the role of these animal species in SARS-CoV-2 transmission and the potential for zoonotic spillover events.

This study comprehensively investigated the binding of SARS-CoV-2 RBD to ACE2 orthologs from a wide range of animal species, revealing a broad host range. The findings highlight potential intermediate hosts and emphasize the need for continuous surveillance of animal populations to mitigate future outbreaks. Future research should focus on detailed epidemiological studies of SARS-CoV-2 in these susceptible species and exploration of the specific molecular determinants of binding affinity and viral entry.

The study relied on in vitro binding assays and pseudovirus transduction, which may not fully reflect the complexity of in vivo infection. The sample size of some animal species was limited, potentially affecting the generalizability of the findings. Further in vivo studies are necessary to confirm the susceptibility of identified species and explore the dynamics of SARS-CoV-2 transmission within different animal populations. The study did not address potential post-binding events influencing infection, such as viral entry and replication.