Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2

The study addresses which animal species are susceptible to SARS-CoV-2 via ACE2 receptor usage, aiming to narrow potential intermediate hosts and understand cross-species transmission. Context includes the emergence of SARS-CoV-2, its likely bat origin, and uncertainty around intermediate hosts (e.g., pangolins, mink, cats/ferrets). Since viral entry requires receptor binding, characterizing interactions between SARS-CoV-2 RBD and ACE2 orthologs from diverse species is critical to assess host range and spillover risk. The purpose is to analyze binding and entry mediated by ACE2 from selected animals and to determine structural basis using cat ACE2.

Background covers coronavirus diversity and human-infecting CoVs (SARS-CoV, MERS-CoV, SARS-CoV-2), with bats as reservoirs and prior zoonoses involving civets and camels. Phylogenetic evidence suggests bat origin of SARS-CoV-2 (e.g., RaTG13 at 96.2% identity; RmYN02 at 93.3% with S1/S2 insertion). Potential intermediate hosts proposed include mink (computational prediction) and pangolins (pangolin CoVs similar to SARS-CoV-2). Experimental data indicate cats and ferrets are permissive and cats can transmit to naïve cats; serology in Wuhan cats post-outbreak supports exposure. These studies motivate examining ACE2 ortholog interactions to evaluate susceptibility.

- Species selection and sequence analysis: 26 animals across 11 orders (Primates, Lagomorpha, Rodentia, Pholidota, Carnivora, Perissodactyla, Artiodactyla, Chiroptera, Insectivora, Afrotheria, Galliformes) plus human. Five bat species and pangolin included due to CoV relevance. ACE2 amino acid sequences aligned; phylogenetic tree constructed with MEGA X. Twenty key hACE2 residues involved in SARS-CoV-2 RBD binding compared across orthologs to assess substitutions and conservation (e.g., F28, D355, R357 conserved). - Flow cytometry (FACS) binding assays: HEK293T cells transiently expressing eGFP-tagged ACE2 orthologs incubated with His-tagged SARS-CoV-2 RBD, SARS-CoV RBD, or SARS-CoV-2 NTD (negative control). Binding detected with anti-His/APC antibody and analyzed by fluorescence shifts. - Mutagenesis: To test potential glycosylation effects at residue 82, N82M substitutions introduced into rat and greater horseshoe bat ACE2; binding to SARS-CoV-2/SARS-CoV RBD assessed by FACS. - Surface plasmon resonance (SPR): mFc-tagged ACE2 orthologs captured on CMS chips pre-immobilized with anti-mouse IgG. Serial dilutions of SARS-CoV-2 RBD, SARS-CoV RBD, or SARS-CoV-2 NTD flowed over to determine binding kinetics/affinities; raw and fitted curves analyzed to estimate KD (or apparent affinity). - Pseudovirus entry assay: BHK21 cells expressing ACE2 orthologs infected with SARS-CoV-2 or SARS-CoV pseudoviruses carrying a luciferase reporter. Luciferase activity measured at 24 h post-infection; relative transduction normalized to hACE2. - Structural analysis: Cryo-electron microscopy structure determination of cat ACE2 (cACE2) in complex with SARS-CoV-2 RBD at 3 Å resolution to define molecular interactions and compare with hACE2 binding mode.

- Broad ACE2 compatibility: SARS-CoV-2 RBD bound ACE2 from multiple species, including monkey (Primates), rabbit (Lagomorpha), pangolin (Pholidota), horse (Perissodactyla), most Carnivora (cat, fox, dog, raccoon dog), and most Artiodactyla (pig, wild Bactrian camel, bovine, goat, sheep). Minimal or no binding observed for Rodentia (guinea pig, mouse, rat), Insectivora (European hedgehog), Afrotheria (lesser hedgehog tenrec), and Galliformes (chicken). Bat ACE2s varied: little brown bat and fulvous fruit bat showed minimal binding; Rhinolophus species (greater, Chinese, least horseshoe bats) showed undetectable interaction in FACS. - SARS-CoV vs SARS-CoV-2: Overall similar binding patterns, but notable exceptions: civet and alpaca ACE2 bound SARS-CoV RBD (clear FACS shifts) while showing undetectable or very weak interaction with SARS-CoV-2 RBD. Fox and dog ACE2 bound SARS-CoV RBD even stronger than SARS-CoV-2 RBD binding to hACE2. - SPR affinities: Relative to hACE2, monkey ACE2 showed equivalent affinity to SARS-CoV-2 RBD. Weaker SARS-CoV-2 RBD binding observed for fox and pig (~2-fold), pangolin, bovine, rabbit, cat, dog, raccoon dog (~3–4-fold), horse, goat, sheep (~6–7-fold). Wild Bactrian camel, little brown bat, and fulvous fruit bat showed >10-fold weaker binding. Civet ACE2 showed no measurable binding to SARS-CoV-2 RBD by SPR, aligning with FACS. Alpaca ACE2 exhibited weak interaction with SARS-CoV-2 RBD (apparent K ~16.5 µM). - Pseudovirus entry: ACE2 orthologs that bound SARS-CoV-2 RBD generally mediated SARS-CoV-2 pseudovirus transduction in BHK21 cells; relative transduction efficiencies varied across species and were normalized to hACE2 (heatmap presented). - Sequence correlates: Among 20 key hACE2 residues for RBD binding, residue substitutions across orthologs ranged from 0 to 10; monkey ACE2 had identical residues to hACE2, while European hedgehog, lesser hedgehog tenrec, and chicken had 10 substitutions each, consistent with poor binding. F28, D355, R357 were fully conserved across 27 species. Residues equivalent to Q24, D30, H34, M82 were highly variable. - Glycosylation at 82: Introducing N82M in rat and greater horseshoe bat ACE2 did not restore binding to SARS-CoV or SARS-CoV-2 RBD, suggesting glycan at 82 alone does not explain lack of binding in these species. - Structural insight: Cryo-EM of cACE2–SARS-CoV-2 RBD at 3 Å revealed a binding mode similar to hACE2, supporting the molecular basis for feline susceptibility. - Host range comparison: SARS-CoV appears to have a slightly broader ACE2 usage across species than SARS-CoV-2.

The findings demonstrate that SARS-CoV-2 can utilize ACE2 receptors from a broad array of mammals, including pets (cats, dogs), domestic livestock (pigs, cattle, sheep, goats), and certain wild animals (pangolin, fox, raccoon dog, camel), implying potential for cross-species transmission and the importance of surveillance in these hosts. The lack of detectable binding and pseudovirus entry via ACE2 orthologs from rodents, some insectivores, and chicken suggests lower susceptibility in those species via ACE2-mediated entry. Differences between SARS-CoV and SARS-CoV-2 in ACE2 usage—such as civet and alpaca compatibility for SARS-CoV but not SARS-CoV-2—highlight virus-specific receptor interaction determinants. The structural similarity of cat ACE2 binding to SARS-CoV-2 RBD with hACE2 provides molecular grounding for reported feline susceptibility. Overall, the data address the research question by linking ACE2 sequence variation to binding affinity and entry efficiency, narrowing candidate intermediate hosts and identifying susceptible species for monitoring.

This work maps the ACE2-mediated host range of SARS-CoV-2 across 26 animal species, integrates biochemical (FACS, SPR) and functional (pseudovirus) assays, and provides structural elucidation of cat ACE2 engagement, revealing a broadly permissive set of mammalian ACE2 receptors. It refines candidate intermediate hosts and underscores the need for surveillance in susceptible domestic and wild animals to mitigate spillover and reverse zoonosis. Future research should: (1) validate findings with live virus infections and in vivo studies across key species; (2) expand species coverage, especially within bats and mustelids; (3) analyze additional receptor cofactors and tissue expression patterns; (4) resolve more ACE2–RBD complex structures from diverse species to pinpoint determinants of specificity; and (5) monitor evolutionary changes in viral RBD that may alter host range.

The study predominantly uses in vitro assays with overexpressed ACE2 (FACS, SPR) and pseudovirus entry in cell lines, which may not fully reflect in vivo susceptibility or tissue tropism. Live virus challenge data are not presented. Species sampling, while broad, is not exhaustive and includes limited representation of bat diversity. The structural analysis is limited to the cat ACE2–RBD complex; structural insights for other species are inferred but not directly determined. Binding measurements for some species are weak or near detection limits (e.g., alpaca), complicating precise affinity estimation.