key: cord-0705259-2yb0iako authors: Kumar, Ashutosh; Pandey, Sada N.; Pareek, Vikas; Narayan, Ravi K.; Faiq, Muneeb A.; Kumari, Chiman title: Predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals using ACE2 protein sequence homology date: 2020-10-09 journal: Zoo Biol DOI: 10.1002/zoo.21576 sha: b0c686fa65fe2b720a15a0a6f5bcae4ac4e0b509 doc_id: 705259 cord_uid: 2yb0iako The article is presenting a bioinformatics based method predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals. Recently, there were reports of cats and ferrets, dogs, minks, golden hamster, rhesus monkeys, tigers, and lions testing for SARS‐CoV‐2 RNA which indicated for the possible interspecies viral transmission. Our method successfully predicted the susceptibility of these animals for contracting SARS‐CoV‐2 infection. This method can be used as a screening tool for guiding viral RNA testing for domestic and wildlife animals at risk of getting COVID‐19. We provide a list of the animals at risk of developing COVID‐19 based on the susceptibility score. The ongoing epidemic of COVID-19 is a global concern for human health, but its impact on domestic and wildlife animals is largely unevaluated. The epidemic was believed to have started from the wet wildlife market in Wuhan in the People's Republic of China, but until now, no study has located the actual source of the virus. Genomic analyses suggest that bat is the most likely origin of this virus . The virus has been detected from the pangolins, but there is no concrete evidence that this animal can act as an intermediate host (Lam et al., 2020) . Currently, very limited data is available that evaluates the susceptibility of the domestic and wildlife stocks for SARS-CoV-2 infection using laboratory testing. There have been reports for positive tests for SARS-CoV-2 RNA in cats and ferrets, dogs, minks, golden hamster, rhesus monkey, tigers, and lions (Imai et al., 2020; Kim et al., 2020; Shi et al., 2020 ; United States Department of Agriculture [USDA], 2020; Yu et al., 2020) . Cats do show disease symptoms and transmit the infection to other animals of their species . In all the cases of pet or zoo animals testing positive, the source of infection was either confirmed or suspected to be a human (American Veterinary Medical Association, 2020). SARS-CoV-2 infection in humans depends on the binding of its spike protein to a human cell receptor angiotensin-converting enzyme 2 (ACE2) which is expressed across the animal species as a cell surface receptor (Bibiana et al., 2020) . Evolutionary conservation of protein sequence of ACE2 across vertebrate species, and more specifically, in hominids and primates, remains strong plausibility (Braun et al., 2020) . Recent studies have decoded the receptorbinding domain (RBD) of SARS-CoV-2 spike protein and demonstrated its structural binding with the N-terminal peptidase domain of human ACE2 (hACE2). Receptor-binding motif (RBM) on SARS-CoV-2 RBD makes direct contacts with amino acid residues at hACE2 (Lan et al., 2020; Wan et al., 2020) . We assumed that a homology to hACE2, more particularly to viral RBD-binding interface, of ACE2 protein sequences of animal species could be used in predicting their susceptibility for contracting SARS-CoV-2 infection. The complete protein sequence of hACE2 (Q9BYF1.2) was retrieved from the open-access protein sequence data base Uniprot.org. Crystal structures of hACE2 and SARS-CoV-2 RBD were created based on X-RAY diffraction data retrieved from RCSB protein data bank, PBD1D-6M0J (https://www.rcsb.org/structure/6m0j) using software: PyMOL built from Schrodinger, Inc. Amino acid residues at hACE2/RBD interface, and conserved RBD-binding hotspots at hACE2, were annotated in consultation with published literature (Lan et al., 2020; Othman et al., 2020; Wan et al., 2020) . Further, the NCBI protein blast tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi?PROGRAM=blastp) was used to analyze open-access protein sequence data of ACE2 for the species in animal kingdom available at NCBI databases. To minimize the prediction error, a twostep homology search was performed. First, we performed a comparative analysis of the variability of hACE2 with that of wildlife and domestic animal species in complete protein sequences. The accession numbers of the species which showed significant homology (E value ≤ 0) for the complete sequence were selected for further analysis for the second step. In the second step, we narrowed down our homology search to a subrange of 36-53 amino acid residues of hACE2 (), which contain conserved hotspots for binding of SARS-CoV-2 RBD (Othman et al., 2020) . A fixed query cover of 100% was applied for the sequence homology match in the second analysis. The final ACE2 sequence homology scores along with the statistical significance value (E value ≤ 1E−08) were used to determine the susceptibility ranking of the animal for contracting SARS-CoV-2 infection. A higher sequence homology score with E-value closure to zero signified higher susceptibility ranking for the species. Details of NCBI protein blast and statistical methods predicting significance can be consulted at https://www. ncbi.nlm.nih.gov/books/NBK20261/. (Table S1 ) and a partial sequence at the RBD interface (a subrange of 36-53 amino acid residues) ( Figure 1b and Table 1) were noted across the animal kingdom. In the search for complete sequence homology as well as a subrange of 36-53 amino acid residues at the RBD interface, hominids and other primates showed the highest homology (up to 100%) with hACE2, followed by Zoonotic spillover of human infectious diseases is not uncommon. selected wildlife and domestic animals warranted for caution that the epidemic can have possible spread in animal settings like zoos and wildlife sanctuaries, including a threat of viral transmission from domestic animals (CDC, 2020; Tiwari et al., 2020; Yoo & Yoo, 2020) . The species-specific protein sequence homology to hACE2 may be a binding hotspots of hACE2 with that of the animal species. On the basis of the sequence homology, we predicted the highest susceptibility for hominids and other primates, followed by carnivores, rodents, and artiodactyles (ungulates), however, sequence match varied widely among the species in any order of the animal kingdom. A similar pattern of susceptibility prediction was recently reported by Damas et al., (2020) who compared ACE2 sequence homology across species, however, their species list varied from us, which can be explained by differences of data sources. Damas et al., (2020) also noted conservation of ACE2 protein sequence among evolutionarily linked species; this has been reflected in our study also, as we find the highest sequence homology to hACE2 in the members of order hominids and primate who are closest to humans, and further across mammalians, and other categories of vertebrates. A lesser sequence homology was found with the species which are remotely related to humans. Our susceptibility predictions for cats, dogs, golden hamster, rhesus monkey, tigers, and lions get confirmation from the recent reports in literature which had performed PCR-based laboratory testing for SARS-CoV-2 RNA in the selective animals (Table 1, footnote b ) (Imai et al., 2020; Kim et al., 2020; Shi et al., 2020; United States Department of Agriculture USDA, 2020; Yu et al., 2020) . However, a viral RNA-based susceptibility information is currently highly limited, especially for the animals inhabiting in natural settings. For certain species, such as American mink and ferret, which was recently reported to get SARS-CoV-2 infection ; USDA, 2020); although we found their significant homology to hACE2 (Table S1 ), we couldn't get homology score for SARS-CoV-2 RBD-binding hotspots in absence of complete protein sequence of ACE2 for these species. However, we found significant homology for SARS-CoV-2 RBD-binding hotspots for the species which are closely related to them, such as, European mink and stoat for which the complete protein sequences for ACE2 were available (Table 1) . Our two-step comparison for sequence homology (for complete sequence and SARS-CoV-2 RBD-binding hotspots, respectively) has reduced the chances of false inclusions and provides a susceptibility rank which may help to identify the most vulnerable animal species for contracting SARS-CoV-2 infection. Many of the wild animals noted in the list, such as giant panda, white rhinoceros, are endangered, which raises immediate concern to protect them from the reach of the COVID-19 pandemic. Laboratory testing of viral RNA for wildlife animals in zoos and sanctuaries is limited by many practical factors, in that sense software-based tool to predict COVID-19 susceptibility in the animals presents a very easy and cheap option for COVID-19 prevention and containment in these settings. However, there remains a need to further confirm their susceptibility through laboratory testing of viral RNA. In the present study, we considered only viral host cell entry receptor ACE2 homology for predicting the susceptibility, however, SARS-CoV-2 host cell entry and replication, in addition, may also depend on many other factors like tissue proteases TMPRSS2 or CTSL, and ADAM-17 , thus, a hACE2 homology may not be sufficient, and a viral infectivity testing of the listed animals will be necessary to confirm their susceptibility for contracting SARS-CoV-2 infection. Also, some animals may not develop clinical symptoms or transmit the virus even after contracting SARS-CoV-2 infection, hence their identification through viral RNA testing accompanied by clinical observation will be necessary to understand their susceptibility risk. Additionally, our susceptibility prediction list (Table 1) has only provided an assessment for the animal species for which ACE2 protein sequence (more particularly from amino acid residues 36-53) is available in the NCBI data base (except, Panthera leo, for which ACE2 sequence has been referred from Alexander et al., 2020) . However, this tool can be suitably used to screen remaining animals if their ACE2 protein sequence is made available. SARS-CoV-2 in animals Which animals are at risk? 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(2020, September) Confirmed cases of SARS-CoV-2 in Animals in the United States Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS coronavirus COVID-19 and veterinarians for one health, zoonotic-and reverse-zoonotic transmissions Age-related rhesus macaque models of COVID-19 A pneumonia outbreak associated with a new coronavirus of probable bat origin Expression of the SARS-CoV-2 ACE2 receptor in the human airway epithelium The authors declare that there are no conflict of interests. Sequence alignment data generated in this study have been provided as Supporting Information Files 2 and 3. ACE2 protein sequences used for this study can be retrieved from NCBI protein (https://www.ncbi.nlm.nih.gov/protein) and Uniprot.org (https://www.uniprot.org/) databases using species-specific accession numbers provided in Tables 1 and S1. https://orcid.org/0000-0003-1589-9568