Received: 4 February 2021 | Revised: 3 March 2021 | Accepted: 4 March 2021 DOI: 10.1002/ajp.23255 RESEARCH ARTICLE Variation in predicted COVID‐19 risk among lemurs and lorises Amanda D. Melin1,2,3 | Joseph D. Orkin4 | Mareike C. Janiak5 | 4 4 6 Alejandro Valenzuela | Lukas Kuderna | Frank Marrone III | Hasinala Ramangason1 | Julie E. Horvath7,8,9,10 | Christian Roos11 | 12 13,14 15,16,17 Andrew C. Kitchener | Chiea Chuen Khor | Weng Khong Lim | Jessica G. H. Lee18 | Patrick Tan13,15,17 | Govindhaswamy Umapathy19 | 20 20 21 Muthuswamy Raveendran | R. Alan Harris | Ivo Gut | Marta Gut21 | Esther Lizano4 | Tilo Nadler22 | Dietmar Zinner23,24,25 | 26 19 27 Minh D. Le | Sivakumara Manu | Clément J. Rabarivola | Alphonse Zaramody27 | Nicole Andriaholinirina27 | Steig E. Johnson1 | 28,29,30 28,30 31,32 Erich D. Jarvis | Olivier Fedrigo | Dongdong Wu | Guojie Zhang33,34,35 | Kyle Kai‐How Farh36 | Jeffrey Rogers20 | 4,37,38,39 4,37,38 Tomas Marques‐Bonet | Arcadi Navarro | David Juan4 | Paramjit S. Arora6 | James P. Higham40,41 1 Department of Anthropology and Archaeology, University of Calgary, Alberta, Canada 2 Department of Medical Genetics, University of Calgary, Alberta, Canada 3 Alberta Children's Hospital Research Institute, University of Calgary, Alberta, Canada 4 Experimental and Health Sciences Department (DCEXS), Institut de Biologia Evolutiva, Universitat Pompeu Fabra‐CSIC, Barcelona, Spain 5 School of Science, Engineering & Environment, University of Salford, Salford, UK 6 Department of Chemistry, New York University, New York, USA 7 Genomics & Microbiology Research Laboratory, North Carolina Museum of Natural Sciences, Raleigh, North Carolina, USA 8 Department of Biological and Biomedical Sciences, North Carolina Central University, Durham, North Carolina, USA 9 Department of Evolutionary Anthropology, Duke University, Durham, North Carolina, USA 10 Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, USA 11 Gene Bank of Primates and Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göettingen, Germany 12 Department of Natural Sciences, National Museums Scotland and School of Geosciences, University of Edinburgh, Edinburgh, UK 13 Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 14 Singapore Eye Research Institute, Singapore National Eye Centre, Singapore 15 SingHealth Duke‐NUS Institute of Precision Medicine, Singapore Health Services, Singapore 16 SingHealth Duke‐NUS Genomic Medicine Centre, Singapore Health Services, Singapore 17 Cancer and Stem Cell Biology Program, Duke‐NUS Medical School, Singapore 18 Department of Conservation, Research and Veterinary Services, Wildlife Reserves Singapore, Singapore 19 CSIR‐Laboratory for the Conservation of Endangered Species, Centre for Cellular and Molecular Biology, Hyderabad, India 20 Human Genome Sequencing Center and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA 21 Universitat Pompeu Fabra (UPF), Barcelona, Spain Amanda D. Melin & Joseph D. Orkin contributed equally to this study. Am J Primatol. 2021;83:e23255. wileyonlinelibrary.com/journal/ajp © 2021 Wiley Periodicals LLC | 1 of 12 https://doi.org/10.1002/ajp.23255 2 of 12 | MELIN ET AL. 22 Cuc Phuong Commune, Nho Quan District, Ninh Binh Province, Vietnam 23 Cognitive Ethology Laboratory, German Primate Center, Leibniz Institute for Primate Research, Goettingen, Germany 24 Leibniz Science Campus Primate Cognition, Goettingen, Germany 25 Department of Primate Cognition, Georg‐August‐University, Goettingen, Germany 26 Department of Environmental Ecology, University of Science and Central Institute for Natural Resources and Environmental Studies, Vietnam National University, Hanoi, Vietnam 27 Life Sciences and Environment, Technology and Environment of Mahajanga, University of Mahajanga, Mahajanga, Madagascar 28 The Vertebrate Genomes Lab, The Rockefeller University, New York, New York, USA 29 Laboratory of Neurogenetics of Language, The Rockefeller University, New York, United States 30 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA 31 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China 32 Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China 33 Villum Center for Biodiversity Genomics, Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark 34 China National Genebank, BGI‐Shenzhen, Shenzhen, China 35 Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China 36 Artificial Intelligence Lab, Illumina Inc, San Diego, California, USA 37 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain 38 CNAG‐CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 39 Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, Barcelona, Spain 40 Department of Anthropology, New York University, New York, New York, USA 41 New York Consortium in Evolutionary Primatology, New York, New York, USA Correspondence Amanda D. Melin, Department of Abstract Anthropology and Archaeology, University of The novel coronavirus SARS‐CoV‐2, which in humans leads to the disease COVID‐ Calgary, 2500 University Dr, Calgary, Alberta T2N 1N4, Canada. 19, has caused global disruption and more than 2 million fatalities since it first Email: amanda.melin@ucalgary.ca emerged in late 2019. As we write, infection rates are at their highest point globally James P. Higham, Department of and are rising extremely rapidly in some areas due to more infectious variants. The Anthropology, New York University, 25 primary target of SARS‐CoV‐2 is the cellular receptor angiotensin‐converting Waverly Pl, New York, NY 10003, USA. Email: jhigham@nyu.edu enzyme‐2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, Funding information CERCA Programme del Departament the anticipated risk is not equal across the Order. Furthermore, some taxonomic d'Economia i Coneixement de la Generalitat de groups show high ACE2 amino acid conservation, while others exhibit high varia- Catalunya, Grant/Award Number: GRC 2017 SGR 880; Secretaria d'Universitats i Recerca; bility at this locus. As an example of the latter, analyses of strepsirrhine primate Spanish Ministry of Science and Innovation ‐ ACE2 sequences to date indicate large variation among lemurs and lorises compared Instituto de Salud Carlos III; Rockefeller University; Centro de Excelencia Severo to other primate clades despite low sampling effort. Here, we report ACE2 gene and Ochoa; Natural Sciences and Engineering protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 Research Council of Canada (NSERC Discovery Grant); Generalitat de Catalunya genera. Our study reinforces previous results while finding additional variability in (Departament de Salut, Departament other strepsirrhine species, and suggests several clades of lemurs have high po- d'Empresa i Coneixement); National Institutes of Health, Grant/Award Number: tential susceptibility to SARS‐CoV‐2 infection. Troublingly, some species, including R35GM130333; CERCA, the rare and endangered aye‐aye (Daubentonia madagascariensis), as well as those in Grant/Award Numbers: BFU2017‐86471‐P (MINECO/FEDER, UE); European Research the genera Avahi and Propithecus, may be at high risk. Given that lemurs are en- Council (ERC) ‐ European Union's Horizon demic to Madagascar and among the primates at highest risk of extinction globally, 2020 research and innovation programme, Grant/Award Number: 864203; Smart Growth further understanding of the potential threat of COVID‐19 to their health should be Operating Program (2014–2020); a conservation priority. All feasible actions should be taken to limit their exposure MINECO/FEDER, UE, Grant/Award Number: CGL2017‐82654‐P; Unidad de Excelencia to SARS‐CoV‐2. María de Maeztu, Grant/Award Number: MELIN ET AL. | 3 of 12 AEI (CEX2018‐000792‐M); Canada Research Chairs Program; European Regional Development Fund, Grant/Award Numbers: MINECO/FEDER, BIO2015‐71792‐P; Howard Hughes Medical Institute; Obra Social "La Caixa"; Natural Environment Research Council, Grant/Award Number: NERC NE/T000341/1; Howard Hughes International Early Career 1 | INTRODUCTION These findings raise questions about the variability and molecular evolution of the ACE2 gene across Strepsirrhini, as well as about the On Friday, January 15, 2021, the two‐millionth human death offi- potential susceptibility to initial infection by SARS‐CoV‐2 of different cially attributed to COVID‐19 was documented by the Johns Hop- species across the suborder. Here, we expand substantially on previous kins University Coronavirus Resource Center (Dong et al., 2020; reports of strepsirrhine ACE2 variation (Melin et al., 2020). We report Santora & Wolfe, 2021). Since this date, the rates of infection by the 71 ACE2 gene sequences, including 66 from unpublished strepsirrhine virus responsible for this disease, SARS‐CoV‐2, have increased in genomes, spanning 39 lemuriform and 12 lorisiform species. For residue most countries. As we write, we are reaching new global highs in variants that have not been previously identified and assessed, active cases and witnessing the spread of new, more transmissible we model the interactions between the translated ACE2 protein and variants (Mahase, 2020; World Health Organization, 2020). As co- the receptor‐binding domain of the SARS‐CoV‐2 spike protein to pre- ordinated efforts within and across institutions, countries, and con- dict the susceptibility of species to initial infection by SARS‐CoV‐2. In tinents seek to identify treatments, develop vaccines, and curb the doing so, we seek to improve our understanding of ACE2 variation and spread of this highly contagious virus, attention has also turned to evolution, and to identify which strepsirrhine species are likely to be the potential risks posed to nonhuman species (Damas et al., 2020; most at risk from the COVID‐19 pandemic. We recognize that disease Liu et al., 2021; Melin et al., 2020; Wu et al., 2020). Zoonotic transfer development, progression, and pathogenesis in any given species will of diseases from humans to nonhuman primates poses a major risk also be impacted by factors influencing the efficacy of viral cellular given the many physiological and genetic similarities shared within entry and taxon‐specific immune responses (Hoffmann et al., 2020; Li the Order Primates, and is a potentially grave risk to already en- et al., 2021; Lukassen et al., 2020). Nonetheless, our hope is that our dangered and fragmented populations (Gillespie & Leendertz, 2020). analysis of the initial susceptibility of different lemur and loris species to In recent studies, the susceptibility of primates and other mammals SARS‐CoV‐2 infection will help to inform decisions on how best to to potential SARS‐CoV‐2 infection has been assessed by analysis of the proceed with strepsirrhine research and management programs. gene sequences that code for the primary viral target, angiotensin‐ converting enzyme‐2 (ACE2; Damas et al., 2020; Delgado Blanco et al., 2020; Liu et al., 2021; Melin et al., 2020). Receptor‐virus inter- 2 | ME THO D S action models, including by members of the present authorship, have highlighted the likely susceptibility of many species, especially of apes 2.1 | Study species and monkeys of Africa and Asia (Parvorder Catarrhini). Meanwhile, monkeys in the Americas (Parvorder Platyrrhini) are predicted to ex- We examine the ACE2 gene sequence of 71 individual strepsirrhines— hibit lower susceptibility (Liu et al., 2021; Melin et al., 2020). One 66 newly sequenced individuals as part of the Primate Variation striking feature of these analyses is the uniformity within these par- Genome Consortium (in preparation; Table S1), plus five obtained from vorders. Across the identified primary viral binding sites, catarrhines publicly available genomes: Otolemur garnettii (Northern greater galago), exhibit one set of amino acid residues, and platyrrhines another. Sur- accession no: XM_003791864.2, gene ID: 100951881; Propithecus co- prisingly, although gene sequences are only publicly available for a few quereli (Coquerel's sifaka), accession no: XM_012638731.1, gene ID: strepsirrhine species (four lemurs and one galago), ACE2 variation in 105805773; Microcebus murinus (gray mouse lemur) accession no: that suborder far exceeds variation present in the rest of the primate XM_020285237.1, gene ID: 105882317; Eulemur flavifrons (blue‐eyed taxa examined to date (24 species spanning 21 genera and including black lemur), accession no: LGHW01000591.1, scaffold 590 (gene tarsiers (1), platyrrhines (6), and catarrhines (14)). Of particular concern identified via BLAST); Daubentonia madagascariensis, (aye‐aye) accession is the high sequence similarity at binding sites to human ACE2 of some no: PVJZ01006595.1, scaffold 13170, (gene identified via BLAST). In lemur ACE2 proteins, including aye‐ayes (Daubentonia madagascariensis) total, we analyze the ACE2 gene and protein sequences of 51 species and Coquerel's sifakas (Propithecus coquereli), which exhibited residues (39 Lemuriformes and 12 Lorisiformes) and 19 genera (12 Lemur- that are far more similar to those of humans and other catarrhines than iformes and 7 Lorisiformes; Table 1 and S1). The number of individuals to those present in platyrrhines (monkeys of the Americas; Melin sampled per species ranges from 1 to 4, with 13 species having at least et al., 2020). 2 individuals sampled. The number of samples per genus ranges from 4 of 12 | MELIN ET AL. 1 to 17, with 13 genera having at least 2 individuals sampled (Table S1). Massova & Kollman, 1999). Alanine scanning is a commonly used In addition, we model the impact of residues at binding sites recently method, and alanine is chosen because it is the smallest residue that reported for Indri indri ACE2 protein (Damas et al., 2020), adding an- may be incorporated without significantly impacting the protein other genus of lemur to our survey. Detailed statistics for these newly backbone conformation (Kortemme et al., 2004). We defined critical generated genomes will be published upon their full release. ACE2 gene residues as those that, upon mutation to alanine, decrease the and protein sequences used in this study are included in Supplementary binding energy by a threshold value ΔΔGbind ≥ 1.0 kcal/mol. Nine Materials. We confirm that this study followed all applicable laws and sites meet this criterion (Table S3). To be conservative, we also ex- regulations of the countries in which it was conducted, that it was amined amino acid variation at additional sites that were identified approved by all appropriate institutional committees, and that it con- as important by different but complementary methods: cryo‐EM and formed to the American Society of Primatologists Principles for the X‐ray crystallography structural analysis (Lan et al., 2020; Shang Ethical Treatment of Non Human Primates. et al., 2020; Wang et al., 2020; Yan et al., 2020). While some of these sites overlap with the critical sites we identified using alanine scanning, three do not—alanine scanning also identifies these as 2.2 | Gene alignments binding sites, but with ΔΔGbind < 1.0 kcal/mol. To be conservative in the present analyses, as in Melin et al., 2020, we added these three We mapped reads from whole‐genome sequence (WGS) data to the sites to our nine sites for a total of 12 critical sites (Table S3). All closest available annotated reference assembly from among the set of computational alanine scanning mutagenesis analyses were per- partially unpublished (unp.) references (Daubentonia madagascariensis, formed using Rosetta (Jochim & Arora, 2010; Kortemme et al., 2004; (unp.) Galago moholi (unp.), Propithecus coquereli (GCF_000956105.1), Raj et al., 2013). Lemur catta (unp.), Loris tardigradus (unp.), Microcebus murinus (unp.), To model how variation in the ACE2 amino acid sequences Nycticebus pygmaeus (unp.) and Otolemur garnettii (unp.). Briefly, after across species affects the relative binding energy of the ACE2/SARS‐ removing adapter sequences using cutadapt, we mapped the reads CoV‐2 interaction, we used the SSIPe program (Huang et al., 2020). using bwa mem and processed and sorted alignments using samtools. This algorithm mutates selected residues and compares the resulting We removed duplicates using biobambam, and added read groups for binding energy to that of human ACE2 bound to the SARS‐CoV‐2 variant calling using picard. We called variants using GATK4 Haploty- RBD as a benchmark (PDB 6M0J). We modeled the full suite of peCaller (v 4.1.6) following best practice pipelines (https://gatk. amino acid changes occurring at critical binding sites for all unique broadinstitute.org). After applying a set of standard hard filters ACE2 sequences. We further examined the predicted effect of each (Table S2), we extracted the coding regions of ACE2 gene sequences individual amino acid change (in isolation) on protein‐binding affinity and introduced homozygous alternative calls to create the putative to better understand each residue's contribution to variation in coding sequence of each individual. We extracted and aligned ACE2 binding affinity. gene sequences from the variant call files, which were then translated into protein sequences. The consensus sequences were manually in- spected and corrected where needed to remove gene‐flanking regions. 3 | RES ULTS We manually verified the absence of indels and premature stop codons for each individual. We then aligned these 66 amino acid sequences We examined variation along the length of the ACE2 protein se- using MAFFT (default settings) with those extracted from publicly quence for the 66 newly sequenced individuals and the ACE2 se- available genomes (Melin et al., 2020). The full nucleotide and amino quences from the five genomes available at NCBI. Sequences are acid alignments used here are provided as tab‐delimited text files in the conserved within, but are variable across, strepsirrhine genera. The Supporting Information. Following alignment, we examined amino acid mean pairwise amino acid sequence identity along the length of the sequence variation within and across species along the length of the ACE2 protein within genera is 99.25% (mean amino acid substitu- ACE2 protein, and specifically at the sites that are critical for SARS‐ tions = 5.85). The mean amino acid sequence identity between lemur CoV‐2 binding. genera was 91.67%, and between lorisiform genera was 92.72%. When we focused solely on the critical binding sites, the pairwise amino acid identity at binding sites within genera was 100%, in- 2.3 | Variation in ACE2 sequences at critical sites dicating an absence of any amino acid variation among species in the and impact on SARS‐CoV‐2 binding same genus in our study. Differences between genera are also pre- sent at the critical binding sites (Figure 1), especially among lemurs, Our method for identifying critical contact sites between the ACE2 where the mean pairwise amino acid sequence identity is 83.18%. protein and the receptor‐binding domain (RBD) of the SARS‐CoV‐2 The mean pairwise amino acid sequence identity at binding sites spike protein is detailed in Melin et al. (2020). Briefly, we conducted between lorisiform genera is 95.04%. alanine scanning mutagenesis to assess the contribution of each We found three novel ACE2 variants not previously reported human ACE2 residue to protein‐protein complex formation with the for any primate at three critical binding sites: H24 (Arctocebus, SARS‐CoV‐2 RBD (Bogan & Thorn, 1998; Kortemme & Baker, 2002; Perodicticus), F83 (Galagoides), and Q353 (Cheirogaleus). MELIN ET AL. | 5 of 12 T A B L E 1 Results of computational protein‐protein interaction models predicting the impact of amino acid changes, relative to human ACE2 residues, at critical binding sites with SARS‐CoV‐2 receptor binding domain (RBD). Impacts of changes across the full complement of critical binding sites are presented in (A), single residue replacements are presented in (B) (A) Estimated decrease in Species Mutations (relative to human sequence) ΔΔG (kcal/mol)a binding efficiencyb Homo sapiens 0 Avahi, Daubentonia, Propithecus M82T +0.9 5 fold Eulemur, Hapalemur, Lemur, Prolemur, Q24E, M82T +1.4 10 fold Varecia Indri H34N, M82N +2.7 100 fold Monkeys (Americas) Y41H, Q42E, M42T +3.5 400 fold Galago, Loris, Nycticebus, Otolemur H34R, D38E, Y41H, M82T +3.8 600 fold Lepilemur, Microcebus, Mirza D30E, H34N, Y41H, M82T +4.0 800 fold Arctocebus, Perodicticus Q24H, H34R, D38E, Y41H, M82T +4.2 1200 fold Galagoides H34R, D38E, Y41H, M82T, Y83F +4.4 1700 fold Cheirogaleus D30E, H34N, Y41H, M82T, K353Q +5.0 4500 fold Carlito H34Q, Y41H, M82S, K353N +5.5 10000 fold (B) Mutation ΔΔG (kcal/mol)a Q24L −0.7 Q24H 0.3 Q24E 0.5 D30E 0.7 H34R 0.8 H34N 1.0 H34L −0.5 H34Y −0.1 D38E 0.6 Y41H 1.9 Q42E 0.9 M82T 0.9 M82N 1.8 Y83F 0.6 K353Q 0.9 a Approximate predicted decrease in binding affinity of individual ACE2 proteins and SARS‐CoV‐2 RBD. b Mutations were analyzed with SSIPe server (https://zhanglab.ccmb.med.umich.edu/SSIPe/) and PDB file 6M0J. The remaining binding site variation is consistent with previous proteins are modeled to have a higher binding affinity to reports. The combination of residues (E24, T82) previously SARS‐CoV‐2 than the human (catarrhine) form (Table 1A). Among reported for Eulemur flavifrons, is also found in other species of the 12 critical sites, the substitutions causing the starkest drop in Eulemur, as well as in Hapalemur, Lemur, and Prolemur. The critical viral‐receptor binding affinity relative to the human sequence are binding site composition found in Indri indri (Damas et al., 2020) is Y41H and M82N (Table 1B). The former substitution is found in not found in any of the genera we sequenced here. The residues all lorisoids and in sportive, dwarf, mouse, and giant mouse at binding sites 37, 42, 355, and 357 are invariant across all lemurs. The latter substitution is only identified in Indri indri, strepsirrhines examined. None of the strepsirrhine ACE2 although a different substitution at the same site occurs in all 6 of 12 | MELIN ET AL. F I G U R E 1 Amino acid composition of the ACE2 protein at sites that are critical for interacting with the SARS‐CoV‐2 receptor binding domain across examined strepsirrhine genera, with respect to the human ACE2 sequence. The phylogenetic relationship among studied species is presented centrally, with branch lengths representing approximate evolutionary distances. (ACE2 = angiotensin‐converting enzyme‐2) other strepsirrhines (M82T). Other mutations had lesser effects 4 | DISCUSS ION (Table 1B). Looking at taxon‐specific predictions based on the entire com- We report ACE2 gene and protein sequences for 19 genera of plement of amino acids at critical binding sites, the species predicted strepsirrhine primates, spanning 51 species and 71 individuals, and to be most susceptible to SARS‐CoV‐2 infection are in the genera examine these together with the Indri indri ACE2 protein. We confirm Avahi, Propithecus, and Daubentonia (Table 1A). These taxa differ previous reports of the amino acid residue composition at viral from humans at only one critical binding site, M82T, which is pre- binding sites for Daubentonia, Propithecus, Eulemur, Microcebus, and dicted to lower the binding affinity between the ACE2 receptor and Otolemur (Damas et al., 2020; Melin et al., 2020). Additionally, we the SARS‐CoV‐2 virus by five‐fold. These genera are followed by identified three novel variants at the following key binding sites: species in the genera Eulemur, Lemur, Prolemur, and Varecia, which H24, F8, and Q353. These variants are modeled to be protective, are differ in one additional (Q24E) substitution, which should further not found in species reported in the previous analyses of a small lower the binding affinity by two‐fold. In potentially promising re- subset of strepsirrhine species (Melin et al., 2020), and were also not sults, we predict that the lorises, galagos, and dwarf, mouse, giant found among previous analyses of primates more generally. Relative mouse, and sportive lemurs are far less susceptible to infection than to variation seen in catarrhines and platyrrhines, strepsirrhine ACE2 humans. This is primarily due to a Y41H mutation (Figure 2), al- variation across genera at critical binding sites is remarkably high, though additional changes in amino acids at binding sites further especially among lemurs. In addition to reporting new ACE2 se- lower the affinity between their ACE2 and the RBD of the SARS‐ quences spanning many strepsirrhine species, we also provide the CoV‐2 spike protein. The decreases in the modeled binding affinity first examination of intraspecific variation in ACE2 sequences out- range from 0.9 ΔΔG (kcal/mol) (Avahi, Propithecus, Daubentonia; side of humans and vervet monkeys (Cao et al., 2020; Schmitt predicted most susceptible) to 5.0 ΔΔG (kcal/mol) (Cheirogaleus, et al., 2020; Stawiski et al., 2020). Unlike Schmitt et al. (2020), who predicted least susceptible), relative to human ACE2 (Table 1A). found an intraspecific polymorphism at a binding site (D30G), which MELIN ET AL. | 7 of 12 might impact susceptibility of vervets to SARS‐CoV‐2 at the in- dividual level, we find that ACE2 proteins are highly conserved within species and within genera, at least for the taxa examined. However, our intraspecific and intrageneric sample sizes (maximally n = 4 and n = 17, respectively) are small, and low‐to‐moderate levels of variation at ACE2 binding sites might be discovered as sampling increases. Still, our results broadly indicate that members of the same species and closely related species are likely to share similar initial susceptibility to SARS‐CoV‐2 infection. At higher taxonomic levels, there does appear to be a phylogenetic effect on susceptibility to SARS‐CoV‐2 infection among the strepsirrhines. Within families, there is broad sequence similarity at ACE2 critical binding sites. Accordingly, all unsampled species in the families Lemuridae and Indriidae are likely to be at high risk. The amino acid conservation within the family Cheirogaleidae (Cheirogaleus, Microcebus, Mirza) suggests that members of the genera Allocebus and Phaner, not sampled here, are likely at low risk. Similarly, we did not sample species in the genera Sciurocheirus, Euoticus, or Paragalago, but if they follow patterns of variation seen among other members of the family Galagidae (Galagoides, Galago, Otolemur) they should be at relatively low risk. However, given the difference in risk assessments within some members of the same family, for example, between Indri and the other Indriidae (Avahi and Propithecus), we caution the applica- tion of risk assessments across genera, especially when we do not have samples for representative species. As with all studies based on predictive modeling, our results require experimental validation and should be interpreted with caution, especially those results which predict that some strepsir- rhines might be at a lower risk. Additional limitations include that our study examined variation at sites identified to be critical for SARS‐ CoV‐2 viral binding, but did not assess the impact of residues that are not in direct contact with the virus and which may still affect binding allosterically. In addition, we did not examine genetic varia- tion or model the function of the protease (TMPRSS2), which facil- itates viral entry post binding (Hoffmann et al., 2020), and which is anticipated to impact disease progression. We also emphasize that our approach investigates the likely initial susceptibility of species to SARS‐CoV‐2 infection. The severity of viral infection responses may differ between species and is related to variation in immune and F I G U R E 2 Model of human ACE2 in complex with SARS‐CoV‐ other responses (Lukassen et al., 2020). Nonetheless, the results of in 2 RBD is presented in Panel (a). Three of the key ACE2 interfacial vivo infection studies conducted on haplorhine primates and other residues are highlighted in Panel (b). The dashed lines indicate mammals strongly support the predictions of protein‐protein inter- predicted hydrogen bonding interactions. Interactions at three of action models about the susceptibility of different species to SARS‐ the critical binding sites (41, 42, and 82) are shown for humans CoV‐2 and the development of COVID‐19‐like symptoms (Blair and other catarrhines (i.e., apes and monkeys in Africa and Asia). et al., 2020; Lu et al., 2020; Rockx et al., 2020; Shan et al., 2020; Shi Changes in amino acids diminish these binding interactions. For example, substitution of tyrosine with histidine at site 41 in Panel et al., 2020), supporting the applicability of our results. An additional (c) decreases the viral‐RBD binding affinity by removal of the tangible contribution of our study is that it provides novel sequence potential hydrogen‐bonding interactions with T500 and N501. data that can be used in site‐directed mutagenesis to recreate taxon‐ (ACE2 = angiotensin‐converting enzyme‐2; RBD = receptor‐ specific ACE2 proteins for cellular assays (Guy et al., 2005). At the binding domain) same time, results predicting high susceptibility among a large number of genera are sufficiently alarming as to warrant special care and attention when interacting with these species in wild and captive management settings, including zoological parks, where humans 8 of 12 | MELIN ET AL. frequently come into contact with vulnerable species (e.g., in walk‐ bodied lemurs (e.g., mouse lemurs)—to SARS‐CoV‐2 infection may be through lemur exhibits). substantially lower. Although the lorisoids as a group are more The predictions of our study suggest that, among the strepsir- geographically widespread and (in relative terms) of lower con- rhines, the lemurs of the families Indriidae, Daubentoniidae, and to a servation concern than the lemurs, many remain threatened and face lesser extent Lemuridae, are likely to be particularly vulnerable to critical challenges to their survival. Their cryptic, nocturnal habits SARS‐CoV‐2 infection. Lemurs are considered to be among the most may make the impact of emerging infectious diseases especially threatened vertebrates globally, with over 94% of extant species difficult to monitor in wild populations. Nonetheless, lower predicted being threatened with extinction (IUCN, 2021; Schwitzer susceptibility to SARS‐CoV‐2 is a potentially positive result. et al., 2014). High rates of deforestation which result from changing Our results are also likely to be of interest and significance for land‐use patterns coupled with high human population growth are zoos and captive research facilities around the world that house among the most potent threats to lemur populations (Elmqvist lemurs, lorises, and galagos. Given the close contact with humans in et al., 2007; Harper et al., 2007). Besides habitat loss, these human‐ such environments (often indoors), extra precautions may be ne- induced disturbances are exposing wild lemur populations to novel cessary, especially when interacting with the likely most at‐risk interactions with humans and domestic animals, and in so doing in- lemurs. These may include many of the measures suggested for creasing risks of disease outbreaks (Barrett et al., 2013). Current those interacting with great ape and other catarrhine populations knowledge of the disease ecology of wild lemurs suggests that po- (Gilardi et al., 2015; Melin et al., 2020), such as: regular testing for pulations that are found in disturbed habitats and those living in SARS‐CoV‐2, requiring face masks for human researchers and areas with a high volume of tourists are at elevated risk of bearing caretakers, imposing quarantines on all individuals ahead of con- pathogens found in humans, livestock, and other domestic animals tact, and disinfecting clothes and footwear. The risk of COVID‐19 (Bublitz et al., 2015; Junge et al., 2011; Rasambainarivo et al., 2013). to nonhuman primates has been demonstrated in clinical trials (Lu Furthermore, lemurs in general, including many of the larger‐bodied et al., 2020) and recently exemplified when members of a captive species which are predicted to be most at risk of infection by SARS‐ gorilla group tested positive for SARS‐CoV‐2 after exposure to an CoV‐2, are highly vulnerable to new diseases because they are asymptomatic keeper, and exhibited respiratory symptoms similar considered to be immunologically naïve, and are unlikely to persist to COVID‐19 (San Diego Zoo, 2021). It is more difficult to assess through a major epidemic outbreak (Junge, 2007). the onset of and potential for other symptoms, such as anosmia, but Historically, conservationists in Madagascar have strived to im- loss of olfactory abilities could be devastating to lemurs and other plement integrative conservation programs to protect the biological primates which rely on their sense of smell to communicate, avoid uniqueness of the island while leveraging the sustainable develop- predators, and select foods. Regardless of where species fall in the ment of local communities (e.g., Birkinshaw et al., 2013; continuum of potential risk that we have presented here, we stress Corson, 2017; Dolins et al., 2010; Wright et al., 2012). In addition to that it is prudent to take all feasible precautions when interacting the considerable conservation obstacles exacerbated by frequent with any primate. political unrest and extreme poverty (Schwitzer et al., 2014), in- Over the past year, scientists have mobilized with remarkable itiatives in Madagascar now have the concern of a viral pandemic in speed and effectiveness to address the COVID‐19 pandemic's im- the human population, which appears likely to pose a direct and pacts on humans, from developing and rolling out new tests, to de- serious risk to many lemurs. Recently, there has been an emergence signing and trialing vaccine candidates. The pandemic also raises of integrated conservation programs that include a human health major new challenges for field primatologists and other biologists, component (e.g., Garchitorena et al., 2018; Mohan & Shellard, 2014), zookeepers, conservationists, and all those interested in the survival a necessary shift in a country with one of the lowest levels of and welfare of primates (Bales, 2020; Douglas et al., 2020; Olival healthcare system financing in the world (Barmania, 2015). This is et al., 2020). Conservation efforts have already been severely im- particularly urgent given that COVID‐19 could potentially infect up peded by the pandemic. National and global lock‐downs have made it to 30% of the human population (Evans et al., 2020). Furthermore, difficult or impossible for conservationists, primatologists, and wild- Madagascar lacks a legal framework to guide best research practices life patrol teams to enter their field sites. Governments are pre- to limit exposure of lemur populations to diseases (in contrast, e.g., to occupied with efforts to curb the pandemic, and in some cases great apes; see Gilardi et al., 2015). It is important that all stake- conservation funding support has been reduced due to financial holders involved in the conservation of lemurs coordinate to draft difficulties and new priorities. While avoiding all contact with wild such guidelines, which should include safeguards against the close primates may be ideal from a zoonotic disease containment per- contact between people and lemurs that occur as part of the suc- spective, it is unlikely to be possible in practice, nor in the overall cessful and widespread community‐based conservation programs. interest of their conservation (Reid, 2020; Trivedy, 2020). While we Such a “One Health” approach is especially important during a global hope that our results will serve to inform conservation efforts, re- pandemic. At a minimum, these concerns should be carefully con- solving the pressures that have resulted from the pandemic will re- sidered for the species identified as most susceptible to SARS‐CoV‐2. quire input from stakeholders with complementary ethical, scientific, In potentially more promising news, our results indicate that the and socioeconomic perspectives. It is our hope that as a community, vulnerability of the lorisoids—and indeed of many of the small‐ we can rise to the challenge. MELIN ET AL. | 9 of 12 A C K N O W L E D GM E N T S original draft (equal); writing‐review & editing (equal). Alejandro We thank the people in the Primate Genome Variation Consortium Valenzuela: funding acquisition (supporting); investigation (equal); and BGI Primate Reference Consortium for allowing us to publish software (lead); writing review & editing (supporting). Lukas partial gene information early due to the urgency for COVID‐19 Kuderna: funding acquisition (supporting); investigation (equal); research. We thank all persons and groups who shared samples for methodology (equal); software (lead); writing original draft (sup- this study, including many samples from the Duke Lemur Center. porting); writing review & editing (supporting). Frank Marrone III: This is Duke Lemur Center publication #1472. ADM is supported by Formal analysis (lead); funding acquisition (supporting); methodology the Natural Sciences and Engineering Research Council of Canada (supporting); visualization (lead); writing‐review & editing (support- (NSERC Discovery Grant) and Canada Research Chairs Program. ing). Hasinala Ramangason: funding acquisition (supporting); writing MCJ's postdoctoral appointment is supported by funding from the original draft (equal); writing review & editing (supporting). Julie E. Natural Environment Research Council (NERC NE/T000341/1). IG Horvath: funding acquisition (supporting); resources (supporting); and MG acknowledge the support of the Spanish Ministry of Science writing review & editing (supporting). Christian Roos: funding ac- and Innovation through the Instituto de Salud Carlos III and the quisition (supporting); resources (supporting); writing review & 2014–2020 Smart Growth Operating Program, to the EMBL part- editing (supporting). Andrew Kitchener: funding acquisition (sup- nership and cofinancing with the European Regional Development porting); resources (supporting); writing review & editing (support- Fund (MINECO/FEDER, BIO2015‐71792‐P). We also acknowledge ing). Chiea Chuen Khor: funding acquisition (supporting); resources the support of the Centro de Excelencia Severo Ochoa, and the (supporting); writing review & editing (supporting). Weng Khong Lim: Generalitat de Catalunya through the Departament de Salut, De- funding acquisition (supporting); resources (supporting); writing re- partament d'Empresa i Coneixement and the CERCA Programme. view & editing (supporting). Jessica G. H. Lee: funding acquisition TMB is supported by funding from the European Research Council (supporting); resources (supporting); writing review & editing (sup- (ERC) under the European Union's Horizon 2020 research and in- porting). Patrick Tan: funding acquisition (supporting); resources novation program (grant agreement No. 864203), BFU2017‐86471‐ (supporting); writing review & editing (supporting). Govindhaswamy P (MINECO/FEDER, UE), “Unidad de Excelencia María de Maeztu”, Umapathy: funding acquisition (supporting); resources (supporting); funded by the AEI (CEX2018‐000792‐M), Howard Hughes Interna- writing‐review & editing (supporting). Muthuswamy Raveendran: tional Early Career, Obra Social "La Caixa" and Secretaria d'Uni- funding acquisition (supporting); investigation (supporting); writing versitats i Recerca and CERCA Programme del Departament review & editing (supporting). R. Alan Harris: funding acquisition d'Economia i Coneixement de la Generalitat de Catalunya (GRC (supporting); investigation (supporting); resources (supporting); 2017 SGR 880). PSA thanks the National Institutes of Health writing review & editing (supporting). Ivo Gut: funding acquisition (R35GM130333) for financial support. EL is supported by CGL2017‐ (supporting); investigation (supporting); resources (supporting); 82654‐P (MINECO/FEDER, UE). EDJ and OF's contributions were writing review & editing (supporting). Marta Gut: funding acquisition supported by funds from Howard Hughes Medical Institute and the (supporting); investigation (supporting); writing review & editing Rockefeller University. Chris Smith drew the images for Figure 1. The (supporting). Esther Lizano: funding acquisition (supporting); in- authors would like to thank the Veterinary and Zoology staff at vestigation (supporting); writing review & editing (supporting). Tilo Wildlife Reserves Singapore for their help in obtaining the tissue Nadler: funding acquisition (supporting); resources (supporting); samples, as well as the Lee Kong Chian Natural History Museum for writing review & editing (supporting). Dietmar Zinner: funding ac- storage and provision of the tissue samples. Finally, we thank two quisition (supporting); resources (supporting); writing review & reviewers for quick and constructive comments, and Karen Bales for editing (supporting). Minh D. Le: funding acquisition (supporting); her supportive editorial oversight. resources (supporting); writing review & editing (supporting). Siva- kumara Manu: Funding acquisition (supporting); resources (sup- C O NF L IC T O F IN T E R ES T S porting); writing‐review & editing (supporting). Clément J. The authors declare that there are no conflict of interests. Rabarivola: Funding acquisition (supporting); resources (supporting); writing‐review & editing (supporting). Alphonse Zaramody: funding AUTHOR CONTRIBUTIONS acquisition (supporting); resources (supporting); writing review & Amanda Melin: conceptualization (lead); formal analysis (support- editing (supporting). Nicole Andriaholinirina: Funding acquisition ing); funding acquisition (supporting); methodology (lead); project (supporting); resources (supporting); writing‐review & editing (sup- administration (lead); supervision (lead); validation (lead); writing‐ porting). Steig Johnson: Funding acquisition (supporting); resources original draft (lead); writing‐review & editing (lead). Joseph D. Orkin: (supporting); writing original draft (supporting); writing review & conceptualization (equal); data curation (lead); formal analysis (lead); editing (supporting). Erich D. Jarvis: Funding acquisition (supporting); funding acquisition (supporting); investigation (lead); project admin- investigation (supporting); resources (supporting); writing review & istration (supporting); software (supporting); visualization (lead); editing (supporting). Olivier Fedrigo: funding acquisition (support- writing‐original draft (equal); writing‐review & editing (equal). ing); investigation (supporting); resources (supporting); writing re- Mareike C. Janiak: data curation (lead); formal analysis (lead); view & editing (supporting). Dongdong Wu: funding acquisition funding acquisition (supporting); investigation (equal); writing‐ (supporting); investigation (supporting); resources (supporting); 10 of 12 | MELIN ET AL. writing review & editing (supporting). Guojie Zhang: funding acqui- Tomas Marques‐Bonet https://orcid.org/0000-0002-5597-3075 sition (supporting); investigation (supporting); resources (support- Arcadi Navarro https://orcid.org/0000-0003-2162-8246 ing); writing review & editing (supporting). Kyle Kai‐How Farh: David Juan https://orcid.org/0000-0003-1912-9667 funding acquisition (lead); resources (supporting); writing review & Paramjit S. Arora https://orcid.org/0000-0001-5315-401X editing (supporting). Jeffrey Rogers: funding acquisition (lead); James P. Higham https://orcid.org/0000-0002-1133-2030 resources (supporting); writing original draft (supporting); writing review & editing (supporting). Tomas Marques‐Bonet: con- REFER E NC ES ceptualization (supporting); funding acquisition (lead); project ad- Bales, K. L. (2020). Special issue: Primates and COVID‐19. American ministration (lead); resources (lead); supervision (lead); writing Journal of Primatology, 82(8), e23174. Barmania, S. (2015). Madagascar's health challenges. The Lancet, original draft (supporting); writing review & editing (supporting). 386(9995), 729–730. Arcadi Navarro: funding acquisition (supporting); methodology Barrett, M. A., Brown, J. L., Junge, R. E., & Yoder, A. D. (2013). Climate (equal); supervision (lead); writing review & editing (supporting). change, predictive modeling and lemur health: Assessing impacts of David Juan: funding acquisition (supporting); methodology (equal); changing climate on health and conservation in Madagascar. Biological Conservation, 157, 409–422. supervision (equal); writing review & editing (supporting). Paramjit S. Birkinshaw, C., Lowry, P. P., II, Raharimampionona, J., & Aronson, J. Arora: formal analysis (lead); funding acquisition (supporting); (2013). 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