Evolution and origins of SARS- CoV-2 and related coronaviruses Introduction to Evolution and Scientific Inquiry Spring 2020, Dr. Spielman Zoonosis Image from April 2019

• Virus that normally infects one animal species infects a NEW/DIFFERENT species • Sometimes from a new mutation • Sometimes from an existing mutation • Sometimes no mutation at all – it just can infect multiple species Viruses bind host receptors or other cell-surface proteins

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000217 ACE2: Angiotension-converting enzyme 2

• ACE constricts blood flow • ACE inhibitors relax veins to improve blood flow. BRIEF COMMUNICATION Used to treat… https://doi.org/10.1038/s41591-020-0868-6 • Heart disease • Angiotension SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate • Diabetes immune genes

• Migraines Waradon Sungnak! !1 , Ni Huang1, Christophe Bécavin! !2, Marijn Berg3,4, Rachel Queen5, Monika Litvinukova1,6, Carlos Talavera-López1, Henrike Maatz6, Daniel Reichart7, • Chronic kidney diseases Fotios Sampaziotis! !8,9,10, Kaylee B. Worlock11, Masahiro Yoshida! !11, Josephine L. Barnes11 and HCA Lung Biological Network*

We investigated SARS-CoV-2 potential tropism by survey- associated with SARS-CoV-2 pathogenesis at cellular resolution, inghttps:// expressionwww.nature.com of viral entry-associated/articles/s41591 genes in single-cell using-020 single-cell-0868 RNA- 6sequencing (scRNA-seq) datasets from healthy RNA-sequencing data from multiple tissues from healthy donors generated by the Human Cell Atlas consortium and other human donors. We co-detected these transcripts in specific resources to inform and prioritize the use of precious, limited clinical respiratory, corneal and intestinal epithelial cells, potentially material that is becoming available from COVID-19 patients. explaining the high efficiency of SARS-CoV-2 transmission. We investigated gene expression of ACE2 in multiple scRNA-seq These genes are co-expressed in nasal epithelial cells with datasets from different tissues, including those of the respiratory genes involved in innate immunity, highlighting the cells’ tree, cornea, retina, esophagus, ileum, colon, heart, skeletal muscle, potential role in initial viral infection, spread and clearance. spleen, liver, placenta/decidua, kidney, testis, pancreas, prostate The study offers a useful resource for further lines of inquiry gland, brain, skin and fetal tissues. We note that studies may lack with valuable clinical samples from COVID-19 patients and we specific cell types due to their sparsity, the challenges associated provide our data in a comprehensive, open and user-friendly with isolation or analysis methodology. Moreover, expression may fashion at www.covid19cellatlas.org. be under-detected due to technical dropout effects. Thus, while The coronavirus disease 2019 (COVID-19) is caused by positive (presence) results are highly reliable, absence should be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. interpreted with care. Detection of the virus was first reported in Wuhan2, China and has ACE2 expression was generally low in all analyzed datasets. since spread worldwide, emerging as a global pandemic3. Consistently with independent studies10,11, ACE2 was expressed in In symptomatic patients, nasal swabs have yielded higher viral cells from multiple tissues, including airways, cornea, esophagus, loads than throat swabs4. The same distribution was observed in an ileum, colon, liver, gallbladder, heart, kidney and testis (Fig. 1a; first asymptomatic patient4, implicating the nasal epithelium as a portal column). TMPRSS2 was highly expressed with a broader distribu- for initial infection and transmission. Cellular entry of coronavi- tion (Fig. 1a; second column), suggesting that ACE2, rather than ruses depends on the binding of the spike (S) protein to a specific TMPRSS2, may be a limiting factor for viral entry at the initial infec- cellular receptor and subsequent S protein priming by cellular tion stage. Cells from the respiratory tree, cornea, esophagus, ileum, proteases. Similarly to SARS-CoV5,6, SARS-CoV-2 employs ACE2 colon, gallbladder and common bile duct expressed both genes in as a receptor for cellular entry. The binding affinity of the S pro- the same cell (Fig. 1a; third column). We also assessed ACE2 and tein and ACE2 was found to be a major determinant of SARS-CoV TMPRSS2 expression in developmental datasets from fetal tissues, replication rate and disease severity4,7. Viral entry also depends on including liver, thymus, skin, bone marrow, yolk sac and lung, and TMPRSS2 protease activity and cathepsin B/L activity may be able found little to no expression of ACE2 in all but fetal liver and thymus to substitute for TMPRSS27. (Fig. 1a) where there was no co-expression with TMPRSS2 (data ACE2 and TMPRSS2 have been detected in both nasal and bron- not shown) except for a cluster of medullary thymic epithelial cells chial epithelium by immunohistochemistry8. Gene expression of ACE2 (Fig. 1a). ACE2 expression is noticeable in certain cell types in pla- and TMPRSS2 has been reported to occur largely in alveolar epithelial centa/decidua without TMPRSS2 (Fig. 1a). Additional fetal data type II cells9–11, which are central to SARS-CoV pathogenesis, whereas across relevant tissues and stages are needed to determine the gen- a different study reported the absence of ACE2 in the upper airway12. erality of these findings. To clarify the expression patterns of ACE2 and TMPRSS2, we ana- To further characterize specific epithelial cell types expressing lyzed their expression and the expression of other genes potentially ACE2, we evaluated ACE2 expression within the lung and airway

1Wellcome Sanger Institute, Cambridge, UK. 2Université Côte d’Azur, CNRS, IPMC, Sophia-Antipolis, France. 3Department of Pathology and Medical Biology, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands. 4Groningen Research Institute for Asthma and COPD, University Medical Centre Groningen, University of Groningen, Groningen, the Netherlands. 5Bioinformatics Core Facility, Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle-upon-Tyne, UK. 6Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany. 7Department of Genetics, Harvard Medical School, Boston, MA, USA. 8Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. 9Department of Medicine, Addenbrookes Hospital, Cambridge, UK. 10Cambridge Liver Unit, Cambridge University Hospitals, Cambridge, UK. 11UCL Respiratory, Division of Medicine, University College London, London, UK. *A list of authors and their affiliations appears at the end of the paper. e-mail: [email protected]; [email protected]

NATURE MEDICINE | www.nature.com/naturemedicine Structure of ACE2 bound to SARS-CoV-2

https://doi.org/10.1101/2020.02.19.956235 Recall “Red Queen Hypothesis” (Evolutionary Arms Race)

“Now, here, you see, it takes all the running you can do, to keep in the same place.” -Lewis Caroll https://en.wikipedia.org/wiki/Red_Queen_hypothesis https://jvi.asm.org/content/90/7/3280 correspondence

of laboratory escapes of SARS-CoV28. sequence helped reveal key RBD mutations Published online: 17 March 2020 We must therefore examine the possibility and the polybasic cleavage site. https://doi.org/10.1038/s41591-020-0820-9 of an inadvertent laboratory release of The genomic features described here SARS-CoV-2. may explain in part the infectiousness and References In theory, it is possible that SARS-CoV-2 transmissibility of SARS-CoV-2 in humans. 1. Zhou, P. et al. Nature https://doi.org/10.1038/s41586-020-2012-7 (2020). acquired RBD mutations (Fig. 1a) Although the evidence shows that SARS- 2. Wu, F. et al. Nature https://doi.org/10.1038/s41586-020-2008-3 during adaptation to passage in cell CoV-2 is not a purposefully manipulated (2020). culture, as has been observed in studies of virus, it is currently impossible to prove 3. Gorbalenya, A. E. et al. bioRxiv https://doi. 11 org/10.1101/2020.02.07.937862 (2020). SARS-CoV . The finding of SARS-CoV- or disprove the other theories of its origin 4. Jiang, S. et al. Lancet https://doi.org/10.1016/S0140- like coronaviruses from pangolins with described here. However, since we observed 6736(20)30419-0 (2020). nearly identical RBDs, however, provides all notable SARS-CoV-2 features, including 5. Dong, E., Du, H. & Gardner, L. Lancet Infect. Dis. https://doi. org/10.1016/S1473-3099(20)30120-1 (2020). a much stronger and more parsimonious the optimized RBD and polybasic cleavage 6. Corman, V. M., Muth, D., Niemeyer, D. & Drosten, C. Adv. Virus explanation of how SARS-CoV-2 acquired site, in related coronaviruses in nature, we Res. 100, 163–188 (2018). these via recombination or mutation19. do not believe that any type of laboratory- 7. Wan, Y., Shang, J., Graham, R., Baric, R. S. & Li, F. J. Virol. https:// The acquisition of both the polybasic based scenario is plausible. doi.org/10.1128/JVI.00127-20 (2020). 8. Walls, A. C. et al. bioRxiv https://doi. cleavage site and predicted O-linked More scientific data could swing the org/10.1101/2020.02.19.956581 (2020). glycans also argues against culture-based balance of evidence to favor one hypothesis 9. Wrapp, D. et al. Science https://doi.org/10.1126/science.abb2507 scenarios. New polybasic cleavage sites over another. Obtaining related viral (2020). 10. Letko, M., Marzi, A. & Munster, V. Nat. Microbiol. https://doi. have been observed only after prolonged sequences from animal sources would be org/10.1038/s41564-020-0688-y (2020). passage of low-pathogenicity avian influenza the most definitive way of revealing viral 11. Sheahan, T. et al. J. Virol. 82, 2274–2285 (2008). virus in vitro or in vivo17. Furthermore, origins. For example, a future observation 12. Nao, N. et al. MBio 8, e02298-16 (2017). 13. Chan, C.-M. et al. Exp. Biol. Med. 233, 1527–1536 (2008). a hypothetical generation of SARS-CoV-2 of an intermediate or fully formed polybasic 14. Follis, K. E., York, J. & Nunberg, J. H. Virology 350, 358–369 by cell culture or animal passage would cleavage site in a SARS-CoV-2-like virus (2006). have required prior isolation of a progenitor from animals would lend even further 15. Menachery, V. D. et al. J. Virol. https://doi.org/10.1128/JVI.01774- 19 (2019). virus with very high genetic similarity, support to the natural-selection hypotheses. 16. Alexander, D. J. & Brown, I. H. Rev. Sci. Tech. 28, 19–38 (2009). which has not been described. Subsequent It would also be helpful to obtain more 17. Ito, T. et al. J. Virol. 75, 4439–4443 (2001). generation of a polybasic cleavage site would genetic and functional data about SARS- 18. Bagdonaite, I. & Wandall, H. H. Glycobiology 28, 443–467 (2018). 19. Cui, J., Li, F. & Shi, Z.-L. Nat. Rev. Microbiol. 17, 181–192 (2019). have then required repeated passage in cell CoV-2, including animal studies. The 20. Almazán, F. et al. Virus Res. 189, 262–270 (2014). culture or animals with ACE2 receptors identification of a potential intermediate 21. Zhang, T., Wu, Q. & Zhang, Z. bioRxiv https://doi. similar to those of humans, but such work host of SARS-CoV-2, as well as sequencing org/10.1101/2020.02.19.950253 (2020). has also not previously been described. of the virus from very early cases, would 22. Yamada, Y. & Liu, D. X. J. Virol. 83, 8744–8758 (2009). 23. Rambaut, A. Virological.org http://virological.org/t/356 (2020). Finally, the generation of the predicted similarly be highly informative. Irrespective 24. Huang, C. et al. Lancet https://doi.org/10.1016/S0140- O-linked glycans is also unlikely to have of the exact mechanisms by which SARS- 6736(20)30183-5 (2020). occurred due to cell-culture passage, as CoV-2 originated via natural selection, 25. Dudas, G., Carvalho, L. M., Rambaut, A. & Bedford, T. eLife 7, e31257 (2018). such features suggest the involvement of an the ongoing surveillance of pneumonia 26. Wang, N. et al. Virol. Sin. 33, 104–107 (2018). immune system18. in humans and other animals is clearly of 27. Ge, X.-Y. et al. Nature 503, 535–538 (2013). utmost importance. ❐ 28. Lim, P. L. et al. N. Engl. J. Med. 350, 1740–1745 (2004). 29. Wong, M. C., Javornik Cregeen, S. J., Ajami, N. J. & Petrosino, J. F. Conclusions bioRxiv https://doi.org/10.1101/2020.02.07.939207 (2020). 1,2 In the midst of the global COVID-19 Kristian G. Andersen , 30. Liu, P., Chen, W. & Chen, J.-P. Viruses 11, 979 (2019). public-health emergency, it is reasonable Andrew Rambaut! !3, W. Ian Lipkin4, https://www.nature.com/articles/s41591-020-0820-9 to wonder why the origins of the pandemic Edward C. Holmes! !5 and Robert F. Garry6,7 Acknowledgements matter. Detailed understanding of how an 1Department of Immunology and Microbiology, We thank all those who have contributed sequences animal virus jumped species boundaries to !e Scripps Research Institute, La Jolla, CA, USA. to the GISAID database (https://www.gisaid.org/) and analyses to Virological.org (http://virological.org/). correspondence infect humans so productively will help in 2Scripps Research Translational Institute, La We thank M. Farzan for discussions, and the Wellcome 3 the prevention of future zoonotic events. Jolla, CA, USA. Institute of Evolutionary Biology, Trust for support. K.G.A. is a Pew Biomedical Scholar For example, if SARS-CoV-2 pre-adapted in University of Edinburgh, Edinburgh, UK. 4Center and is supported by NIH grant U19AI135995. A.R. is another animal species, then there is the risk for Infection and Immunity, Mailman School of supported by the Wellcome Trust (Collaborators Award of future re-emergence events. In contrast, Public Health of Columbia University, New York, NY, 206298/Z/17/Z―ARTIC network) and the European 5 Research Council (grant agreement no. 725422― if the adaptive process occurred in humans, USA. Marie Bashir Institute for Infectious Diseases ReservoirDOCS). E.C.H. is supported by an ARC The proximal origin of SARS-CoV-2 then even if repeated zoonotic transfers and Biosecurity, School of Life and Environmental Australian Laureate Fellowship (FL170100022). R.F.G. is occur, they are unlikely to take off without Sciences and School of Medical Sciences, !e supported by NIH grants U19AI135995, U54 HG007480 the same series of mutations. In addition, University of Sydney, Sydney, Australia. 6Tulane and U19AI142790. identifying the closest viral relatives of University, School of Medicine, Department of Competing interests Microbiology and Immunology, New Orleans, LA, To the Editor — Since the first reports of While the analyses above suggest that low-pathogenicity avianSARS-CoV-2 influenza circulating viruses in animals will R.F.G. is co-founder of Zalgen Labs, a biotechnology 7 greatly assist16 studies of viral function. USA. Zalgen Labs, Germantown, MD, USA. company that develops countermeasures to emerging novel pneumonia (COVID-19) in Wuhan, SARS-CoV-2 may bind human ACE2 with into highly pathogenicIndeed, forms the availability. The of the RaTG13 bat e-mail: [email protected] viruses. Hubei province, China1,2, there has been high affinity, computational analyses predict acquisition of polybasic cleavage sites by considerable discussion on the origin of that the interaction is not ideal7 and that HA has also been observed after repeated the causative virus, SARS-CoV-23 (also the RBD sequence is different from those passage in cell culture or through animals17. referred to as HCoV-19)4. Infections with shown in SARS-CoV to be optimal for The function of the predicted O-linked SARS-CoV-2 are now widespread, and as receptor binding7,11. Thus, the high-affinity glycans is unclear, but they could create a of 11 March 2020, 121,564 cases have been binding of the SARS-CoV-2 spike protein ‘mucin-like domain’ that shields epitopes confirmed in more than 110 countries, with to human ACE2 is most likely the result of or key residues on the 452SARS-CoV-2 spike NATURE MEDICINE | VOL 26 | APRIL 2020 | 450–455 | www.nature.com/naturemedicine 4,373 deaths5. natural selection on a human or human-like protein18. Several viruses utilize mucin- SARS-CoV-2 is the seventh coronavirus ACE2 that permits another optimal binding like domains as glycan shields involved known to infect humans; SARS-CoV, MERS- solution to arise. This is strong evidence immunoevasion18. Although prediction CoV and SARS-CoV-2 can cause severe that SARS-CoV-2 is not the product of of O-linked glycosylation is robust, disease, whereas HKU1, NL63, OC43 and purposeful manipulation. experimental studies are needed 229E are associated with mild symptoms6. to determine if these sites are used in Here we review what can be deduced about 2. Polybasic furin cleavage site and SARS-CoV-2. the origin of SARS-CoV-2 from comparative O-linked glycans. The second notable analysis of genomic data. We offer a feature of SARS-CoV-2 is a polybasic Theories of SARS-CoV-2 origins perspective on the notable features of the cleavage site (RRAR) at the junction of It is improbable that SARS-CoV-2 emerged SARS-CoV-2 genome and discuss scenarios S1 and S2, the two subunits of the spike8 through laboratory manipulation of a by which they could have arisen. Our (Fig. 1b). This allows effective cleavage by related SARS-CoV-like coronavirus. As analyses clearly show that SARS-CoV-2 is furin and other proteases and has a role noted above, the RBD of SARS-CoV-2 is not a laboratory construct or a purposefully in determining viral infectivity and host optimized for binding to human ACE2 with manipulated virus. range12. In addition, a leading proline is also an efficient solution different from those inserted at this site in SARS-CoV-2; thus, previously predicted7,11. Furthermore, if Notable features of the SARS-CoV-2 the inserted sequence is PRRA (Fig. 1b). genetic manipulation had been performed, genome The turn created by the proline is predicted one of the several reverse-genetic systems Our comparison of alpha- and to result in the addition of O-linked glycans available for betacoronaviruses would betacoronaviruses identifies two notable to S673, T678 and S686, which flank the probably have been used19. However, the genomic features of SARS-CoV-2: (i) on the cleavage site and are unique to SARS-CoV-2 genetic data irrefutably show that SARS- basis of structural studies7–9 and biochemical (Fig. 1b). Polybasic cleavage sites have CoV-2 is not derived from any previously experiments1,9,10, SARS-CoV-2 appears to be not been observed in related ‘lineage B’ used virus backbone20. Instead, we propose optimized for binding to the human receptor betacoronaviruses, although other human two scenarios that can plausibly explain ACE2; and (ii) the spike protein of SARS- betacoronaviruses, including HKU1 (lineage the origin of SARS-CoV-2: (i) natural CoV-2 has a functional polybasic (furin) A), have those sites and predicted O-linked selection in an animal host before zoonotic cleavage site at the S1–S2 boundary through glycans13. Given the level of genetic variation transfer; and (ii) natural selection in humans the insertion of 12 nucleotides8, which in the spike, it is likely that SARS-CoV-2-like following zoonotic transfer. We also discuss additionally led to the predicted acquisition viruses with partial or full polybasic cleavage whether selection during passage could have of three O-linked glycans around the site. sites will be discovered in other species. given rise to SARS-CoV-2. The functional consequence of the 1. Mutations in the receptor-binding polybasic cleavage site in SARS-CoV-2 1. Natural selection in an animal host domain of SARS-CoV-2. The receptor- is unknown, and it will be important to before zoonotic transfer. As many early binding domain (RBD) in the spike protein determine its impact on transmissibility cases of COVID-19 were linked to the is the most variable part of the coronavirus and pathogenesis in animal models. Huanan market in Wuhan1,2, it is possible genome1,2. Six RBD amino acids have been Experiments with SARS-CoV have shown that an animal source was present at this shown to be critical for binding to ACE2 that insertion of a furin cleavage site at the location. Given the similarity of SARS- receptors and for determining the host S1–S2 junction enhances cell–cell fusion CoV-2 to bat SARS-CoV-like coronaviruses2, range of SARS-CoV-like viruses7. With without affecting viral entry14. In addition, it is likely that bats serve as reservoir hosts coordinates based on SARS-CoV, they are efficient cleavage of the MERS-CoV spike for its progenitor. Although RaTG13, Y442, L472, N479, D480, T487 and Y4911, enables MERS-like coronaviruses from bats sampled from a Rhinolophus affinis bat1, is which correspond to L455, F486, Q493, to infect human cells15. In avian influenza ~96% identical overall to SARS-CoV-2, its S494, N501 and Y505 in SARS-CoV-27. Five viruses, rapid replication and transmission spike diverges in the RBD, which suggests of these six residues differ between SARS- in highly dense chicken populations selects that it may not bind efficiently to human CoV-2 and SARS-CoV (Fig. 1a). On the for the acquisition of polybasic cleavage ACE27 (Fig. 1a). basis of structural studies7–9 and biochemical sites in the hemagglutinin (HA) protein16, Malayan pangolins (Manis javanica) experiments1,9,10, SARS-CoV-2 seems to which serves a function similar to that illegally imported into Guangdong province have an RBD that binds with high affinity to of the coronavirus spike protein. Acquisition contain coronaviruses similar to SARS- ACE2 from humans, ferrets, cats and other of polybasic cleavage sites in HA, by CoV-221. Although the RaTG13 bat virus species with high receptor homology7. insertion or recombination, converts remains the closest to SARS-CoV-2 across the

450 NATURE MEDICINE | VOL 26 | APRIL 2020 | 450–455 | www.nature.com/naturemedicine “Full” (not at all full) diversity of coronaviruses

Research from 2018 https://doi.org/10.1038/s41579-018-0118-9 Other humanREVIEWS coronaviruses are also from

zoonosis Early 2020 information

Origin and evolution of pathogenic coronaviruses

Jie Cui 1, Fang Li2 and Zheng- Li Shi 1* Abstract | Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS- CoV) are two highly transmissible and pathogenic viruses that emerged in humans at the beginning of the 21st century. Both viruses likely originated in bats, and genetically diverse coronaviruses that are related to SARS-CoV and MERS- CoV were discovered in bats worldwide. In this Review , we summarize the current knowledge on the origin and evolution of these two pathogenic coronaviruses and discuss their receptor usage; we also highlight the diversity and potential of spillover of bat- borne coronaviruses, as evidenced by the recent spillover of swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs.

Severe acute respiratory Coronaviruses cause respiratory and intestinal infections of Viruses). This subfamily consists of four genera — syndrome in animals and humans1. They were not considered to Alphacoronavirus, Betacoronavirus, Gammacoronavirus A serious form of pneumonia be highly pathogenic to humans until the outbreak of and Deltacoronavirus — on the basis of their phylo- that is characterized by diffuse severe acute respiratoryResearch syndrome (SARS) in 2002from and genetic 2018 relationships and genomic structures (FIG. 1). alveolar damage and that has 2–5 the potential to progress to 2003 in Guangdong province, China , as the coronavi- The alphacoronaviruses and betacoronaviruses infect acute respiratory distress. ruses that circulated before that time in humans mostly only mammals. The gammacoronaviruses and deltacoro- caused mild infections in immunocompetent people. Ten naviruses infect birds, but some of them can also infect Ty p e I I p n e u m o c y t e s yearshttps://doi.org/10.1038/s41579 after SARS, another highly pathogenic coronavirus, mammals24.- Alphacoronaviruses018-0118 and-9 betacoronaviruses Epithelial cells that line the lung alveoli; type II cells are Middle East respiratory syndrome coronavirus (MERS- usually cause respiratory illness in humans and gastro- 6 round and produce surfactants CoV) emerged in Middle Eastern countries . SARS enteritis in animals. The two highly pathogenic viruses, to lower the surface tension of coronavirus (SARS- CoV) uses angiotensin- converting SARS- CoV and MERS- CoV, cause severe respiratory water and allow the membrane enzyme 2 (ACE2) as a receptor and primarily infects cil- syndrome in humans, and the other four human coro- to separate, thereby increasing iated bronchial epithelial cells and type II pneumocytes7,8, naviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and the capability to exchange gases. whereas MERS- CoV uses dipeptidyl peptidase 4 (DPP4; HKU1) induce only mild upper respiratory diseases in also known as CD26) as a receptor and infects unciliated immunocompetent hosts, although some of them bronchial epithelial cells and type II pneumocytes9–11. can cause severe infections in infants, young children SARS- CoV and MERS- CoV were transmitted directly and elderly individuals1,28,29. Alphacoronaviruses and to humans from market civets and dromedary camels, betacoronaviruses can pose a heavy disease burden on respectively12–14, and both viruses are thought to have orig- livestock; these viruses include porcine transmissible inated in bats15–21. Extensive studies of these two important gastroenteritis virus32, porcine enteric diarrhoea virus coronaviruses have not only led to a better understanding (PEDV)33 and the recently emerged swine acute diar- of coronavirus biology but have also been driving coro- rhoea syndrome coronavirus (SADS- CoV)34. On the navirus discovery in bats globally21–31. In this Review, basis of current sequence databases, all human corona- 1CAS Key Laboratory of we focus on the origin and evolution of SARS- CoV and viruses have animal origins: SARS- CoV, MERS- CoV, Special Pathogens and MERS-CoV. Specifically, we emphasize the ecological dis- HCoV- NL63 and HCoV-229E are considered to have Biosafety, Wuhan Institute of Virology, Chinese Academy tribution, genetic diversity, interspecies transmission and originated in bats; HCoV- OC43 and HKU1 likely orig- 28,29 of Sciences, Wuhan, Hubei, potential for pathogenesis of SARS- related coronaviruses inated from rodents . Domestic animals may have China. (SARSr-CoVs) and MERS-related coronaviruses (MERSr- important roles as intermediate hosts that enable virus 2Department of Veterinary CoVs) found in bats, as this information can help prepare transmission from natural hosts to humans. In addition, https://doi.org/10.1093/femspd/ftaa006 and Biomedical Sciences, countermeasures against future spillover and pathogenic domestic animals themselves can suffer disease caused College of Veterinary infections in humans with novel coronaviruses. by bat- borne or closely related coronaviruses: genomic Medicine, University of Minnesota, Saint Paul, MN, sequences highly similar to PEDV were detected in 35–38 USA. Coronavirus diversity bats , and SADS- CoV is a recent spillover from 34 *e- mail: [email protected] Coronaviruses are members of the subfamily Corona- bats to pigs (FIG. 2). Currently, 7 of 11 ICTV- assigned https://doi.org/10.1038/ virinae in the family Coronaviridae and the order Alphacoronavirus species and 4 of 9 Betacoronavirus spe- s41579-018-0118-9 Nidovirales (International Committee on Taxonomy cies were identified only in bats (FIG. 3). Thus, bats are

NATURE REVIEWS | MICROBIOLOGY VOLUME 17 | MARCH 2019 | 181 Other human coronaviruses are also from zoonosis

Ghana bat coronavirus Human 229E sample

Human NL63 samples

North American bat coronavirus

https://jvi.asm.org/content/86/23/12816 Research from 2012 SARS-CoV-2 emergence “shouldn’t” surprise us

2012

https://www.nature.com/articles/nrmicro.2016.81 SARS-CoV-2 emergence “shouldn’t” surprise us Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats Susanna K. P. Lau*†‡§, Patrick C. Y. Woo*†‡§, Kenneth S. M. Li*, Yi Huang*, Hoi-Wah Tsoi*, Beatrice H. L. Wong*, Samson S. Y. Wong*†‡, Suet-Yi Leung¶, Kwok-Hung Chan*, and Kwok-Yung Yuen*†‡§ʈ

*Department of Microbiology, †Research Centre of Infection and Immunology, ‡State Key Laboratory of Emerging Infectious Diseases, and ¶Department of Pathology, University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China

Communicated by Lap-Chee Tsui, University of Hong Kong, Hong Kong Special Administrative Region, China, August 8, 2005 (received for review June 22, 2005) Research from 2005 Although the finding of severe acute respiratory syndrome coro- Kong and propose that this virus be named bat SARS CoV navirus (SARS-CoV) in caged palm civets from live animal markets (bat-SARS-CoV). in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that Methods the civet may have served only as an amplification host for Wild Animal Surveillance and Sample Collection. The study was SARS-CoV. In a surveillance study for CoV in noncaged animals approved by the HKSAR’s Department of Agriculture, Fisher- from the wild areas of the Hong Kong Special Administration ies, and Conservation and the University of Hong Kong Com- Region, we identified a CoV closely related to SARS-CoV (bat-SARS- mittee on the Use of Live Animals in Teaching and Research. CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe From summer 2004 to spring 2005, 127 bats, 60 rodents, and 20 bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and anal- monkeys from 11 locations in the HKSAR were captured (Fig. ysis of three bat-SARS-CoV genomes from samples collected at 1a and Table 2, which is published as supporting information on Fig. 1. Surveillance for bat-SARS-CoV in the wild areas of the HKSAR. (a) Map of HKSAR showing locations of wild animal surveillance indicated by stars. Areas different dates showedbelonging that bat-SARS-CoVto Shenzhen of mainland China is closely are shaded. Red related stars represent to the twothe locations PNAS with bats positive web for site). bat-SARS-CoV, Nasopharyngeal both being water tunnels. and Other anal swabs and blood sampling sites include abandoned mines, sea caves and forested areas. SZDM, Shenzhen Dongmen market, the nearest wildlife market with SARS-CoV-infected SARS-CoV from humanscivets. and (b civets.) A group of Phylogenetic Chinese horseshoe bats analysis roosting in ashowed water tunnel in the HKSAR.samples The common were namecollected describes the horseshoe-shaped by a veterinary area of thick skin surgeon. Swabs were https://doi.org/10.1073/pnas.0506735102that bat-SARS-CoV formedsurrounding a distinct the nostrils that cluster covers the with upper lipSARS-CoV (close-up view). as taken with sterile swabs and kept in viral transport medium at group 2b CoV, distantly related to known group 2 CoV. Most 4°C before processing (14). Where possible, blood was collected amplification of cDNA ends by using the 5Ј͞3Ј RACE kit (Roche formed by using 1:2,000 horseradish peroxidase-conjugated pro- differences between theDiagnostics). bat-SARS-CoV Sequences andwere assembled SARS-CoV and manually genomes edited to fortein Gserological and 3,3Ј,5,5Ј-tetramethylbenzidine, studies. both from Zymed. were observed in the spikeproduce genes, complete ORF sequences 3 and of ORF the three 8, which viral genomes, are the which Each sample was tested in duplicate, and the mean absorbance have been deposited into GenBank (Table 3, which is published for each serum was calculated. regions where most variationsas supporting also information were onobserved the PNAS web between site). The hu-nucleo- RNA Extraction, RT-PCR, and DNA Sequencing. Nasopharyngeal and man and civet SARS-CoVtide genomes. and deduced In amino addition, acid sequences the presence were compared of awith analSpecificity swabs of Recombinant were tested bat-SARS-CoV for N CoV Protein-Based RNA Western by RT-PCR. Viral RNA those of other CoV in GenBank (Table 3) by multiple sequence Blot Analysis and EIA. To evaluate the specificity of the recombi- 29-bp insertion in ORF 8alignment of bat-SARS-CoV using CLUSTALW software genome, (www.ebi.ac.uk not in͞ mostclustalw). extractionnant N protein-based was performedWestern blot assay by and using EIA, convalescent the QIAamp viral RNA mini Phylogenetic tree construction was performed by using the kithuman (Qiagen, serum samples Valencia, from patients CA) with recent according infections by to the manufacturer’s human SARS-CoV genomes,neighbor-joining suggests method that withit hasGROWTREE a commonsoftware ances- (Genetics HCoV-OC43 (n ϭ 13), HCoV-229E (n ϭ 9), HCoV-NL63 (n ϭ tor with civet SARS-CoV.Computer Antibody Group, against Madison, recombinant WI) using Jukes–Cantor bat-SARS- correction. instructions.5), and CoV-HKU1 cDNA (n ϭ 11), was positive generated for specific antibodies by using random hexamers Prediction of signal peptides and cleavage sites was performed andagainst the the respective SuperScript CoV, were II subject kit (Invitrogen) to Western blot assay as described in ref. 7. CoV nucleocapsid proteinby was using SIGNALP detectedsoftware, in 84% transmembrane of Chinese domains horse- by using and EIA against recombinant N protein of bat-SARS-CoV. shoe bats by using an enzymeTMPRED immunoassay.and TMHMM software, Neutralizing potential N-glycosylation antibody sites by CoV screening was performed by using conserved primers using SCANPROSITE software, and protein family analysis by using Neutralization Assays. Because attempts to passage bat-SARS- to human SARS-CoV alsoPFAM wasand detectedINTERPROSCAN insoftware bats (15–21). with lower viral (5CoVЈ-GGTTGGGACTATCCTAAGTGTGA-3 in cell cultures were not successful, neutralization assays for Ј and 5Ј-CCAT- loads. Precautions should be exercised in the handling of these CATCAGATAGAATCATCATA-3human SARS-CoV were carried out as described inЈ)targetedtoa440-bpfrag- ref. 9. Sequencing of Complete Spike (S) Genes of bat-SARS-CoV. The Animal sera serially diluted from 1:20 to 1:640 were mixed with animals. complete S genes of bat-SARS-CoV from 14 positive samples, ment100 tissue of culture the pol 50%gene infective as describeddose of SARS-CoV in ref. isolate 7. Standard precautions with adequate amount of RNA available, were sequenced by wereHKU-39849. taken Human to sera avoid from PCRSARS patients contamination, with neutralizing and no false-positive using primers targeted to S. The nucleotide and deduced amino antibody titer of 1:160 were included as a positive control. After oronaviruses (CoV)acid are sequences found were in acompared wide varietywith those of of SARS-CoV animals by wasincubation observed for1hat in37°C, negative the mixture controls. was inoculated The sequencesin of the PCR multiple alignment. productstriplicate onto were 96-well compared plates of FRhK-4 with cell known cultures. Resultssequences of the pol genes Cin which they can cause respiratory, enteric, hepatic, and were recorded after 3 days of incubation at 37°C. neurological diseases ofWestern varying Blot Analysis severity. Using Recombinant As a Nucleocapsid result of(N) Protein the of of CoV in GenBank. bat-SARS-CoV. Cloning and purification of (His)6-tagged recom- Quantitative RT-PCR. Quantitaive RT-PCR was performed on anal unique mechanism of viralbinant N replication, protein of bat-SARS-CoV CoV werehave performed a high as described fre- swabs of bats positive for bat-SARS-CoV by RT-PCR or anti- quency of recombinationin ref.(1). 22. Their Primers tendency (5Ј-CGCGGATCCGATGTCTGATAATG- for recombination Viralbody by Cultures. EIA. cDNAAttempts was amplified to in isolateSYBR Green bat-SARS-CoV I fluores- were made by GACCC-3Ј and 5Ј-CGGAATTCTTATGCCTGAGTAGAA- inoculatingcence reactions (Roche) RT-PCR-positive using specific primers specimens (5Ј-TGTGACA- to FRhK-4, HRT-18G, and high mutation ratesTCA-3 mayЈ)wereusedtoamplifytheNgeneofbat-SARS-CoVby allow them to adapt to new hosts GAGCCATGCCTAA-3Ј and 5Ј-ATCTTATTACCATCAGTT- and ecological niches. AmongRT-PCR. CoV Western that blot infect analysis, humans, using 900 ng including of purified bat- Huh-7,GAAAGA-3 VeroЈ)asdescribedinref.7.Aplasmidwiththetarget E6, C6͞36 and Caco-2 cells, and chicken embry- SARS-CoV (His)6-tagged N protein and sera at 1:1,000 dilution, onatedsequence was eggs. used for Viral generating replication the standard curve.was At detected the end by observation for human CoV 229E (HCoV-229E),was performed as human described in CoV ref. 22. OC43 Antigen–antibody (HCoV- inter- of the assay, PCR products (280-bp fragment of pol)were action was detected with 1:4,000 horseradish peroxidase- cytopathicsubjected to melting effects curve and analysis quantitative (65–95°C, 0.1°C͞s) RT-PCR to con- described below. OC43), severe acute respiratoryconjugated protein syndrome G (Zymed) CoV and an enhanced (SARS-CoV), chemilumines- firm the specificity of the assay. human CoV NL63 (HCoV-NL63),cence fluorescence system and (Amersham CoV HKU1 Pharmacia). (CoV- Results HKU1), SARS-CoV causes the most severe disease, with Ͼ700 Complete Genome Sequencing and Genome Analysis. The complete Enzyme Immunoassay (EIA) Using Recombinant N Protein of bat-SARS- genomeWild Animal Surveillance, of bat-SARS-CoV Identification of bat-SARS-CoV, was sequenced and Viral by using RNA ex- fatalities reported sinceCoV. theSera SARS from eight epidemic bats of four in different 2003 species, (2–8). six rodents, Cultures. Atotalof414nasopharyngealandanalswabsfrom127 The isolation of SARS-CoVand two monkeys from negative caged for animals, bat-SARS-CoV including antibody by tractedbats, 60 rodents, from and three 20 monkeys anal were swabs obtained from from rural three areas bats (B24, B41, and Western blot analysis were used to set up the baseline for the B43)in the HKSAR as template. (Fig. 1a and RNA Table was 2). RT-PCR converted for a 440-bp to cDNA by a combined Himalayan palm civets andEIA a performed raccoon as dog, described from in ref. wild 22. live Nunc markets immunoplates fragment of pol gene of CoV was positive in anal swabs from 29 coated with 20 ng of purified (His)6-tagged recombinant bat- random-primingbats. Sequencing results suggestedand oligo(dT)-priming the presence of three differ- strategyMICROBIOLOGY described in ref. in mainland China suggested that these animals are the reservoir SARS-CoV N protein per well were used. Detection was per- 7.ent A CoV total (Fig. 4, of which 63 is sets published of primers, as supporting available information on on request, were used for the origin of the SARS epidemic (9). However, subsequent for PCR. The 5Ј end of the viral genome was confirmed by rapid studies suggested thatLau theet al. civet may have served only as an PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 ͉ 14041 amplification host for SARS-CoV and provided the environment Downloaded by guest on April 22, 2020 for major genetic variations permitting efficient animal-to- Abbreviations: CoV, coronavirus; HCoV, human CoV; BCoV, bovine CoV; SARS, severe acute human and human-to-human transmissions (10–13). Because respiratory syndrome; HKSAR, Hong Kong Special Administrative Region; S, spike; N, civets are often mixed with different species in overcrowded nucleocapsid; EIA, enzyme immunoassay; ACE2, angiotensin-converting enzyme 2. conditions at markets, we conducted a surveillance study for Data deposition: The sequences reported in this paper have been deposited in the GenBank CoV in noncaged animals from the wild areas of the Hong Kong database (accession nos. DQ022305, DQ084199, and DQ084200). Special Administrative Region (HKSAR). In this report, we §S.K.P.L., P.C.Y.W., and K.-Y.Y. contributed equally to this work. describe the identification and molecular characterization of a ʈTo whom correspondence should be addressed. E-mail: [email protected]. SARS-CoV-related virus from Chinese horseshoe bats in Hong © 2005 by The National Academy of Sciences of the USA

14040–14045 ͉ PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506735102 Downloaded by guest on April 22, 2020 SARS-CoV-2 emergence “shouldn’t” surprise us

Evidence for ACE2-Utilizing Coronaviruses (CoVs) Related to Severe Acute Respiratory Syndrome CoV in Bats

Ann Demogines,a Michael Farzan,b and Sara L. Sawyera Department of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA,a and Department of Microbiology and Immunobiology, Harvard Medical School, New England Primate Research Center, Southborough, Massachusetts, USAb

In 2002, severe acute respiratory syndrome (SARS)-coronavirus (CoV) appeared as a novel human virus with high similarity to bat coronaviruses. However, while SARS-CoV uses the human angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry, no coronavirus isolated from bats appears to use ACE2. Here we show that signatures of recurrent positive selection in the bat ACE2 gene map almost perfectly to known SARS-CoV interaction surfaces. Our data indicate that ACE2 utilization preceded the emergence of SARS-CoV-like viruses from bats. Downloaded from

Researchell-surface receptors from often 2012 play a key role in defining viral host that made it compatible with the human ortholog of its cell-sur- Crange. New diseases can emerge when existing viruses evolve face receptor, angiotensin-converting enzyme 2 (ACE2) (19, 21, the ability to bind the ortholog of their cell-surface receptor in a 30, 31). However, one key observation has driven the field to favor new species (1, 25, 35). Indeed, the principal genetic component alternate, more complex theories of emergence. The observation defining host range in coronaviruses is the spike protein on the is that while SARS-CoV and closely related viruses from the civet

surface of the virus and, in particular, its receptor-binding domain can use ACE2 as a receptor, no bat coronavirus has been shown to http://jvi.asm.org/ (RBD) (5, 14). It is believed that the severe acute respiratory syn- use bat, human, or any other orthologs of ACE2 (2, 27). Further, drome (SARS) epidemic resulted from the zoonotic transmission of a coronavirus from bats to humans (15, 18, 32). The central role of the RBD in the SARS-coronavirus (CoV) zoonosis was crystal- lized in an experiment in which a bat coronavirus became infec- Received 13 February 2012 Accepted 13 March 2012 tious in primate cells when it was altered to contain the RBD of Published ahead of print 21 March 2012 human SARS-CoV (2). Address correspondence to Sara L. Sawyer, [email protected]. Bats are thought to have initially infected one or more species Supplemental material for this article may be found at http://jvi.asm.org/. on April 22, 2020 by guest of small mammals, such as the palm civet (6, 13, 20, 37). One Copyright © 2012, American Society for Microbiology. All Rights Reserved. DOI:theory10.1128/JVI.00311 is that this intermediate host provided-12 a selective environ- doi:10.1128/JVI.00311-12 ment that drove the coronavirus RBD to acquire point mutations

TABLE 1 Positive selection of bat ACE2 codons 1 to 358 Model comparisonb M1a vs M2a M7 vs M8 M8a vs M8 ␻o and codon dN/dS value modela 2⌬lnL P value 2⌬lnL P value 2⌬lnL P value (% of codons)c Residues under positive selectiond 0.4, f61 52.7 P Ͻ 0.0001 56.5 P Ͻ 0.0001 52.8 P Ͻ 0.0001 4.3 (11) Q24**, T27*, K31*, H34*, M82*, L91*, T92, N159*, V212, D213*, D216*, E231*, S280, V298, A301, E329 0.4, f3 ϫ 4 56.3 P Ͻ 0.0001 56.4 P Ͻ 0.0001 56.1 P Ͻ 0.0001 4.3 (11) Q24**, T27*, K31*, H34*, M82*, L91**, T92, N159*, V212*, D213*, D216*, E231*, S280, V298*, A301, E329 1.6, f61 52.7 P Ͻ 0.0001 56.3 P Ͻ 0.0001 52.8 P Ͻ 0.0001 4.3 (11) Q24**, T27*, K31*, H34*, M82*, L91*, T92, N159*, V212, D213*, D216*, E231*, S280, V298, A301, E329 1.6, f3 ϫ 4 56.3 P Ͻ 0.0001 56.4 P Ͻ 0.0001 56.1 P Ͻ 0.0001 4.3 (11) Q24**, T27*, K31*, H34*, M82*, L91**, T92, N159*, V212*, D213*, D216*, E231*, S280, V298*, A301, E329 a Initial seed value for ␻ (dN/dS) and model of codon frequency (f61 or f3 ϫ 4). b Twice the difference in the natural logs of the likelihoods (2⌬lnL) of the two models being compared. This value is used in a likelihood ratio test along with the degrees of freedom. In all cases (M1a versus M2a, M7 versus M8, and M8a versus M8), a model that allows positive selection is compared to a null model. The P value indicates the confidence with which the null model can be rejected. c dN/dS value of the class of codons evolving under positive selection in M8 and the percentage of codons falling in that class. d Residues corresponding to codons assigned to the class with a dN/dS ratio of Ͼ1 in M8 (P Ͼ 0.90 by naive empirical Bayes [NEB]). Coordinates correspond to the human protein, although the human sequence was not used in this analysis. Bat numerical coordinates are identical with the exception of three species with single codon insertions or deletions (see alignment in Fig. S1 in the supplemental material). *, P Ͼ 0.95; **, P Ͼ 0.99. Three additional codons were identified in the analysis of the full-length gene (see Table S2 in the supplemental material).

6350 jvi.asm.org Journal of Virology p. 6350–6353 June 2012 Volume 86 Number 11 SARS-CoV-2 emergence “shouldn’t” surprise us

LETTER Research from 2013 doi:10.1038/nature12711

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

Xing-Yi Ge1*, Jia-Lu Li1*, Xing-Lou Yang1*, Aleksei A. Chmura2, Guangjian Zhu2, Jonathan H. Epstein2, Jonna K. Mazet3, Ben Hu1, Wei Zhang1, Cheng Peng1, Yu-Ji Zhang1, Chu-Ming Luo1, Bing Tan1, Ning Wang1, Yan Zhu1, Gary Crameri4, Shu-Yi Zhang5, Lin-Fa Wang4,6, Peter Daszak2 & Zheng-Li Shi1

The 2002–3 pandemic caused by severe acute respiratory syndrome in Kunming, Yunnan Province, China (Extended Data Table 1). A total coronavirus (SARS-CoV) was one of the most significant public health of 117 anal swabs or faecal samples were collected from individual bats Editorialevents in recent historySummary1.AnongoingoutbreakofMiddleEastrespira- using a previously published method5,14. A one-step reverse transcrip- tory syndrome coronavirus2 suggests that this group of viruses remains tion (RT)-nested PCR was conducted to amplify the RNA-dependent Aakeythreatandthattheirdistributioniswiderthanpreviouslyrecog- SARS-like virus in bats RNA polymerase (RdRP) motifs A and C, which are conserved among nized. Although bats have been suggested to be the natural reservoirs alphacoronaviruses and betacoronaviruses15. Peterof both viruses Daszak3–5, attemptsand to isolate colleagues the progenitor virus identify of SARS- twoTwenty-seven novel of the coronaviruses 117 samples (23%) were classed as positive by CoV from bats have been unsuccessful. Diverse SARS-like corona- PCR and subsequently confirmed by sequencing. The species origin of fromviruses (SL-CoVs) Chinese have nowhorseshoe been reported frombats bats that in China, areall closely positive samples related was confirmed to to severe be R. sinicus by cytochrome b Europe and Africa5–8, but none is considered a direct progenitor sequence analysis, as described previously16. A higher prevalence was acuteof SARS-CoV respiratory because of their phylogenetic syndrome disparity fromcoronavirus this virus observed (SARS in samples-CoV collected), in the October cause (30% in 2011 and 48.7% in and the inability of their spike proteins to use the SARS-CoV cellular 2012) than those in April (7.1% in 2011) or May (7.4% in 2012) (Extended receptor molecule, the human angiotensin converting enzyme II Data Table 1). Analysis of the S protein RBD sequences indicated the of(ACE2) a pandemic9,10. Here we report whole-genome during sequences2002 ofand two novel 2003. bat presence of seven different strains of SL-CoVs (Fig. 1a and Extended Theycoronaviruses also from isolate Chinese horseshoe a live bats (family:virus Rhinolophidae) from theseData Figs1bats and 2).that Inadditionto has RBD high sequences, which closelymatched in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more previously described SL-CoVs (Rs672, Rf1 and HKU3)5,8,17,18, two novel closely related to SARS-CoV than any previously identified bat coro- strains (designated SL-CoV RsSHC014 and Rs3367) were discovered. sequencenaviruses, particularly identity in the receptor to binding SARS domain-CoV of theand spike thatTheir full-length can infect genome sequences human were determined, cells and both were protein. Most importantly, we report the first recorded isolation of found to be 29,787 base pairs in size (excluding the poly(A) tail). The usinga live SL-CoV ACE2, (bat SL-CoV-WIV1) the same from batreceptor faecal samples inthat Vero isoverall used nucleotide by sequenceSARS identity-CoV of these. The two genomes with human E6 cells, which has typical coronavirus morphology, 99.9% sequence SARS-CoV (Tor2 strain) is 95%, higher than that observed previously resultsidentity to Rs3367 provide and uses ACE2the from strongest humans, civets andevidence Chinese for batto SL-CoVs date in Chinathat (88–92%) horseshoe5,8,17,18 or Europe (76%)6 (Extended horseshoe bats for cell entry. Preliminary in vitro testing indicates Data Table 2 and Extended Data Figs 3 and 4). Higher sequence iden- batsthat WIV1 are also hasnatural a broad species reservoirs tropism. Our results of provideSARS the-CoVtities. were observed at the protein level between these new SL-CoVs strongest evidence to date that Chinese horseshoe bats are natural and SARS-CoVs (Extended Data Tables 3 and 4). To understand the reservoirs of SARS-CoV, and that intermediate hosts may not be evolutionary origin of these two novel SL-CoV strains, we conducted necessary for direct human infection by some bat SL-CoVs. They also recombination analysis with the Recombination Detection Program highlight the importance of pathogen-discovery programs targeting 4.0 package19 using available genome sequences of bat SL-CoV strains high-risk wildlife groups in emerging disease hotspots as a strategy (Rf1, Rp3, Rs672, Rm1, HKU3 and BM48-31) and human and civet for pandemic preparedness. representative SARS-CoV strains (BJ01, SZ3, Tor2 and GZ02). Three The 2002–3 pandemic of SARS1 and the ongoing emergence of the breakpoints were detected with strong P values (,10220)andsupported Middle East respiratory syndrome coronavirus (MERS-CoV)2 demon- by similarity plot and bootscan analysis (Extended Data Fig. 5a, b). Break- strate that CoVs are a significant public health threat. SARS-CoV was points were located at nucleotides 20,827, 26,553 and 28,685 in the shown to use the human ACE2 molecule as its entry receptor, and this Rs3367 (and RsSHC014) genome, and generated recombination frag- is considered a hallmark of its cross-species transmissibility11.Thereceptor ments covering nucleotides 20,827–26,533 (5,727 nucleotides) (inclu- binding domain (RBD) located in the amino-terminal region (amino ding partial open reading frame (ORF) 1b, full-length S, ORF3, E and acids 318–510) of the SARS-CoV spike (S) protein is directly involved partial M gene) and nucleotides 26,534–28,685 (2,133 nucleotides) in binding to ACE2 (ref. 12). However, despite phylogenetic evidence (including partial ORF M, full-length ORF6, ORF7, ORF8 and partial that SARS-CoV evolved from bat SL-CoVs, all previously identified Ngene).Phylogeneticanalysisusingthe major and minor parental regions SL-CoVs have major sequence differences from SARS-CoV in the RBD suggested that Rs3367, or RsSHC014, is the descendent of a recombination of their S proteins, including one or two deletions6,9. Replacing the RBD of lineages that ultimately lead to SARS-CoV and SL-CoV Rs672 (Fig. 1b). of one SL-CoV S protein with SARS-CoV S conferred the ability to use The most notable sequence differences between these two new SL- human ACE2 and replicate efficiently in mice9,13. However, to date, no CoVs and previously identified SL-CoVs is in the RBD regions of their SL-CoVs have been isolated from bats, and no wild-type SL-CoV of bat S proteins.First, they have higher amino acid sequence identity to SARS- origin has been shown to use ACE2. CoV (85% and 96% for RsSHC014 and Rs3367, respectively). Second, We conducted a 12-month longitudinal survey (April 2011–September there are no deletions and they have perfect sequence alignment with 2012) of SL-CoVs in a colony of Rhinolophus sinicus at a single location the SARS-CoV RBD region (Extended Data Figs 1 and 2). Structural

1Center for Emerging Infectious Diseases, State Key Laboratory of Virology, Wuhan Institute of Virology of the Chinese Academy of Sciences, Wuhan 430071, China. 2EcoHealth Alliance, New York, New York 10001, USA. 3One Health Institute, School of Veterinary Medicine, University of California, Davis, California 95616, USA. 4CSIRO Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia. 5College of Life Sciences, East China Normal University, Shanghai 200062, China. 6Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, Singapore 169857. *These authors contributed equally to this work.

28 NOVEMBER 2013 | VOL 503 | NATURE | 535 ©2013 Macmillan Publishers Limited. All rights reserved Characterization of a Novel Betacoronavirus Related to Middle East Respiratory Syndrome Coronavirus in European Hedgehogs

Victor Max Corman,a René Kallies,a Heike Philipps,b Gertraude Göpner,b Marcel Alexander Müller,a Isabella Eckerle,a Sebastian Brünink,a Christian Drosten,a Jan Felix Drexlera ‹Institute of Virology, University of Bonn Medical Centre, Bonn, Germanya; Igel-Schutz-Initiative e.V., Laatzen, Germanyb

Bats are known to host viruses closely related to important human coronaviruses (HCoVs), such as HCoV-229E, severe-acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV). As RNA viruses may coevolve with their hosts, we sought to investigate the closest sister taxon to bats, the Eulipotyphla, and screened European hedgehogs (Erinaceus europaeus) from Germany for CoV by nested reverse transcriptase PCR. A novel betacoronavirus species in a phylogenetic sister relationship to MERS-CoV and clade c bat CoVs was detected and characterized on the whole-genome

level. A total of 58.9% of hedgehog fecal specimens were positive for the novel CoV (EriCoV) at 7.9 log10 mean RNA copies per ml. EriCoV RNA concentrations were higher in the intestine than in other solid organs, blood, or urine. Detailed analyses of the full hedgehog intestine showed the highest EriCoV concentrations in lower gastrointestinal tract specimens, compatible with viral replication in the lower intestine and fecal-oral transmission. Thirteen of 27 (48.2%) hedgehog sera contained non-neutral- SARSizing antibodies-CoV against-2 MERS-CoV. emergence The animal origins of this“shouldn’t” betacoronavirus clade that includes surprise MERS-CoV may us thus include both bat and nonbat hosts.

he Coronaviridae subfamily Coronavirinae contains the four BtCoV HKU4-1 NC 009019 BtCoV HKU4-3 EF065507 genera Alpha-, Beta-, Gamma- and Deltacoronavirus (1, 2). 1 BtCoV 133/2005 NC 008315 CharacterizationT of a Novel Betacoronavirus Related to Middle East BtCoV HKU4-4 EF065508 1 BtCoV HKU4-2 EF065506 Betacoronaviruses are further discriminated into clades a to d. BtCoV HKU5-3 EF065511 Respiratory Syndrome Coronavirus in European Hedgehogs BtCoV HKU5-1 NC 009020 Until recently, five human coronaviruses (HCoVs) were known, 1 BtCoV HKU5-5 EF065512 1 BtCoV HKU5-2 EF065510 Victor Maxnamely, Corman,a René the Kallies alphacoronaviruses,a Heike Philipps,b Gertraude Göpner HCoV-229E,b Marcel Alexander and Müller HCoV-NL63,a Isabella Eckerle,a BtCoV/UKR-G17/UKR/2011 KC243392 Sebastian Brünink,a Christian Drosten,a Jan Felix Drexlera BtCoV/8-724/ROU/2009 KC243390 and the betacoronaviruses HCoV-OC43, HCoV-HKU1 (both BtCoV/8-691/ROU/2009 KC243391 Clade c ‹Institute of Virology, University of Bonn Medical Centre, Bonn, Germanya; Igel-Schutz-Initiative e.V., Laatzen, Germanyb 0.98 1 BtCoV/VM314/NED/2008 GQ259977 clade a), and Severe acute respiratory syndrome coronavirus (SARS- MERS-CoV/London KC164505 Bats are known to host viruses closely related to important human coronaviruses (HCoVs), such as HCoV-229E, severe-acute MERS-CoV/EMC/2012 JX869059 CoV, clade b) (3–8). In 2012, a highly pathogenic novel HCoV ErinaceusCoV/2012-68/GER/2012 respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV). As RNA viruses may 1 1 ErinaceusCoV/2012-216/GER/2012 coevolveResearch withtermed their from hosts,Middle we sought2014 East to investigate respiratory the closest sister syndrome taxon to bats, coronavirus the Eulipotyphla, and(MERS- screened European ErinaceusCoV/2012-174/GER/2012 hedgehogs (Erinaceus europaeus) from Germany for CoV by nested reverse transcriptase PCR. A novel betacoronavirus species ErinaceusCoV/2012-51/GER/2012 CoV) emerged (9–11). MERS-CoV belongs to the Betacoronavirus BtCoV/KW2E-F93/GHA/2010 JX899383 in a phylogenetic sister relationship to MERS-CoV and clade c bat CoVs was detected and characterized on the whole-genome 1 BtCoV/KW2E-F53/GHA/2010 JX899384 level. A totalclade of 58.9% c, of which hedgehog previously fecal specimens contained were positive for only the novel bat CoV CoVs (EriCoV) (BtCovs) at 7.9 log10 mean (12– RNA copies per BtCoV/KW2E-F82/GHA/2010 JX899382 ml. EriCoV RNA concentrations were higher in the intestine than in other solid organs, blood, or urine. Detailed analyses of the 1 SARS-CoV NC 004718 Clade b BtCoV HKU9-1 NC 009021 Clade d full hedgehog17). intestine Because showed of the thehighest high EriCoV number concentrations of in bat lower CoVs gastrointestinal newly tract described specimens, compatible in with HCoV-OC43 NC 005147 Clade a viral replicationthe aftermath in the lower intestine of SARS, and fecal-oral it was transmission. assumed Thirteen that of 27 all (48.2%) mammalian hedgehog sera CoVs contained non-neutral- HCoV-NL63 NC 005831 izing antibodies against MERS-CoV. The animal origins of this betacoronavirus clade that includes MERS-CoV may thus include0.04 both bat andoriginated nonbat hosts. in the order Chiroptera (18). The majority of these FIG 1 Betacoronavirus phylogeny, including the novel viruses from European novel bat CoVs were found in insectivorous bats (18). Therefore, hedgehogs. Bayesian phylogeny of an 816-nucleotide RdRp gene sequence he Coronaviridae subfamily Coronavirinae contains the four BtCoV HKU4-1 NC 009019 we speculated that other insectivorous mammals couldBtCoV HKU4-3 also EF065507 har- fragment corresponding to positions 14822 to 15637 in MERS-CoV strain genera Alpha-, Beta-, Gamma- and Deltacoronavirus (1, 2). 1 BtCoV 133/2005 NC 008315 T BtCoV HKU4-4 EF065508 bor CoVs. This might specifically apply to the animal1 BtCoV order HKU4-2 EF065506Euli- EMC/2012 (GenBank accession no. JX869059). The novel Erinaceus viruses Betacoronaviruses are further discriminated into clades a to d. BtCoV HKU5-3 EF065511 BtCoV HKU5-1 NC 009020 Until recently, five human coronaviruses (HCoVs) were known, 1 BtCoV HKU5-5 EF065512 are shown in red, and MERS-CoVs in blue. potyphla, which includes hedgehogs, moles, solenodons,1 BtCoV HKU5-2 EF065510 and namely, the alphacoronaviruses HCoV-229E and HCoV-NL63 BtCoV/UKR-G17/UKR/2011 KC243392 BtCoV/8-724/ROU/2009 KC243390 and the betacoronavirusesshrews, because HCoV-OC43, this and HCoV-HKU1 the order (both Chiroptera are phylogeneticallyBtCoV/8-691/ROU/2009 KC243391 Clade c 0.98 1 BtCoV/VM314/NED/2008 GQ259977 clade a), and Severe acute respiratory syndrome coronavirus (SARS- MERS-CoV/London KC164505 MERS-CoV/EMC/2012 JX869059 CoV, claderelated b) (3–8). In (19 2012,). a For highly this pathogenic reason, novelwe HCoV analyzed fecal samplesErinaceusCoV/2012-68/GER/2012 from 248 1 1 ErinaceusCoV/2012-216/GER/2012 termed Middle East respiratory syndrome coronavirus (MERS- ErinaceusCoV/2012-174/GER/2012 European hedgehogs (Erinaceus europaeus) for CoVs.ErinaceusCoV/2012-51/GER/2012 A novel be- PCR amplicons. For phylogenetic analyses, sequences from the PCR CoV) emerged (9–11). MERS-CoV belongs to the Betacoronavirus BtCoV/KW2E-F93/GHA/2010 JX899383 1 BtCoV/KW2E-F53/GHA/2010 JX899384 DOI: 10.1128/JVI.00311-12 BtCoV/KW2E-F82/GHA/2010 JX899382 screening assays were extended to an 816-nucleotide (nt) RdRp fragment clade c, whichtacoronavirus previously contained clade only bat c CoVs species (BtCovs) was (12– found and1 describedSARS-CoV NCusing 004718 mo- Clade b BtCoV HKU9-1 NC 009021 Clade d 17). Because of the high number of bat CoVs newly described in HCoV-OC43 NC 005147 (Clade22 a). In addition, carcasses from 27 hedgehogs that died in the animal lecular and immunologic tools. HCoV-NL63 NC 005831 the aftermath of SARS, it was assumed that all mammalian CoVs 0.04 originated in the order Chiroptera (18). The majority of these shelter during their stay were collected and stored at Ϫ20°C until dissec- FIG 1 Betacoronavirus phylogeny, including the novel viruses from European novel bat CoVs were found in insectivorous bats (18). Therefore, hedgehogs. Bayesian phylogeny of an 816-nucleotide RdRp genetion. sequence Samples from the brain, heart, lung, liver, kidney, spleen, and intes- we speculatedMATERIALS that other insectivorous AND mammals METHODS could also har- fragment corresponding to positions 14822 to 15637 in MERS-CoV strain bor CoVs. This might specifically apply to the animal order Euli- EMC/2012 (GenBank accession no. JX869059). The novel Erinaceustineviruses were taken. The intestines of five additional CoV-positive animals potyphla,Sample which includes collection, hedgehogs, processing, moles, solenodons, and and screeningare shown in for red, and coronavirus MERS-CoVs in blue. RNA. Fe- were cleaned and dissected in 10 portions taken in equal intervals imme- shrews, because this and the order Chiroptera are phylogenetically related (19cal). For samples this reason, from we analyzed European fecal samples hedgehogs from 248 (Erinaceus europaeus) kept in an diately after the stomach and until the anal orifice. Europeananimal hedgehogs (shelterErinaceus ineuropaeus northern) for CoVs. Germany A novel be- becausePCR amplicons. of poor For phylogenetic physical analyses, condition sequences from the PCR tacoronavirus clade c species was found and described using mo- screening assays were extended to an 816-nucleotide (nt) RdRp fragment lecular andor immunologic injuries tools. were sampled noninvasively( and22). In stored addition, carcasses in RNAlater from 27 hedgehogs (Qiagen, that died in the animal Hilden, Germany) at Ϫ20°C until furthershelter duringinvestigation. their stay were collected For the and stored initial at Ϫ20°C until dissec- tion. Samples from the brain, heart, lung, liver, kidney, spleen, andReceived intes- 13 June 2013 Accepted 3 October 2013 MATERIALSCoV AND screening, METHODS fecal samples of 10 individualtine were taken.animals The intestines were of pooled. five additional RNA CoV-positive animals Sample collection, processing, and screening for coronavirus RNA. Fe- were cleaned and dissected in 10 portions taken in equal intervalsPublished imme- ahead of print 16 October 2013 cal samplespurification from European hedgehogs andCoV (Erinaceus detection europaeus) kept using in an twodiately different after the stomach nested and until reverse the anal orifice. tran- animal shelterscription-PCR in northern Germany (RT-PCR) because of poor physicalassays condition targeting the RNA-dependent RNA poly- Address correspondence to Christian Drosten, [email protected], or Jan or injuries were sampled noninvasively and stored in RNAlater (Qiagen, Felix Drexler, [email protected]. Hilden, Germany) at Ϫ20°C until further investigation. For the initial merase (RdRp) gene were done as describedReceived previously13 June 2013 Accepted (16,320 October, 21 2013). Indi- CoV screening, fecal samples of 10 individual animals were pooled. RNA Copyright © 2014, American Society for Microbiology. All Rights Reserved. Published ahead of print 16 October 2013 purificationvidual and CoV detectionspecimens using two in different positive nested pools reverse tran- were identified using a strain-specific scription-PCR (RT-PCR) assays targeting the RNA-dependent RNA poly- Address correspondence to Christian Drosten, [email protected], ordoi:10.1128/JVI.01600-13 Jan real-time RT-PCR (oligonucleotide sequencesFelix Drexler, [email protected]. available upon request) merase (RdRp) gene were done as described previously (16, 20, 21). Indi- vidual specimensbased in positive on the pools nucleotide were identified using sequences a strain-specific obtainedCopyright from © 2014, American sequencing Society for Microbiology. of initial All Rights Reserved. real-time RT-PCR (oligonucleotide sequences available upon request) doi:10.1128/JVI.01600-13 based on the nucleotide sequences obtained from sequencing of initial

January 2014January Volume 88 2014 Number 1 Volume 88 Number 1 Journal of Virology p. 717–724 Journaljvi.asm.org of Virology717 p. 717–724 jvi.asm.org 717 SARS-CoV-2 emergence “shouldn’t” surprise us

LETTERS

“The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting Research from 2015 enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of A SARS-like cluster of circulating bat coronaviruses SARS-CoV." shows potential for human emergence

Vineet D Menachery1, Boyd L Yount Jr1, Kari Debbink1,2, Sudhakar Agnihothram3, Lisa E Gralinski1, Jessica A Plante1, Rachel L Graham1, Trevor Scobey1, Xing-Yi Ge4, Eric F Donaldson1, Scott H Randell5,6, Antonio Lanzavecchia7, Wayne A Marasco8,9, Zhengli-Li Shi4 & Ralph S Baric1,2

The emergence of severe acute respiratory syndrome the afflicted regions5. Although public health measures were able to coronavirus (SARS-CoV) and Middle East respiratory stop the SARS-CoV outbreak4, recent metagenomics studies have syndrome (MERS)-CoV underscores the threat of cross-species identified sequences of closely related SARS-like viruses circulating transmission events leading to outbreaks in humans. Here we Translation:in Chinese bat populations that Bat may pose acoronaviruses future threat1,6. However, are “out there” that can, examine the disease potential of a SARS-like virus, SHC014- sequence data alone provides minimal insights to identify and prepare CoV, which is currently circulating in Chinese horseshoe bat byfor futurechance prepandemic viruses., ALREADY Therefore, to examine theinfect emergence humans. They just haven’t populations1. Using the SARS-CoV reverse genetics system2, potential (that is, the potential to infect humans) of circulating bat we generated and characterized a chimeric virus expressing CoVs, we built a chimeric virus encoding a novel, zoonotic CoV spike the spike of bat coronavirus SHC014 in a mouse-adapted doneprotein—from so the RsSHC014-CoV*yet*. sequence that was isolated from SARS-CoV backbone. The results indicate that group 2b Chinese horseshoe bats1—in the context of the SARS-CoV mouse- viruses encoding the SHC014 spike in a wild-type backbone adapted backbone. The hybrid virus allowed us to evaluate the ability can efficiently use multiple orthologs of the SARS receptor of the novel spike protein to cause disease independently of other human angiotensin converting enzyme II (ACE2), replicate necessary adaptive mutations in its natural backbone. Using this efficiently in primary human airway cells and achieve in vitro approach, we characterized CoV infection mediated by the SHC014 titers equivalent to epidemic strains of SARS-CoV. Additionally, spike protein in primary human airway cells and in vivo, and tested in vivo experiments demonstrate replication of the chimeric the efficacy of available immune therapeutics against SHC014-CoV. virus in mouse lung with notable pathogenesis. Evaluation of Together, the strategy translates metagenomics data to help predict available SARS-based immune-therapeutic and prophylactic and prepare for future emergent viruses. modalities revealed poor efficacy; both monoclonal antibody The sequences of SHC014 and the related RsWIV1-CoV show that https://and vaccine approachesdoi.org failed/10.1038/nm.3985 to neutralize and protect from these CoVs are the closest relatives to the epidemic SARS-CoV strains infection with CoVs using the novel spike protein. On the basis (Fig. 1a,b); however, there are important differences in the 14 resi- of these findings, we synthetically re-derived an infectious dues that bind human ACE2, the receptor for SARS-CoV, including full-length SHC014 recombinant virus and demonstrate robust the five that are critical for host range: Y442, L472, N479, T487 and viral replication both in vitro and in vivo. Our work suggests a Y491 (ref. 7). In WIV1, three of these residues vary from the epidemic potential risk of SARS-CoV re-emergence from viruses currently SARS-CoV Urbani strain, but they were not expected to alter binding circulating in bat populations. to ACE2 (Supplementary Fig. 1a,b and Supplementary Table 1). This fact is confirmed by both pseudotyping experiments that meas- The emergence of SARS-CoV heralded a new era in the cross-species ured the ability of lentiviruses encoding WIV1 spike proteins to enter transmission of severe respiratory illness with globalization leading to cells expressing human ACE2 (Supplementary Fig. 1) and by in vitro rapid spread around the world and massive economic impact3,4. Since replication assays of WIV1-CoV (ref. 1). In contrast, 7 of 14 ACE2- then, several strains—including influenza A strains H5N1, H1N1 and interaction residues in SHC014 are different from those in SARS-CoV, H7N9 and MERS-CoV—have emerged from animal populations, including all five residues critical for host range (Supplementary causing considerable disease, mortality and economic hardship for Fig. 1c and Supplementary Table 1). These changes, coupled with

1Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 3National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas, USA. 4Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China. 5Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 6Cystic Fibrosis Center, Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 7Institute for Research in Biomedicine, Bellinzona Institute of Microbiology, Zurich, Switzerland. 8Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. 9Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. Correspondence should be addressed to R.S.B. ([email protected]) or V.D.M. ([email protected]). Received 12 June; accepted 8 October; published online 9 November 2015; corrected online 20 November 2015 (details online); doi:10.1038/nm.3985

1508 VOLUME 21 | NUMBER 12 | DECEMBER 2015 NATURE MEDICINE Phylogenetics identifies origins of SARS-CoV2

SARS-CoV and SARS-CoV2 with other bat SARS-like viruses Betacoronaviruses and alphacoronavirus outgroup

CONSENSUS STATEMENT https://doi.org/10.1038/s41564-020-0695-z

The species Severe acute respiratory syndrome- related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses*

The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for develop- ing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome corona- viruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS- CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus–host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

pon a viral outbreak, it is important to rapidly establish consider their inherent genetic variability, which often results in two whether the outbreak is caused by a new or a previously or more viruses with non-identical but similar genome sequences Uknown virus (Box 1), as this helps decide which approaches being regarded as variants of the same virus. This immediately and actions are most appropriate to detect the causative agent, con- poses the question of how much difference to an existing group is trol its transmission and limit potential consequences of the epi- large enough to recognize the candidate virus as a member of a new, demic. The assessment of virus novelty also has implications for distinct group. This question is answered in best practice by evalu- virus naming and, on a different timescale, helps to define research ating the degree of relatedness of the candidate virus to previously priorities in virology and public health. identified viruses infecting the same host or established monophy- For many human virus infections such as influenza virus1 or letic groups of viruses, often known as genotypes or clades, which norovirus2 infections, well-established and internationally approved may or may not include viruses of different hosts. This is formally methods, standards and procedures are in place to identify and addressed in the framework of the official classification of virus tax- name the causative agents of these infections and report this infor- onomy and is overseen and coordinated by the ICTV4. Viruses are mation promptly to public health authorities and the general public. clustered in taxa in a hierarchical scheme of ranks in which the spe- In outbreaks involving newly emerged viruses, the situation may cies represents the lowest and most populous rank containing the be different, and appropriate procedures to deal with these viruses least diverged groups (taxa) of viruses (Box 2). The ICTV maintains need to be established or refined with high priority. a Study Group for each virus family. The Study Groups are respon- Here, we present an assessment of the genetic relatedness of the sible for assigning viruses to virus species and taxa of higher ranks, newly identified human coronavirus3, provisionally named 2019- such as subgenera, genera and subfamilies. In this context they play nCoV, to known coronaviruses, and detail the basis for (re)naming an important role in advancing the virus species concept and high- this virus severe acute respiratory syndrome coronavirus 2 (SARS- lighting its significance5. CoV-2), which will be used hereafter. Given the public interest in nam- Virus nomenclature is a formal system of names used to label ing newly emerging viruses and the diseases caused by these viruses viruses and taxa. The fact that there are names for nearly all viruses in humans, we will give a brief introduction to virus discovery and within a species is due to the historical perception of viruses as classification — specifically the virus species concept — and the roles causative agents of specific diseases in specific hosts, and to the way of different bodies, such as the World Health Organization (WHO) we usually catalogue and classify newly discovered viruses, which and the International Committee on Taxonomy of Viruses (ICTV), in increasingly includes viruses that have not been linked to any known this process. We hope this will help readers to better understand the disease in their respective hosts (Box 1). The WHO, an agency of the scientific approach we have taken to arrive at this name, and we will United Nations, coordinates international public health activities also discuss implications of this analysis and naming decision. aimed at combating, containing and mitigating the consequences of communicable diseases—including major virus epidemics—and Classifying and naming viruses and virus species is responsible for naming disease(s) caused by newly emerging Defining the novelty of viruses is one of the topics that virus human viruses. In doing so, the WHO often takes the traditional classification deals with. The classification of RNA viruses needs to approach of linking names of specific diseases to viruses (Box 1) and

*A list of authors and their affiliations appears at the end of the paper.

536 NATURE MICROBIOLOGY | VOL 5 | MARCH 2020 | 536–544 | www.nature.com/naturemicrobiology Studying PRECISE origins requires LOTS of sampling

Most recent common ancestor:

RaTG13 - SARS-CoV-2: ~57 years • ~96% sequence identity overall • ~93% identity in Spike gene

Rhinolophus affinis “Between May and October 2019, we collected a total of 302 samples from 227 bats from Mengla County, Yunnan Province in southern China. These bats belonged to 20 different species…. The samples comprised multiple tissues, including patagium (n=219), lung (n=2) and liver (n=3), and feces (n=78).” Study on last slide estimates 37 year divergence since RmYN02. Lots of recombination in the sarbecoviruses Virus recombination (influenza-specific image) • Leads to DIFFERENT phylogenies across regions of the genome

https://www.khanacademy.org/science/biology/biology-of-viruses/virus-biology/a/evolution-of-viruses a) Phylogenetic tree of the full-length virus genome. (b) the S gene. (c) the RBD. https://doi.org/10.1101/2020.03.02.974139

Phylogeographic analysis of the current outbreak

https://www.biorxiv.org/content/10.1101/2020.04.20.050039v1

Follow me now to nextstrain.org ! • https://twitter.com/nextstrain/status/1253051831426723841?s=12

Recommended follows on twitter (a very limited and kind of biased set…) • Me! @stephspiel • NextStrain @nextstrain • Trevor Bedford @trvrb • Richard Neher @richardneher • Emma Hodcroft @firefoxx66 • Jesse Bloom @bloomlab • Sam Scarpino @svscarpino • Lauren Meyers’ Lab @meyerslab • Akiko Iwasaki @VirusesImmunity • Andy Rambaut* @arambaut • Eddie Holmes @edwardholmes • Oliver Pybus @EvolveDotZoo • Florian Krammer @florian_krammer • Marc Lipsitch @mlipsitch • Ellie Murray @epiellie • Angela Rasmussen @angie_rasmussen • Carl Bergstrom @CT_Bergstrom • Carl Zimmer @carlzimmer • Ed Yong @edyong209 • We rate dogs @dog_rates wait how’d this get here what’s happening