R e v i e w s Ecology, evolution and spillover of coronaviruses from bats Manuel Ruiz-Aravena 1,12, Clifton McKee2,12, Amandine Gamble3, Tamika Lunn4, Aaron Morris5, Celine E. Snedden3, Claude Kwe Yinda6, Julia R. Port6, David W. Buchholz7, Yao Yu Yeo 7, Christina Faust8, Elinor Jax5, Lauren Dee5, Devin N. Jones1, Maureen K. Kessler 1,11, Caylee Falvo1, Daniel Crowley1, Nita Bharti 8, Cara E. Brook9, Hector C. Aguilar7, Alison J. Peel 4, Olivier Restif 5, Tony Schountz 10, Colin R. Parrish 7, Emily S. Gurley2, James O. Lloyd-Smith 3, Peter J. Hudson8, Vincent J. Munster 6 and Raina K. Plowright 1 ✉ Abstract | In the past two decades, three coronaviruses with ancestral origins in bats have emerged and caused widespread outbreaks in humans, including severe acute respiratory syn- drome coronavirus 2 (SARS-CoV-2). Since the first SARS epidemic in 2002–2003, the appreciation of bats as key hosts of zoonotic coronaviruses has advanced rapidly. More than 4,000 coronavirus sequences from 14 bat families have been identified, yet the true diversity of bat coronaviruses is probably much greater. Given that bats are the likely evolutionary source for several human corona- viruses, including strains that cause mild upper respiratory tract disease, their role in historic and future pandemics requires ongoing investigation. We review and integrate in fo r mat ion on bat–coronavirus interactions at the molecular, tissue, host and po pu la ti on l ev e ls . We i d en ti fy cr it i- cal ga p s in knowledge of bat coronaviruses, which relate to spillover and pandemic risk, including the pathways to zoonotic spillover, the infection dynamics within bat reservoir hosts, the role of prior adaptation in intermediate hosts for zoonotic transmission and the viral genotypes or traits that predict zoonotic capacity and pandemic potential. Filling these knowledge gaps may help prevent the next pandemic. Planetary health Bats are the reservoir hosts of three of the ten virus planetary health approaches to understanding spillover approaches groups of pandemic concern, as designated by the World as a multilayered process. Here, we focus on bat corona- Ecological approaches to Health Organization: henipaviruses (Nipah virus and viruses and the ecological, evolutionary and epidemio- understanding the impact of Hendra virus), filoviruses (Ebola virus and Marburg logical features that influence the risk of spillover into anthropogenic disruption virus) and coronaviruses1. Common features among humans and subsequent epidemic emergence. of natural systems on human health. these emerging viruses include the ability to cause severe Bats are the second most diverse order of mam- disease in humans but not in the reservoir hosts, rare mals, with more than 1,400 species, and they host an spillovers despite a wide geographical distribution and exceptional diversity of coronaviruses with ancient viral the potential role of bridging hosts that increase oppor- lineages that are spread across all six continents that tunities for human infections. The recent spillovers of bats inhabit. More than 4,800 coronavirus sequences bat coronaviruses to humans are consistent with an have been detected in bats, accounting for more than increasing number of emergent zoonoses from wildlife2,3. 30% of all bat viruses sequenced7. Given that 543 bat Wildlife farming and trade facilitate cross-species species — from a global diversity of 1,435 — have been transmission of viruses by mixing species in stressful sampled for coronaviruses (Supplementary Table 1), the and crowded conditions4–6, while other behaviours, true diversity of bat coronaviruses is likely much greater. including hunting and guano mining, facilitate con- Bats are hosts of ancestral lineages of betacoronaviruses tact with bat-borne pathogens. Those are part of larger from which viruses of public health concern evolved, ✉ patterns of encroachment into wildlife habitats and including severe acute respiratory syndrome corona-e-mail: raina.plowright@ montana.edu increasing pressure from human population expansion virus (SARS-CoV), Middle East respiratory syndrome https://doi.org/10.1038/ and intensifying natural resource use. The COVID-19 coronavirus (MERS-CoV) and SARS-CoV-2. These s41579-021-00652-2 pandemic has highlighted the need for integrated recent cases may just be the latest in a longer history N aTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 299 0123456789();: R e v i e w s of spillover and emergence of bat coronaviruses into subgenera of Alphacoronavirus and Betacoronavirus humans. For example, of the four endemic human corona- (Fig. 3). The apparent absence of coronaviruses in parti- viruses that cause 30% of mild upper respiratory tract cular bat taxa is most likely due to insufficient sampling infections (common cold), two may have originated rather than true absence16. in bats (alphacoronaviruses human coronavirus 229E Sequence similarity among viruses in different (HCoV-229E) and HCoV-NL63)8. Thus, the ancestry hosts has been used to infer viral origins. Viruses with of at least five of the seven coronaviruses capable of high sequence similarity to the three recently emerged human-to-human transmission can be traced back to human coronaviruses — SARS-CoV, SARS-CoV-2 and bat coronaviruses9,10. The other two human corona- MERS-CoV — have all been identified in bats (Figs 2,3). viruses (HCoV-OC43 and HCoV-HKU1) also may have Separate clades of coronaviruses from rhinolophid bats spilled over from animals to humans, with pathways that show up to 92% sequence identity to SARS-CoV17 and may involve rodents and cattle11. Additionally, animal up to 96% sequence identity to SARS-CoV-2 (rEF.10) at coronaviruses might have evolutionary origins in line- the genome level. Additional SARS-related corona- ages from bats, such as the recently emerged corona- viruses (SARSr-CoVs) have been detected in hippo- virus causing severe acute diarrhoea syndrome in pigs12. siderid and molossid bats in Africa, Asia and Europe Serological evidence of exposure of humans to bat corona- (Supplementary Table 1), and it is widely accepted viruses in rural China suggest that spillovers from bats that bats are the natural reservoir of SARSr-CoVs18–20. might occur relatively frequently but are not detected13,14. Similarly, coronaviruses from vespertilionid bats show Here, we review the ecology, evolution and spillover up to 86.5% sequence identity to MERS-CoV at the of bat coronaviruses and assess the current knowledge of genome level16, and related coronaviruses circulate in the determinants of coronavirus spillover and transmis- bats within the families Nycteridae, Emballonuridae sion among recipient hosts — from the ecology of hosts and Molossidae in Africa, Europe, North America and and viruses to single virus–cell interactions. We further Asia (Supplementary Table 1). The absence of related highlight the knowledge gaps that prevent us from pre- sequences in other animals suggests that a progeni- paring for and mitigating coronavirus emergence risk tor of MERS-CoV spilled over from bats into drome- and suggest a research agenda for developing the science dary camels (Camelus dromedarius)21. Viruses related of preventing coronavirus spillover. to the endemic human coronaviruses HCoV-229E (Duvinacovirus) and HCoV-NL63 (Setracovirus) have Distribution of bat coronaviruses been detected in Africa and South-East Asia in hippo- Coronaviruses (order Nidovirales, family Coronaviridae) siderid bats (sharing up to 91% sequence identity at include four genera: Alphacoronavirus and Betacorona- the genome level with HCoV-229E) and rhinonycterid virus, which infect a broad range of mammals, and bats (sharing up to 78% sequence identity across the Gammacoronavirus and Deltacoronavirus, which primar- genome with HCoV-NL63) (Figs 2,3; Supplementary ily infect birds15. Since the emergence of SARS-CoV in data; Supplementary Table 1). 2002, and the evidence that it originated from a bat reser- The wide distribution and high diversity of corona- voir, coronaviruses have been detected in 16% of bat spe- viruses in bats is most likely the result of a long coevo- cies (238) (Supplementary Table 1). Alphacoronaviruses lutionary history. Some coronavirus groups seem to be and betacoronaviruses have been detected in bats from exclusively associated with specific taxonomic groups of 14 of the 21 bat families, in at least 69 countries across six bats. For instance, the subgenus Nobecovirus has been continents (Figs 1,2; TablE 1; Supplementary Table 1). The detected mostly in Old World fruit bats (Pteropodidae). diversity of coronaviruses found in bats is high, with more Further understanding of the biogeography of bats and than 60 coronavirus species (more than 4,000 individual their coronaviruses would reveal key geographical areas sequences) detected from 13 of the 19 known mammalian of risk as well as bat coronavirus dynamics. Author addresses Infection and response in bats. Frequently, reservoir hosts of zoonoses appear tolerant of the pathogenic 1Department of microbiology and Cell Biology, montana State university, Bozeman, effects of infection, whereas humans experience severe mT, uSa. 22 2Department of epidemiology, Johns Hopkins Bloomberg School of Public Health, disease . Whether bat species are universally tolerant Baltimore, mD, uSa. of coronavirus infection remains unclear as few exper- 3Department of ecology and evolutionary Biology, university of California, los angeles, imental coronavirus challenge studies involving bats los angeles, Ca, uSa. have been performed, the putative natural reservoir bat 4Centre for Planetary Health and Food Security, Griffith university, Nathan, QlD, australia. species was often not used and it is unclear whether the 5Department of veterinary medicine, university of Cambridge, Cambridge, uK. infectious doses resembled those of natural exposures 6National Institute of allergy and Infectious Diseases, Hamilton, mT, uSa. (TablE 1; Supplementary Table 2). In bats experimen- 7Department of microbiology and Immunology, College of veterinary medicine, tally infected with coronaviruses, some individuals Cornell university, Ithaca, Ny, uSa. have shown mild tissue damage, including rhinitis23,24 8Department of Biology, Center for Infectious Disease Dynamics, Pennsylvania State and interstitial pneumonia24, with virus or viral RNA university, university Park, Pa, uSa. 9Department of ecology and evolution, university of Chicago, Chicago, Il, uSa. detected in the respiratory tract and/or intestines; how- 10Department of microbiology, Immunology, and Pathology, College of veterinary ever, infected animals did not exhibit evident clinical medicine and Biomedical Sciences, Colorado State university, Fort Collins, Co, uSa. signs of infection. 11Department of ecology, montana State university, Bozeman, mT, uSa. Little is known about the immune responses of bats 12These authors contributed equally: manuel Ruiz-aravena, Clifton mcKee. to coronavirus infections, both adaptive and innate. 300 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: R e v i e w s a Noctilionidae b 2 2 0 Species Furipteridae 2 0 0 500 Thyropteridae 5 1 0 400300 Mormoopidae 18 5 3 200 Phyllostomidae 222 88 19 100 Mystacinidae 2 1 1 0 Myzopodidae 2 0 0 Emballonuridae 55 19 3 Nycteridae 16 7 3 Cistugidae 2 0 0 Vespertilionidae 513 204 91 Miniopteridae 35 19 11 Molossidae 131 40 16 Natalidae 11 2 0 Craseonycteridae 1 1 0 Rhinopomatidae 6 2 1 Megadermatidae 6 5 2 Rhinonycteridae 9 5 4 Hipposideridae 90 33 21 Rhinolophidae 106 50 31 Pteropodidae 201 59 32 ica ica pe ica sia nia ed ed ive me r er ur o r l t m E A f A ce a am mp os i h A h A O N t t S a P r u No So Continent Status Family present, sampled, CoVs detected Family present, sampled, no CoVs detected Family present, not sampled Family not present Fig. 1 | Geographical and taxonomic distribution of reported bat hosts of coronaviruses. a | Biogeographical patterns of bat families, sampling and coronavirus host status. b | Bat taxonomic diversity and coronavirus testing results. Data were compiled from field studies involving sequencing of coronaviruses in wild bats. A list of all reported bat coronavirus hosts based on the reviewed studies can be found in Supplementary Table 1 and Supplementary data. ‘Named’ refers to the number of taxonomically described bat species per family based on the expert-curated Bat Species of the World database. Bat Species of the World database. CoVs, coronaviruses. While serological studies have been used for surveil- are generally key to understanding the epidemiological lance of pathogens such as Nipah virus, Marburg virus history of the population26, but the variability in adaptive and Ebola virus in bats, little serological information is humoral responses in bats suggests caution is required available for most coronaviruses in bats, although anti- in the interpretation of serological data, especially at the body responses may be relatively weak and transient. individual level. For example, limited humoral responses There are even fewer data on coronavirus-specific innate may make it difficult to use serology to identify infections immune responses, or whether those might render a by certain pathogens. robust antibody response less important. For example, In bats, coronaviruses may have tropism for the res- seroconversion of bats after challenge with coronaviruses piratory tract and the gastrointestinal tract. The highest is not always observed24,25. In experimental challenges loads of MERS-CoV RNA and infectious virus in exper- of Egyptian fruit bats (Rousettus aegyptiacus) with bat imentally infected Jamaican fruit bats were detected in SARS-like coronavirus WIV1 (originally isolated from the respiratory tract, with less virus in the intestines and a Chinese rufous horseshoe bat, Rhinolophus sinicus), internal organs24. Intranasal inoculation of Egyptian evidence of viral replication was limited, no bats showed fruit bats with SARS-CoV-2 resulted in transient res- obvious signs of disease and only 2 of 12 individuals piratory infections, with the highest viral loads in the seroconverted (measured by enzyme-linked immuno- respiratory tract on day 4 after inoculation, whereas sorbent assay), although no neutralizing antibodies were oral and faecal viral shedding was observed for up to detected25. When Jamaican fruit bats (Artibeus jamaicen- 12 days23. Long periods of viral shedding in faeces of sis) were challenged with a human isolate of MERS-CoV, 3–11 weeks have been reported in wild bats (Myotis mac- only one of ten bats produced neutralizing antibod- ropus), supporting the importance of a potential faecal– ies, and moderate pathological changes in the lungs oral route of transmission; in that field study, potentially were present and innate antiviral genes (MX1, CCL5 persistent infections could not be distinguished from and ISG56) were modestly upregulated24. It is unclear reinfections27. Viral RNA was also found in the intes- whether these apparently poor antibody responses result tines of Leschenault’s rousette (Rousettus leschenaultii) from weak infection of the bat species challenged — bats orally inoculated with a betacoronavirus isolated perhaps due to suppression of virus replication by the from a lesser short-nosed fruit bat (Cynopterus brachy- innate immune response — or naturally low viral capacity otis), but no infectious virus was isolated from recipient to infect the host species. In-depth seroprevalence studies bats nor was disease observed, suggesting the species is N aTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 301 0123456789();: R e v i e w s Bat species Species 120 90 60 30 1 Sampled species Species 84 63 42 21 1 Coronavirus hosts (alphacoronavirus and betacoronavirus) Species 39 30 20 10 1 Key coronavirus subgenus hosts Duvinacovirus hosts (alphacoronavirus) Species Species 28 6 21 5 14 3 7 2 1 1 Hibecovirus hosts (betacoronavirus) Merbecovirus hosts (betacoronavirus) Species Species 6 13 5 10 3 7 2 4 1 1 Nobecovirus hosts (betacoronavirus) Rhinacovirus hosts (alphacoronavirus) Species Species 14 16 11 12 7 8 4 4 1 1 Sarbecovirus hosts (betacoronavirus) Setracovirus hosts (alphacoronavirus) Species Species 14 2 11 7 4 1 1 302 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: R e v i e w s ◀ Fig. 2 | Geographical distribution of reported bat hosts of coronaviruses. Data on bat was associated with low body condition in Lyle’s flying hosts were compiled from field studies involving sequencing of coronaviruses in wild foxes (Pteropus lylei)40. In the Chinese rufous horse- bats. Where phylogenetic analysis was included in studies, key Alphacoronavirus and shoe bat (Rhinolophus sinicus), low body condition was Betacoronavirus subgenera of bats associated with human or domestic animal infections associated with increased shedding of one variant of or well characterized in bats (for example, Hibecovirus and Nobecovirus) are summarized Sarbecovirus (SARS-related Rhinolophus bat coronavirus (see Supplementary data). Geographical ranges of reported bat host species for any coronaviruses or key subgenera were obtained from the International Union for (SARSr-Rh-BatCoV)), but not of another Rhinolophus 41 Conservation of Nature (IUCN). The plots display the number of bat species based on coronavirus (Rh-BatCoV HKU2) . In Ghana, infec- overlapping geographical ranges. The plots of bat species include 1,317 species with tion by the alphacoronavirus Alpha229E-CoV corre- IUCN range data as of September 2021. Patterns in the left-hand maps indicate that lated with low body condition in Noack’s roundleaf bat sampling of bat species largely reflects the biogeographical patterns of bat diversity, (Hipposideros cf. ruber) but not in the Aba roundleaf with hotspots in Central America, South America, equatorial Africa and South-East Asia. bat (Hipposideros abae). However, hotspots of bat hosts of coronaviruses display important differences: lower Colony size, density and composition could also than expected diversity of hosts in South America and higher diversity of hosts in affect virus prevalence by changing transmission rates South-East Asia. Although biological differences in bat coronavirus interactions with both within and between roosts. Roost composition certain bat families (for example, Rhinolophidae) might explain some of these patterns, affects viral circulation as multiple bat species often roost small sample sizes in some species in the Americas and more intensive sampling in China and South-East Asia likely contribute as well. together and viral infection of different bat hosts will depend on combinations of the host species and the viral strains involved. For example, mixed-species roosts in not a competent host for this virus28. Further evidence Yunnan province, China, exhibited greater prevalence of of tropism of coronaviruses for the gastrointestinal and SARSr-CoVs when Rhinolophus sinicus, a primary host respiratory systems of bats comes from field studies in of SARSr-CoVs, was more abundant in the roost than which coronaviruses have been detected in intestines other species. In the same roost, the lowest prevalence of little brown bats (Myotis lucifugus)29. Additionally, was detected when Aselliscus stoliczkanus was the most expression of cell receptors used by multiple corona- abundant bat species34. Roost size and location, including viruses was high in both the gastrointestinal system whether the roosts are in caves, seem to affect the chance and the respiratory system in fruit bats, whereas it was of spillover of viruses between host species — likely due to present only in the intestines of insectivorous bats30. close physical contact in dense roosts42. In addition Many coronavirus infection studies have used bat to heterogeneity in competence among host species, cell lines (TablE 1; Supplementary Table 2), and mostly heterogeneity in shedding and infectivity (for example, focused on viral receptor binding, cell entry and infec- superspreading and aerosolization capacity) is a feature of tion, providing insights into the ability of specific corona- coronavirus infections in humans43. However, the extent viruses to infect cells from different hosts. Although to which this individual-level heterogeneity explains these studies may provide insights into the spillover coronavirus transmission in bats, variation in prevalence potential of specific viruses, they likely provide limited among roosts and the risk of spillover is unknown. insight into bat susceptibility at the organismal level — Reproductive cycles also influence prevalence and and studies making such inferences should be interpreted transmission of viruses in bat colonies by affecting with caution. For example, a study that used big brown patterns of behaviour and physiological susceptibility. bat (Eptesicus fuscus) kidney cells showed that innate Increased social contacts among different species of antiviral genes, specifically the interferon-β gene, were Chinese horseshoe bats during the mating season and not repressed by MERS-CoV31, and long-term persis- when feeding after hibernation might explain peaks of tent MERS-CoV infections were achieved in these big SARSr-Rh-BatCoV and Rh-BatCoV HKU2 infection brown bat cells. However, whether those viruses cause in spring41. In species that form maternal roosts, for persistent infections in bats cannot be predicted without example, increases in group size coincide with preg- infections of live animals. nancy and gestation, during which time inflammatory immune responses are downregulated, potentially facil- Circulation in bat populations. The prevalence of corona- itating infection and shedding44,45. Periparturient stress viruses — as estimated by the proportion of bats with may also affect viral shedding, as observed in greater Body condition detectable viral RNA in faeces or in faecal or oral swabs — horseshoe bats (Rhinolophus ferrumequinum), Geoffroy’s Proxy for nutritional status shows high temporal and spatial variability (Fig. 4). Overall, bats (Myotis emarginatus)46 and mouse-eared bat (Myotis of an organism. Commonly shedding of coronaviruses tends to peak during summer myotis)47, in which both the proportion of bats shedding measured as the body mass or autumn in Australia and China32–35, dry seasons in virus and viral concentrations increased after parturi- above or below that predicted 16 as a function of skeletal size. central Africa and Asia , and wet seasons in western or tion. Similarly, in relation to reproductive cycles, high south-eastern Africa36,37. Although trends differ among prevalence and concentration of coronaviruses detected Superspreading studies, seasonal variations are consistently observed, in Chinese horseshoe bats (predominantly Rhinolophus Transmission event in which pointing to potential mechanistic roles of resource sinicus) during September and October, are attributed one infected host generates 32,34 several new infections above availability, reproductive cycles and host behaviour. to increases in the number of susceptible juveniles . the average in the population. Although nutritional stress during periods of Cross-sectional surveys of multiple bat species report resource scarcity has been implicated in the shedding higher infection rates or viral shedding in juveniles and Aerosolization of other bat viruses38,39, their influence on coronavirus subadults, supporting age-related differences in suscep- Physical process by which a shedding is unclear, with effects differing by bat species tibility and competence of infection, consistently across pathogen stabilizes in particles and virus variants. In Thailand, increased prevalence species16,40,48,49small enough to be transported . Further field studies of multiple species through air currents. of both alphacoronaviruses and betacoronaviruses across East Africa found that in both age categories, N aTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 303 0123456789();: R e v i e w s shedding was highest during weaning49 — timing that through coordinated and systematic approaches to field relates to behavioural changes, physiological stress and studies that sample individual bats, paired with experi- potential waning of maternal immunity. mental inoculation and transmission studies, and then Although some associations have been seen between integrated with modelling studies aimed at assessing the seasonal factors and circulation of coronaviruses in bats, importance of factors driving infection50. our understanding of the mechanisms is currently insuf- ficient to predict dynamics of shedding (Fig. 4). Many of Co-infections in bats. Co-infections with multiple the associations with seasonal factors may be coinciden- pathog ens can influence transmission to conspecifics tal rather than causal, explaining the lack of consistent and to spillover hosts. Cross-protective immunity from patterns across taxa and geographies. Small sample sizes infection by related pathogens might reduce suscepti- and limited temporal resolution are common issues that bility or transmission, whereas trade-offs in immune hamper statistical power. We could vastly improve our response to one pathogen might increase susceptibility understanding of coronavirus dynamics across species and facilitate transmission of another39,51. Co-infection of bats with multiple coronaviruses at the same time, or co-circulation of multiple virus genotypes within a roost, Table 1 | Summary of 214 original studies on coronaviruses in bats might result in interactions that affect the timing, location Study typea Number of studies Overview and intensity of virus shedding, as has been described in 39 Experimental Bat cell lines: 29 Target cells: brain, embryo, other viral families . As with other putative drivers, the intestine, kidney, lung incidence and effects of coronavirus co-infections on Tested viruses: multiple bat transmission dynamics in bats are not well understood. SARS-related CoVs, BatCoV Co-infections by two coronavirus species36,41,52–57 and by HKU4, BatCoV HKU9, HCoV-229E, coronaviruses and viruses from other families, including HCoV-NL63, MERS-CoV, PEDV, adenoviruses58,59, astroviruses58,60–62, herpesviruses58 and Ro-BatCoV GCCDC1, SADS-CoV, 63 SARS-CoV, SARS-CoV-2, paramyxoviruses , have been described and are likely Scotophilus BatCoV 512, TGEV common. Cases of co-infections (by detection of viral Live bats: 6 Tested hosts and viruses: RNA) involving coronav iruses range from 0.2% to 34.2% 36,52–56 Artibeus jamaicensis (MERS-CoV), in wild bats and are as high as 73% in captive bats64, Eptesicus fuscus (SARS-CoV-2), while up to 88% of virus-positive samples contained Myotis lucifugus (Myl-CoV), multiple viral families60. Frequent co-infection has addi- Rousettus leschenaultii (BatCoV tional important consequences because coronaviruses HKU9), Rousettus aegyptiacus (bat SARS-like CoV WIV1, recombine frequently, providing an opportunity for SARS-CoV-2) the emergence of new variants with altered properties, Longitudinal 14 Countries: Australia, China, including host ranges. Denmark, Germany, Malaysia, A few studies have examined ecological interactions Singapore, South Korea, Thailand between co-infections of coronaviruses and non-viral (n = 8) pathogens, including whether they are competitive, Serially sampled bat families: synergistic or neutral. For instance, a 60-fold increase Pteropodidae, Hipposideridae, in coronavirus (Myotis lucifugus coronavirus) RNA Vespertillionidae, Rhinolophidae was observed in the intestines of bats (Myotis lucifu- (n = 4) gus) co-infected with the fungus that causes white nose Serially sampled species: syndrome (Pseudogymnoascus destructans)65. Systemic Eonycteris spelaea, Hipposideros cervinus, Myotis daubentonii, downregulation of antiviral immune responses due Myotis macropus, Myotis myotis, to Pseudogymnoascus destructans infection was sug- Pteropus lylei, Rhinolophus sinicus, gested as the cause of increased coronavirus replication. Rousettus leschenaultii (n = 8) Similarly, ectoparasite loads have been associated with Surveys Cross-sectional, intraspecies: 14 Sampled countries: primarily in coronavirus infection; infection with Alpha229E-CoV Cross-sectional, interspecies: 123 Asia, Africa and Europe; fewer in almost doubled the risk of infection by BetaBI-CoV the Americas or Oceania (n = 69) in Noack’s roundleaf bat but also correlated positively CoV detection and sequencing only: 29 Sampled bat families: all bat with loads of streblid flies, mites and nycteribiid flies 36. families have been sampled at Longitudinal studies tracking the health and immune Multipathogen detection: 36 least once except Cistugidae, Furipteridae and Myzopodidae status of individual bats, including co-infections, are (n = 18) crucial to understanding the dynamics of bat viruses. Positive bat families: 14 Molecular evolution and host range Sampled bat species: 543 Viral genetic diversity and evolution. Coronaviruses Positive bat species: 238 have the largest genome among the RNA viruses, and BatCoV, bat coronavirus; CoV, coronavirus; HCoV, human coronavirus; MERS-CoV, Middle are subject to both mutation and recombination66. These East respiratory syndrome coronavirus; Myl-CoV, Myotis lucifugus coronavirus; PEDV, porcine epidemic diarrhoea virus; Ro-BatCoV, Rousettus bat coronavirus; SADS-CoV, swine acute processes generate genetic diversity, some of which may diarrhoea syndrome coronavirus; SARS, severe acute respiratory syndrome; SARS-CoV, severe introduce new properties, including altered host ranges, acute respiratory syndrome coronavirus; TGEV transmissible gastroenteritis virus. aStudy types along with increases in the ability to spread in the new were not exclusive, so a study may fit into multiple types depending on the sampling approach and analytical methods. More details are provided in Supplementary Table 2, and all classified host. Approximately two-thirds of the coronavirus studies can be found in Supplementary data. genome encodes an RNA-dependent RNA polymerase 304 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: R e v i e w s Genus/subgenus Notable virus species Hosts species Alphacoronavirus Colacovirus Colacovirus Myl-CoV Bats (Vespertilionidae) Pedacovirus Pedacovirus PEDV Bats (Vespertilionidae), pigs Nyctacovirus Nyctacovirus Bats (Vespertilionidae) Decacovirus Bats (Hipposideridae, Rhinolophidae) Decacovirus Minunacovirus Bats (Miniopteridae) Minunacovirus Myotacovirus Bats (Vespertilionidae) Myotacovirus Duvinacovirus HCoV-229E Bats (Hipposideridae), dromedary Duvinacovirus camels, alpacas, humans Setracovirus HCoV-NL63 Bats (Rhinonycteridae), humans Setracovirus Rhinacovirus SADS-CoV Bats (Rhinolophidae), pigs Rhinacovirus Luchacovirus Rodents (Muridae, Cricetidae) Luchacovirus Minacovirus Ferrets, minks Minacovirus Tegacovirus CCoV, FCoV, TGEV Cats, dogs, pigs Alphacoronavirus SoracovirusTegacovirus Shrews (Suncus murinus) Sunacovirus Shrews (Sorex araneus) Soracovirus Betacoronavirus Sunacovirus Hibecovirus Bats (Hipposideridae) Hibecovirus Sarbecovirus SARS-CoV, SARS-CoV-2 Bats (Rhinolophidae), Malayan pangolins, carnivores (Canidae, Felidae, Sarbecovirus Mustelidae, Viverridae), humans Nobecovirus Nobecovirus Bats (Pteropodidae) Betacoronavirus Merbecovirus Merbecovirus MERS-CoV Bats (Vespertilionidae), dromedary camels, humans Embecovirus Embecovirus BCoV, CRCoV, Rodents (Muridae, Cricetidae), dogs, Deltacoronavirus HCoV-OC43, rabbits, cattle, horses, pigs, sable Gammacoronavirus HCoV-HKU1, antelopes, dromedary camels, MCoV giraffes, humans Deltacoronavirus PorCoV-HKU15 Birds, pigs Gammacoronavirus IBV Birds, cetaceans Fig. 3 | Coronavirus taxonomy and host distribution. The proposed phylogeny has been compiled from analyses of full genomes and/or gene segments. Branch lengths do not reflect evolutionary distance between taxa and are drawn only to clearly illustrate relationships between and within genera. The distribution of bat species hosting highlighted subgenera is given in Fig. 2. The associated table summarizes a selection of important pathogenic virus species within genera and the host species or taxa with reported infections of a virus within a genus. BCoV, bovine coronavirus; CCoV, canine corona- virus; CRCoV, canine respiratory coronavirus; FCoV, feline coronavirus; HCoV, human coronavirus; IBV, infectious bronchitis virus (avian coronavirus); MCoV, murine coronavirus; MERS, Middle East respiratory syndrome; Myl-CoV, Myotis lucifugus coronavirus; PEDV, porcine epidemic diarrhoea virus: PorCoV, porcine coronavirus; SADS-CoV, swine acute diarrhoea syndrome coronavirus; SARS-CoV, severe acute respiratory syndrome coronavirus; TGEV, transmissible gastroenteritis virus. and other non-structural proteins required for replication, host cell, subgenomic rNas are generated, which result while the remaining third encodes four structural pro- from the polymerase jumping to new positions in the teins — the spike, envelope, membrane and nucleocapsid template genome. This may facilitate recombination proteins — as well as accessory proteins67. The genomes of genes from different coronavirus lineages during of coronaviruses replicate via the RNA-dependent RNA co-infection of a host cell when the RNA-dependent polymerase, which is generally error-prone, resulting in RNA polymerase ‘jumps’ from one RNA template mol- mutations during replication68,69. However, the three larg- ecule to another one that may come from a different viral est viral families in the order Nidovirales — Coronaviridae, genome66,78. These recombination processes have been Roniviridae and Mesoniviridae — all encode a 3′–5′ exor- implicated in the cross-species emergence of numerous ibonuclease that improves their RNA replication fidelity, novel coronaviruses, including murine coronavirus79, which may be necessary for maintaining sufficient fitness transmissible gastroenteritis virus80, feline and canine in the large genome70–73. The activity of the exoribonucle- coronaviruses81,82, and six of the seven human corona- ase might differ in different hosts, modulating the level of viruses, HCoV-OC43 (rEF.83), HCoV-NL63 (rEFs8,84), sequence variation. Replication in different host species HCoV-229E8, HCoV-HKU1 (rEF.85), SARS-CoV86,87 and may therefore present heterogeneities in their sequence MERS-CoV88. Interestingly, evidence supports recom- variation, which may influence the emergence of new bination of coronavirus genomes possibly happening variants16,20,74–76. also with RNA viruses from the family Reoviridae89. Recombination of large coronavirus genomes is However, how frequent interfamily recombination Subgenomic RNAs common; recombination creates additional genetic events may happen and their consequences for evolution Fragments of rNa smaller diversity, expands viral evolution and increases the of zoonotic potential are unknown. than the full genome size generated during replication potential for shifts in cell tropism, host range66 and Mutation, recombination and host competence for of coronaviruses in a host cell. pathogenicity77. During coronavirus replication in the infection and co-infection will have generated the current NaTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 305 0123456789();: R e v i e w s 100 a g 75 50 25 0 100 b h 75 50 25 0 100 c i 75 50 25 0 100 d j 75 50 25 0 100 e k 75 50 25 0 1 2 3 4 5 6 7 8 9 10 11 12 100 f Month of sampling 75 50 25 0 1 2 3 4 5 6 7 8 9 10 11 12 Month of sampling Fig. 4 | Prevalence of detection of bat coronaviruses in field samples. Data were obtained from published studies that included two or more sampling events with at least ten samples collected and that reported the virological status of samples (positive and negative). While the data show that prevalence varies in space and time and by bat species (each plot), few studies provide insights into the drivers of prevalence. No field studies have yet combined longitudinal sampling of individuals with collection of extensive metadata on bat ecology, bat health and environmental conditions. Sampling designs differed across studies. Most studies conducted cross-sectional sampling over multiple years. One field study sampled individual bats at multiple sites over time, although data were pooled across three sites40 (panel a). Other studies sampled the same bat species over time across multiple sites or sampled multiple species and individuals in pooled samples across time within a site. These sampling approaches reflect the purpose of the studies — most were focused on characterizing viral diversity, not infection dynamics. Details are presented in Supplementary information. Each plot represents the prevalence of detections per bat species: Pteropus lylei (panel a)40; Eonycteris spelaea (panel b)64; Rousettus leschenaultii (panel c)158; Chaerephon pumilus (panel d)49; Eidolon helvum (panel e)49; Myonycteris angolensis (panel f)49; Rhinolophus cf. clivosus (panel g)49; Myotis daubentonii (panel h)159; Rhinolophus sinicus (panel i)160; Rhinolophus sinicus, Rhinolophus ferrumequinum, Rhinolophus affinis and Aselliscus stoliczkanus (panel j)34; and Myotis myotis (panel k)47. Colours in the plots represent different years within the study: year 1, red; year 2, blue; year 3, green; year 4, purple; and year 5, orange. Black asterisks show sampling events in which no coronavirus was detected. Circles show the mean prevalence, and bars show the 95% confidence intervals estimated by the Wilson method. 306 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: Sample prevalence (%) R e v i e w s diversity of coronaviruses, including that seen in bats16,90. suggesting that zoonotic capacity could emerge in a few Some families of bats appear to have coevolved over mil- evolutionary steps. lions of years with particular subgroups of betacorona- Isolates of bat coronaviruses are difficult to obtain, viruses: rhinolophid bats and SARS-related sarbecoviruses, and therefore their zoonotic capacity is largely unknown, vespertilionid bats and MERS-related merbecoviruses, with many inferences being based on genomic sequences. and pteropodid bats and nobecoviruses (which have not Among 187 studies that examined coronaviruses in been implicated in zoonosis)90. Host switching, resulting primary samples from wild bats, in less than a quar- from successful broad jumps in host range, appear most ter, researchers attempted to recover live viruses in common in the rhinopholid–Sarbecovirus clade16,20,91. one or more cell cultures, yielding only five viral spe- Altogether, the variation in the bat coronaviruses may cies successfully cultured, including one merbecovirus enable them to gain new host and tissue tropisms, and (Tylonycteris BatCoV HKU4), three sarbecoviruses varying transmissibility and infection severity in new related to SARS-CoV (WIV1, WIV16 and Rs4874) and hosts. Indeed, once a virus is established in a new host one sarbecovirus related to SARS-CoV-2 (BANAL-236), population, evolution is expected to enable selection for reported in September 2021 (Supplementary data). lineages with increased fitness in those hosts, includ- High-throughput analyses of sequences and carefully ing exhibiting higher transmissibility, as observed for controlled cell culture experiments and other experiments SARS-CoV-2 in humans92. are needed to assess spillover and zoonotic potential of the coronavirus variants currently circulating in bats1. Host receptor recognition. The capacity of coronaviruses In silico analysis of cell receptors of humans and other to enter a host cell is mediated by the spike protein, species are useful for initial identification of species that which supports both binding to the host cell — through could serve as bridge or reservoir hosts of zoonotic corona- its receptor-binding domain (RBD) — and fusion with viruses, which could promote optimization of resources its membrane67. The RBD attaches to host-cell receptors, for pre-emptive surveillance. For instance, relatively which are membrane proteins or sialic acids. For exam- conserved SARS-CoV-2-binding residues in the ACE2 ple, HCoV-NL63, SARS-CoV and SARS-CoV-2 bind sequences of non-human primates, hooved mammals, angiotensin-converting enzyme 2 (ACE2), MERS-CoV felids and cetaceans suggest those species would be sus- binds dipeptidyl peptidase 4 (DPP4) and HCoV-229E, ceptible to infection100. Several of these predictions have canine coronavirus and several porcine and feline corona- been validated by empirical studies confirming the broad viruses bind alanine aminopeptidase (APN), whereas host range of SARS-CoV-2 (rEFs98,101). However, these HCoV-OC43, HCoV-HKU1 and bovine coronavirus studies also classified horseshoe bats, pangolins, minks bind N-acetyl-9-O-acetylneuraminic acid93–96. and ferrets as less likely to be hosts of SARS-CoV-2, yet The interaction between the RBD of the coronavirus field and laboratory data have revealed their susceptibility spike protein and the host receptor can be thought of as to SARS-CoV-2 or related viruses, highlighting the need a match between a key and a lock, and the specific struc- for empirical validation of model predictions101,102. tures of both the virus RBD and the receptors available It is likely that differences will be seen between in sil- on a potential host cell determine, in part, the capacity ico analysis and empirical analysis of receptor use by virus for infection of different hosts. The functional interac- species in different hosts. Several studies suggest that tions between the viral protein and the host receptor dif- the progenitor viruses of SARS-CoV and SARS-CoV-2 fer, and the wide host range of several coronaviruses can may not use the ACE2 receptor in their original bat be explained by the conservation of cell receptor struc- hosts100,103,104. However, this discrepancy could also tures across animal species, such is the case of ACE2, result from variability in the host receptors with which DPP4 and APN97,98. However, small differences in recep- the viruses have evolved, favouring specific interac- tor structures can also alter receptor affinity and virus tions between the RBD and small numbers of receptor infection efficiency, including both variation in glycosyl- residues, so that progenitor viruses are adapted to their ation profile or amino acid changes93. MERS-CoV spike specific reservoir ACE2, but not to the human ACE2 protein, for instance, binds DPP4 of various species of (rEF.99), which is used to model many interactions100. primates, hooved mammals and bats, but not of ferrets There is naturally high variation among the ACE2 recep- and rodents owing to differences in five amino acids in tors of bat species105, in addition to the high diversity of the receptor97. Thus, direct coronavirus spillover from SARSr-CoVs106. New host infection and adaptation likely bats to other mammals would therefore be regulated by involves mutations in the viral spike protein, and poten- the host-cell receptor structures and viral RBD identity. tially selection in an intermediate (bridge) host, to enable This is a critical aspect for characterization of zoonotic effective binding and use of human ACE2 and facilitate potential of extant bat coronaviruses; however, for res- zoonotic spillover104,107. Such a case is supported by the ervoir bat hosts we know relatively little about their use of human DPP4 by MERS-CoV, where affinity for receptors or interactions with the viruses. It is currently the human receptor may have emerged by evolution of the known from experimental and modelling work that sev- virus in dromedary camels, after the initial spillover eral bat coronaviruses bind to human ACE2 or DPP4; from bats76. Importantly, virus evolution that facilitates however, structural modelling and biochemical data binding of human receptors may diminish the binding indicate differences in binding affinity97–99 and there- affinity of a virus for the receptors of the original res- fore potential for successful infection of human cells. In ervoir hosts108, indicating a host shift that may favour some cases, there is only one amino acid residue dif- sustained human-to-human transmission. Such behaviour ferent between the spike protein RBD and the receptor, is characteristic of pandemic viruses (box 1). NaTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 307 0123456789();: R e v i e w s Box 1 | Pathways to pandemic emergence of bat coronaviruses While the zoonotic potential of an animal virus depends on its ability levels to increase the probability of initial infection (see the figure, and opportunity to infect humans, pandemic potential depends on panel b). human-to-human transmissibility, quantified by the virus’s reproduction another possibility is that a virus circulating in bats would be subcritical in number in humans, R. The critical value for R is 1, the level at which humans but has opportunity to evolve to become supercritical within a each case replaces itself on average. For subcritical viruses, with R < 1, bridge host that shares some key traits (for example, homologous receptor transmission chains inevitably die out. For supercritical viruses, with R > 1, proteins) with humans (see the figure, panel c). a fourth possibility, not epidemics and pandemics are possible139. depicted here, is that a subcritical virus reaches humans and evolves to Novel viruses with pandemic potential can reach humans by several become supercritical before its transmission chains die out161. routes. a virus circulating in bats could have the traits needed for super- In any of these scenarios, epidemiological factors (and simple chance) critical transmission in humans, by chance or due to evolutionary pressures will determine whether the supercritical virus goes on to cause an in the reservoir that fortuitously align with fitness in humans161. epidemic or a pandemic. many such introductions die out, particularly if Such a virus could spill over directly from bats to humans, overcoming transmission is highly heterogeneous43. Reconstruction of outbreak origins ecological barriers of limited spatial overlap and contacts between hinges on the availability of data and samples from the earliest human these species (see the figure, panel a). alternatively, such a virus could cases, and extensive sampling of all host species involved (which often are reach humans via a bridge host that has greater contact with humans than not known with confidence). origins and emergence pathways will remain the reservoir host, and perhaps also serves to amplify the virus to high obscure until such data are obtained and analysed. Coronavirus in bats is supercritical for humans (R > 1) Coronavirus in bats is subcritical for humans (R < 1) a b c Key Ecological Epidemiological Compatibility Zoonotic potential Time Part a of the figure adapted from rEF.140, Springer Nature limited. Once a coronavirus RBD can bind a receptor on a host tissues, including the respiratory tract, kidney, heart and cell, the differing distribution of those receptors in differ- digestive tract, consistent with the respiratory and gastro- ent cell types within a host will influence tissue tropism, intestinal pathology of SARS-CoV and the multisys- impacting pathogenesis and transmission. In humans, temic pathology of SARS-CoV-2 (rEFs109,110). Although ACE2 is expressed primarily in epithelial cells of many detailed expression profiles of ACE2 in other species are 308 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: R e v i e w s lacking, tissue tropism of SARSr-CoVs in several animals the evolution and zoonotic capacity among corona- is consistent with that in humans. SARSr-CoVs have been viruses naturally circulating in bats. However, for detected in the respiratory tract or gastrointestinal tract zoonotic spillovers to occur, humans must be exposed of Malayan pangolins (Manis javanica)111, experimentally to the viruses (box 1), and this can occur through direct inoculated ferrets, felids101,112 and non-human primates113. contact with virus excreted from infected bats or bridge Similarly, DPP4 expression in humans, dromedary cam- hosts, or through other contacts with infected animals, els and fruit bats includes epithelial cells of the respiratory such as slaughtering or butchering. The nature and and gastrointestinal tracts30. In humans, ACE2 expression intensity of the reservoir bat–human interface are criti- is particularly high in the upper respiratory tract, while cal to determining spillover risk. Human behaviour is a DPP4 is expressed mainly in the lower respiratory tract, primary determinant of exposure, which may increase potentially contributing to the greater human-to-human contact with bats or with other animals (bridge hosts) transmissibility of SARS-CoV and SARS-CoV-2 that may expose susceptible humans. Little is known compared with MERS-CoV30,114. Additionally, DPP4 about the specific conditions of coronavirus spillovers, expression is almost entirely restricted to the intes- but human behaviours that may increase viral exposure tines in two vespertilionid bats, the putative reservoir include activities such as bat hunting and consumption, of the MERS-CoV progenitor, suggesting different guano farming and wildlife trading4,5,123. These contacts tropism, and potentially transmission routes, between res- between humans and bats likely occur under physiologi- ervoir and spillover hosts30. Nevertheless, the detection of cally stressful situations that may increase viral shedding coronaviruses in the respiratory and gastrointestinal tracts from bats or bridge hosts and exposure of humans — the of experimentally inoculated and wild-caught bats sup- potential ‘patients zero’ of a new epidemic. Exposures ports the relevance of these two systems for coronavirus often occur in rural areas with limited access to health infections among diverse host species18,24,29,41,78,90. care, so spillovers are detected only when they cause outbreaks or epidemics. For recently emerged human Host proteases and host range. Besides binding to the coronaviruses, some factors are known, including roles cellular receptor, successful infection and replication for bridge hosts in the wildlife trade or among domestic require several consecutive steps, including entry, repli- animals; for example, SARS-CoV likely transferred from cation, potential evasion of the host innate immunity and rhinolophid bats into humans via farmed Himalayan budding. In addition to the receptors, host proteases are civets (Paguma larvata)78,124,125. Alternative pathways of needed to activate (cleave) the virus spike protein to enable direct human exposure to bat coronaviruses have not entry, and this cleavage may be as important as host recep- been explored thoroughly, and studies that specifically tor binding in determining viral zoonotic potential96,115 examine human populations at risk of exposure, such and potentially human-to-human transmissibility92. Spike as guano farmers, bat hunters and wildlife traders, for proteins of coronaviruses have multiple cleavage sites for evidence of bat coronavirus exposure126 and the roles host proteases, which are cleaved at different stages of the of other species in the transmission chain (box 2) are cell infection cycle114. Transmembrane serine proteases required for effective surveillance of, response to and (such as TMPRSS2), trypsin-like proteases and other prevention of future zoonotic coronavirus pandemics. cell-surface proteases participate in spike protein cleavage after viral attachment, whereas lysosomal proteases such Reservoir animal–human interface. Human–bat inter- as cathepsins cleave spike proteins after virus endocytosis. actions differ widely in space, time, nature and inten- By contrast, the furin proprotein convertase — present sity; some bat species rarely encounter humans, whereas in the Golgi apparatus — may be involved in spike pro- tein cleavage during biosynthesis116,117. The distribution Box 2 | Spillover of coronaviruses in other species and activity of these proteases differ among cell types and Coronaviruses have a demonstrated ability for cross- physiological conditions, therefore influencing tissue tro- species transmission involving not only bats and humans, pism and cell entry114,118,119. For instance, the respiratory but also transmission to and among other animal species. tropism of SARS-CoV might be driven by trypsin-like For example, HKu2, a coronavirus related to a virus proteases present in respiratory cells120,121. detected in rhinolophid bats, caused an outbreak of Therefore, the expression patterns of proteases also fatal disease in domestic pigs in China in 2016 (swine directly contribute to host range. For instance, while acute diarrhoea syndrome coronavirus; Fig. 3)12. In 2017, specific bat proteases cleave the spike proteins of both camel (HKu23) and equine coronaviruses were detected MERS-CoV and BatCoV HKU4 and enable entry into in asymptomatic domestic horses in Saudi arabia and 162 bat cells, human proteases cleave only the MERS-CoV oman . In 2020, chicken, duck, pigeon and goose spike proteins122. Understanding how coronavirus spike coronaviruses were observed in live-poultry markets in China, where each of the viruses was found in species proteins adapt to being activated by proteases of new of birds other than its primary host163. In the 1980s, a hosts (for example, to type, activity and distribution) is fatal outbreak of feline infectious peritonitis in cheetahs essential for predicting the potential for changes in host (Acinonyx jubatus) was caused by a feline coronavirus range and tissue tropism, including spillback infection. that circulates in domestic cats164. Within feline corona- viruses, type II feline coronavirus emerged from recombi- Spillback infection Human exposure and spillover nation between type I feline coronavirus and canine also called ‘anthropozoonosis’. The great diversity of bat species in which alphacorona- coronavirus82,165, highlighting the potential role of Transmission of a zoonotic pathogen from humans to viruses and betacoronaviruses have evolved, and the co-infection in new hosts in the emergence of new genetic variability of these RNA viruses, facilitates coronaviruses.animals. N aTuRe RevIeWS | MiCrObiOlOGy volume 20 | may 2022 | 309 0123456789();: R e v i e w s others have frequent contact. For example, humans in not efficiently spreading among humans. It is unknown Oceania, Asia, Africa, South America and Pacific islands whether the antibodies detected arise entirely from have long hunted fruit bats for food127,128. Humans in primary spillover or from a combination of primary Thailand, the Philippines, Indonesia, Mexico and the cases with limited human-to-human transmission139,140. United States harvest guano from bat caves for agricul- Syndromic surveillance at health-care facilities, com- tural fertilizer129. Those long-term bat–human inter- bined with improved unbiased molecular diagnostic actions contrast with the recent increasing emergence tools that could target unknown pathogens, and periodic of highly virulent infections in humans linked to bats. serological surveys of human populations are important Land-use change, animal farming and domestication, tools to provide better understanding of when, where and human expansion into wildlands, among other and how coronavirus spillovers occur. Technologies factors, have been linked to the emergence of infec- such as phage immunoprecipitation sequencing or Virscan tious diseases in general, and most likely play a role in that use coronavirus sequences recovered from multiple spillover of bat-borne viruses3. Changes in the qual- species (including bats) would enable multiantigen test- ity of bats’ habitat may also affect their overall health ing that can reveal undetected past spillovers and other and viral circulation owing to factors such as stress130. epidemics141. Low food availability, mediated by climate change and deforestation, appears to be a driver of shedding of other Spillover through bridging hosts. Besides bats, other ani- viruses in bats, including the zoonotic Hendra virus mals may provide ecological, amplifying or evolutionary and Nipah virus131,132. Coronavirus shedding in horse- opportunities for coronavirus transmission from bats to shoe bats was higher in human-dominated landscapes humans9,78. Once infected from bats, such bridging hosts than in natural landscapes16. In addition, the legal and may promote virus spread to humans through increased illegal wildlife trade results in viruses being transported exposure or high viral loads. This will lead to a higher over longer distances within hosts maintained in stress- probability of human exposure to infectious doses of the ful and unsanitary conditions, likely increasing shedding viruses, as seen for Hendra virus, where the initial spillover and transmission, as demonstrated for coronaviruses in and infection of horses leads to exposure and infection in rodents5 and MERS-CoV in dromedary camels133. humans142, or for Nipah virus, through infection of swine143. In addition, bridging hosts may also enable viral evolution Direct bat-to-human spillover. There are currently no that results in new or enhanced zoonotic capacity78. Farmed well documented cases of direct bat-to-human spill- Himalayan palm civets are thought to have served as bridge over infections by coronaviruses, but this is likely due hosts in the spillover of SARS-CoV from bats to humans, to inadequate surveillance rather than to a true absence and selection for enhanced viral replication in civets may of spillovers. Infections occurring in rural areas or in have favoured viral mutations that increased zoonotic low-resource countries, where human–bat contacts capacity78,124,125. Endemic circulation of MERS-CoV in might be common but access to health care is limited, dromedary camels suggests that transmission of ancestral likely go undetected. Furthermore, infection by some merbecoviruses from bats to camelids occurred decades bat coronaviruses might be asymptomatic in humans or much longer ago, and likely resulted in evolution of or might be mistaken for other common diseases. zoonotic capacity133,144. Thus, MERS-CoV is considered a Even for highly virulent pathogens for which surveil- camelid virus with ancestral origins in bats145–147. lance programmes exist, such as Ebola virus or Nipah The ecological and evolutionary conditions that facil- virus, reported spillover events appear to be the tip itated the spillover of SARS-CoV-2 remain unknown of the iceberg134,135 and are recognized only after sub- for now; however, circulation of closely related sarbeco- stantial human-to-human transmission. In the case of viruses in horseshoe bats in Asia supports an ances- Ebola virus, it takes on average 44 days of undetected tral origin in bats148. Whether the first SARS-CoV-2 transmission before an outbreak is recognized136. transmission event happened directly from bats to Bat coronaviruses face numerous barriers that likely humans or through a bridging host — possibly involv- reduce infection and spread among humans. Those may ing host-specific evolution that increased infectivity for occur at the levels of exposure (lack of bat virus–human humans — is unclear. However, coronaviruses closely Phage immunoprecipitation contact), infection (coronavirus is not compatible with related to SARS-CoV-2 with the capacity to infect sequencing humans) or transmission (virus cannot be efficiently humans cells have circulated widely in bats, supporting Technique in which synthetic transmitted among humans). Perhaps, very few human the possibility of direct bat-to-human transmission106. antigens are displayed in a viral exposures lead to infection, and even fewer to onward In addition to the infection of humans from other particle (T7 phage) enabling assessment of the reactivity transmission. Studies in Asia have found serological reservoirs, humans can also act as bridging hosts for of serum samples against evidence of human exposure to SARS-CoV or related reverse zoonoses. Humans have infected domestic cats, antigens from several viruses viruses in healthy adults in Hong Kong and army dogs, large felids (for example, tigers (Panthera tigris)) simultaneously. recruits in mainland China sampled before the 2002 and farmed American minks (Neovison vison) with VirScan SARS pandemic 137,138. More recent studies of villagers SARS-CoV-2, which could potentially act as reservoirs High-throughput method in the southern Chinese province of Yunnan found low for new variants149. In the specific case of farmed minks, to profile the reactivity seroprevalence of antibodies to SARSr-CoVs13,14. These SARS-CoV-2 can spread at epidemic levels, facilitating of a serum sample against studies suggest that bat-associated coronaviruses are viral adaptation to the new host149. Thus, spillback to antigens from several viruses potentially breaching the exposure and infection bar- other wildlife species might lead to establishment in sec- simultaneously using phage immunoprecipitation riers, although the low seroprevalence (less than 3%) ondary reservoirs. ACE2 sequences of cricetid rodents sequencing. indicates that such cases are rare, and these viruses are suggest many are putatively susceptible to SARS-CoV-2. 310 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: R e v i e w s R Old World Syrian hamsters (Mesocricetus auratus), Chinese Our ability to understand mechanisms leading to reproductive number, the hamsters (Cricetulus griseus) and New World North successful spillover is limited by the apparent rarity number of new infections American deer mice (Peromyscus maniculatus) are cricetid of spillover events as well as by the limited ecological generated by an average rodents that are susceptible to SARS-CoV-2 (rEFs150–153). data available. Assessment of spillover risk requires an infected host in the population. Although many wild and domestic animal species are sus- increased capacity to detect these events, especially those Metapopulation ceptible and could even transmit the virus among them- that are missed by public health surveillance. Serosurveys spatial arrangement of selves (for example, see rEFs149,151,154–156), it is unclear for in humans and potential bridge hosts at risk of exposure populations of a species that these species how transmission dynamics, population size, to bat coronaviruses should be prioritized, and multiplex are connected by migration structure and connectivity, and eventual immunity would serological technologies, such as luminex or VirScan, processes. influence the establishment of continuous or temporary could facilitate wide screening, even when an agent has Luminex reservoirs. This evidence of reverse zoonosis or spillback not been fully characterized141. Human-focused sur- Technology that enables calls for further research to elucidate the potential for veillance, coupled with spatiotemporal information on measurement of multiple other wild animal species becoming new viral reservoirs. bat–virus interactions, viral discovery and functional proteins in a single well (sample). characterization are needed to estimate the magnitude Knowledge gaps and research agenda and frequency of spillover events that might have gone Fundamental knowledge gaps remain about the different undetected in the past. It is urgent to implement this conditions that result in coronaviruses passing from bats field research agenda, targeting high-risk interfaces in into humans. Dynamic integration among field studies, areas of rapid environmental change. modelling, laboratory experiments and human epide- Finally, as we fill the gaps and integrate knowledge miology is required to understand the processes and to across scales and disciplines, we should also develop pro- prevent new coronavirus spillovers and pandemics157. active strategies for spillover prevention, in addition to The extensive study of coronavirus diversity in wild reactive outbreak mitigation. The exponential nature of bats has yet to translate into a more profound under- epidemic growth makes stopping a new pathogen with standing of their zoonotic capacity. For instance, it is efficient person-to-person transmission a difficult task, unknown whether coronaviruses circulating in bat as demonstrated by SARS-CoV-2. As we understand the populations can be transmitted directly to humans and conditions that facilitate spillover, interventions to pre- whether they can be transmitted among humans with vent those conditions will become clearer, and proactive R > 1 without passage through bridging host species. actions may be taken to prevent the next coronavirus Combining the probabilistic ecological drivers of spill- pandemic. over with an understanding of the molecular basis of host range and tropism will lead to a more comprehensive Conclusions understanding of the zoonotic capacity of coronaviruses. Coronaviruses that circulate in bat populations have To accomplish this, a high-throughput characterization spilled over into human populations several times, and of the zoonotic potential of bat coronaviruses using a most likely will continue to be a public health threat. tiered system of in silico, in vitro and in vivo methods The diversity and broad geographical distribution of is needed to understand the potential risk to humans. bats, the ubiquitous shedding of coronaviruses from bat Despite the rapidly growing number of genomic populations and the molecular interactions of corona- sequences of bat coronaviruses, our knowledge of viruses facilitate their zoonotic capacity. However, these the ecology and evolution of these viruses is still low. pathogens cannot cause outbreaks in humans unless Understanding how, when and where viral shedding the conditions for spillover and onward transmission happens will directly inform how we assess the risk of are met. The risk of spillover depends on the level of spillover over time and space, as viral shedding and human exposure, which is increasingly influenced by thus pathogen pressure is the first step in spillover. habitat deterioration and encroachment into wild areas. It remains unclear whether the spatiotemporal patterns Integration of ecological, evolutionary and epidemiolog- of coronavirus prevalence and shedding seen in some bat ical data from bat–virus systems, coupled with human populations are generalizable. To fill this gap, we need epidemiological and health surveillance in high-risk longitudinal studies at multiple scales, from the indi- areas, is urgently needed to improve risk assessment and vidual level to the population and metapopulation lev- predictive capacity. This integration of scientific fields els. 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M.R.-A., C.M., prevent spillover and the next pandemic. Viruses 13, type II strains 79-1683 and 79-1146 originate R.K.P., A.G., C.R.P., E.S.G., J.O.L.-S., P.J.H. and V.J.M. con- 1298 (2021). from a double recombination between feline tributed substantially to discussion of the content. M.R.-A., 158. Obameso, J. O. et al. The persistent prevalence and coronavirus type I and canine coronavirus. J. Virol. C.M., C.F., E.J., L.D., D.N.J. and M.K.K. compiled the data evolution of cross-family recombinant coronavirus 72, 4508–4514 (1998). and performed formal analysis. M.R.-A. and C.M. contributed GCCDC1 among a bat population: a two-year equally. All authors wrote the article and reviewed and/or follow-up. Sci. China Life Sci. 60, 1357–1363 Acknowledgements edited the manuscript before submission. (2017). Work related to this Review was supported by the Defense This study provides evidence of coronavirus Advanced Research Projects Agency (PREEMPT Competing interests evolution in a longitudinally sampled population D18AC00031). M.R.-A., D.N.J., M.K.K., C.F., D.C., N.B., The authors declare no competing interests. of bats. A.J.P., O.R., P.J.H. and R.K.P. were supported by the US 159. Lazov, C. et al. Detection and characterization of National Science Foundation (DEB-1716698). R.K.P. was Peer review information distinct alphacoronaviruses in five different bat supported by the US Department of Agriculture National Nature Reviews Microbiology thanks Jie Cui and Vikram species in Denmark. Viruses 10, 486 (2018). Institute of Food and Agriculture (Hatch project 1015891). Misra for their contribution to the peer review of this work. 160. Hu, D. et al. Genomic characterization and infectivity J.O.L.-S. and A.G. were supported by the UCLA AIDS Institute of a novel SARS-like coronavirus in Chinese bats. and Charity Treks and by the US National Science Foundation Publisher’s note Emerg. Microbes Infect. 7, 1–10 (2018). (DEB-1557022). C.E.S was supported by the US National Springer Nature remains neutral with regard to jurisdictional 161. Pepin, K. M., Lass, S., Pulliam, J. R. C., Read, A. F. Institutes of Health (T32 GM008185-33). C.K.Y., J.R.P. and claims in published maps and institutional affiliations. & Lloyd-Smith, J. O. Identifying genetic markers of V.J.M. are supported by the Intramural Research Program of adaptation for surveillance of viral host jumps. the US National Institute of Allergy and Infectious Diseases, Supplementary information Nat. Rev. Microbiol. 8, 802–813 (2010). US National Institutes of Health. A.J.P. was supported by an The online version contains supplementary material available 162. Hemida, M. G. et al. Coronavirus infections in horses ARC DECRA fellowship (DE190100710). O.R. is supported by at https://doi.org/10.1038/s41579-021-00652-2. in Saudi Arabia and Oman. Transbound. Emerg. Dis. the ALBORADA Trust. E.J. is funded by a research fellowship 64, 2093–2103 (2017). from the Deutsche Forschungsgemeinschaft (438001934). RelateD linKs 163. Zhuang, Q. et al. Surveillance and taxonomic C.E.B. was funded by a postdoctoral fellowship from the Bat species of the world database: https://batnames.org/ analysis of the coronavirus dominant in pigeons in Miller Institute for Basic Research, a Branco Weiss Society in China. Transbound. Emerg. Dis. 67, 1981–1990 Science Fellowship and US National Institutes of Health grant (2020). R01AI129822-01. D.W.B., Y.Y.Y. and H.C.A. were supported © Springer Nature Limited 2021, corrected publication 2022 314 | may 2022 | volume 20 www.nature.com/nrmicro 0123456789();: https://doi.org/10.1038/s41579-021-00652-2 Supplementary information Ecology, evolution and spillover of coronaviruses from bats In the format provided by the authors and unedited Supplementary Table 1. Wild bat hosts of coronaviruses reported in published studies. All coronaviruses were considered in our search, but we highlight links between bat species and key bat coronavirus subgenera associated with human infections (e.g., Sarbecovirus), domestic animal infections (e.g., Rhinacovirus), or are widespread and well characterized (e.g., Nobecovirus) based on sequencing information available in the associated studies. Bat species Bat family Key coronavirus Reference subgenera Emballonura alecto Emballonuridae Nobecovirus 5 Taphozous melanopogon Emballonuridae 1,9 Taphozous perforatus Emballonuridae Merbecovirus 10,11 Aselliscus stoliczkanus Hipposideridae Rhinacovirus 12,145,163,173,174 Sarbecovirus Hipposideros abae Hipposideridae Duvinacovirus 13 Hipposideros armiger Hipposideridae Hibecovirus 1,9,14-17,145,174 Merbecovirus Nobecovirus Rhinacovirus Sarbecovirus Hipposideros bicolor Hipposideridae 1 Hipposideros caffer Hipposideridae Duvinacovirus 1-4,142,163,172 Hibecovirus Sarbecovirus Hipposideros cervinus Hipposideridae 18,163 Hipposideros cf. caffer Hipposideridae Duvinacovirus 19 Hibecovirus Hipposideros cf. ruber Hipposideridae Duvinacovirus 13,20-22 Hibecovirus Hipposideros cineraceus Hipposideridae Rhinacovirus 23,152 Hipposideros curtus Hipposideridae Duvinacovirus 163,172 Hipposideros diadema Hipposideridae 1,5,163 Hipposideros fuliginosus Hipposideridae Hibecovirus 163,172 Hipposideros galeritus Hipposideridae Sarbecovirus 1 Hipposideros gentilis Hipposideridae 169 Hipposideros Hipposideridae 169 khaokhouayensis Hipposideros larvatus Hipposideridae Hibecovirus 1,9,15,26,27,152,163,173,174 Nobecovirus Rhinacovirus Sarbecovirus Hipposideros lekaguli Hipposideridae Nobecovirus 1,9,163 Hipposideros pomona Hipposideridae Hibecovirus 28-30,145,152,163,173,174 Rhinacovirus Sarbecovirus Hipposideros pratti Hipposideridae Hibecovirus 1,31,145 Rhinacovirus Sarbecovirus Hipposideros ruber Hipposideridae Duvinacovirus 1,4,141,142,156,163,172 Hibecovirus Nobecovirus Sarbecovirus Bat species Bat family Key coronavirus Reference subgenera Macronycteris gigas Hipposideridae Duvinacovirus 1,22,142,163,172 (formerly Hipposideros Hibecovirus gigas) Macronycteris vittatus Hipposideridae Duvinacovirus 24,25,32 (formerly Hipposideros Hibecovirus commersoni) Nobecovirus Cardioderma cor Megadermatidae 24,32 Lyroderma lyra Megadermatidae 1,9,163,174 (formerly Megaderma lyra) Miniopterus africanus Miniopteridae 24 Miniopterus australis Miniopteridae 33 Miniopterus fuliginosus Miniopteridae 1,14,30,31,34,35,140,162,171 Miniopterus fuscus Miniopteridae 30,145 Miniopterus inflatus Miniopteridae 1,22,24,142 Miniopterus magnater Miniopteridae 1,9,36-39,163 Miniopterus minor Miniopteridae 2,24,32 Miniopterus mossambicus Miniopteridae 3 Miniopterus natalensis Miniopteridae 7,24 Miniopterus pusillus Miniopteridae 9,36-40,145,163,174 Miniopterus schreibersii Miniopteridae Merbecovirus 8,9,17,30,33,37,41-47,140,145,163,171,174 Rhinacovirus Sarbecovirus Chaerephon plicatus Molossidae Merbecovirus 26,31,48,49,152,169,174 Sarbecovirus Chaerephon pumilus Molossidae Duvinacovirus 1-4,6,24,142,163 Nobecovirus Cynomops abrasus Molossidae 50 Cynomops planirostris Molossidae 50 Eumops glaucinus Molossidae Merbecovirus 51 Molossus currentium Molossidae 52 Molossus molossus Molossidae 53-55 Molossus rufus Molossidae 51,52,54,55 Mops condylurus Molossidae Hibecovirus 1-3,6,142,163,172 Nobecovirus Mops midas Molossidae 3,7,163 Mormopterus Molossidae 3 francoismoutoui Mormopterus jugularis Molossidae 3 Nyctinomops laticaudatus Molossidae Merbecovirus 1,56 Otomops martiensseni Molossidae 24,32,163 Tadarida brasiliensis Molossidae 1,53,56,158 Tadarida teniotis Molossidae Sarbecovirus 8,57 Pteronotus davyi Mormoopidae 54 Pteronotus parnellii Mormoopidae 1,52,56 Pteronotus personatus Mormoopidae 1 Mystacina tuberculata Mystacinidae 58 Nycteris cf. gambiensis Nycteridae Merbecovirus 59 Nycteris macrotis Nycteridae Merbecovirus 141 Bat species Bat family Key coronavirus Reference subgenera Nycteris thebaica Nycteridae Merbecovirus 3 Nycteris tragata Nycteridae 163 Anoura caudifer Phyllostomidae 1,163 Anoura geoffroyi Phyllostomidae 52 Artibeus jamaicensis Phyllostomidae 1,52,56,60 Artibeus lituratus Phyllostomidae 1,51,52,55,56 Artibeus obscurus Phyllostomidae 1,163 Artibeus planirostris Phyllostomidae 1,163 Carollia brevicauda Phyllostomidae 52 Carollia castanea Phyllostomidae 60 Carollia perspicillata Phyllostomidae 1,51,52,56,60,61 Carollia sowelli Phyllostomidae 1,56 Dermanura phaeotis Phyllostomidae 1,56 (formerly Artibeus phaeotis) Desmodus rotundus Phyllostomidae 50,62,63,143,164 Glossophaga soricina Phyllostomidae 1,50,51,55,60,61 Lichonycteris obscura Phyllostomidae 163 Lonchorhina aurita Phyllostomidae 1,56 Mesophylla macconnelli Phyllostomidae 1,163 Phyllostomus discolor Phyllostomidae 52,55 Sturnira erythromos Phyllostomidae 1,163 Sturnira lilium Phyllostomidae 1,51 Acerodon celebensis Pteropodidae Nobecovirus 163 Cynopterus brachyotis Pteropodidae Nobecovirus 1,5,9,27,64,65,163,170 Cynopterus horsfieldii Pteropodidae Nobecovirus 1,163 Cynopterus sphinx Pteropodidae Nobecovirus 1,9,23,27,145,147,163,169 Dobsonia moluccensis Pteropodidae Nobecovirus 66 Dyacopterus spadiceus Pteropodidae Nobecovirus 1 Eidolon dupreanum Pteropodidae Nobecovirus 67 Eidolon helvum Pteropodidae Nobecovirus 1,2,4,6,10,11,24,32,68,141,142,163,172 Eonycteris spelaea Pteropodidae Nobecovirus 1,27,64,69-71,144,145,163,169,174 Epomophorus gambianus Pteropodidae Nobecovirus 1,141,156,163,172 Epomophorus labiatus Pteropodidae Nobecovirus 4,32 Epomops buettikoferi Pteropodidae Nobecovirus 163 Epomops franqueti Pteropodidae Nobecovirus 1,142,163,172 Macroglossus minimus Pteropodidae Nobecovirus 5,72,170 Megaerops ecaudatus Pteropodidae Nobecovirus 163 Megaerops kusnotoi Pteropodidae Nobecovirus 23 Megaerops niphanae Pteropodidae Nobecovirus 1,27 Megaloglossus Pteropodidae Nobecovirus 1,142,163,172 woermanni Micropteropus pusillus Pteropodidae Nobecovirus 1,20,142,163,172 Myonycteris angolensis Pteropodidae Duvinacovirus 1,4,6,141,163 (formerly Lissonycteris Hibecovirus angolensis) Nobecovirus Myonycteris torquata Pteropodidae Nobecovirus 163,172 Nanonycteris veldkampii Pteropodidae Nobecovirus 141 Ptenochirus jagori Pteropodidae Nobecovirus 5,64 Bat species Bat family Key coronavirus Reference subgenera Pteropus alecto Pteropodidae Nobecovirus 1,33,73 Pteropus conspicillatus Pteropodidae Nobecovirus 163 Pteropus lylei Pteropodidae Nobecovirus 74,163 Pteropus medius Pteropodidae Nobecovirus 1,75-77,163 (formerly Pteropus giganteus) Pteropus rufus Pteropodidae Nobecovirus 67 Rousettus aegyptiacus Pteropodidae Nobecovirus 1,2,4,6,24,32,78,141,163,172 Rousettus Pteropodidae Nobecovirus 1,5,27,64,170 amplexicaudatus Rousettus leschenaultii Pteropodidae Merbecovirus 1,23,27-29,40,71,79-81,159,162,163,174 Nobecovirus Rousettus Pteropodidae Nobecovirus 3 madagascariensis Rhinolophus acuminatus Rhinolophidae Sarbecovirus 151,163 Rhinolophus affinis Rhinolophidae Rhinacovirus 1,12,30,47,82,83,145,146,161,163,169,174 Sarbecovirus Rhinolophus blasii Rhinolophidae Rhinacovirus 45,163 Sarbecovirus Rhinolophus cf. clivosus Rhinolophidae Duvinacovirus 6,139 Sarbecovirus Rhinolophus clivosus Rhinolophidae Duvinacovirus 1,4,84 Hibecovirus Rhinacovirus, Sarbecovirus Rhinolophus cornutus Rhinolophidae Sarbecovirus 85,148 Rhinolophus creaghi Rhinolophidae Sarbecovirus 1,163 Rhinolophus darlingi Rhinolophidae 141 Rhinolophus euryale Rhinolophidae Rhinacovirus 8,45,86,163 Sarbecovirus Rhinolophus Rhinolophidae Merbecovirus 1,8,12,17,23,29,31,43-45,57,78,83,86,89- ferrumequinum Nobecovirus 93,140,145,159,160,163,171,174 Rhinacovirus Sarbecovirus Rhinolophus fumigatus Rhinolophidae 2 Rhinolophus hildebrandtii Rhinolophidae Sarbecovirus 32 Rhinolophus hipposideros Rhinolophidae Sarbecovirus 86,94,95,160,165 Rhinolophus landeri Rhinolophidae 2,32 Rhinolophus lepidus Rhinolophidae 163 Rhinolophus lobatus Rhinolophidae Rhinacovirus 3 Rhinolophus macrotis Rhinolophidae Rhinacovirus 17,43,83,91,145 Sarbecovirus Rhinolophus malayanus Rhinolophidae Rhinacovirus 96,152,169,174 Sarbecovirus Rhinolophus marshalli Rhinolophidae Sarbecovirus 169 Rhinolophus megaphyllus Rhinolophidae 33 Rhinolophus mehelyi Rhinolophidae Sarbecovirus 45,163 Rhinolophus monoceros Rhinolophidae Sarbecovirus 14,17,97 Bat species Bat family Key coronavirus Reference subgenera Rhinolophus pearsonii Rhinolophidae Rhinacovirus 17,43,91,174 Sarbecovirus Rhinolophus pusillus Rhinolophidae Rhinacovirus 17,31,46,49,82,83,93,98,99,145,152,153,163,169 Sarbecovirus ,174 Rhinolophus rex Rhinolophidae Rhinacovirus 1,17,82 Sarbecovirus Rhinolophus rhodesiae Rhinolophidae Rhinacovirus 3 Rhinolophus rufus Rhinolophidae Nobecovirus 5 Rhinolophus shameli Rhinolophidae Rhinacovirus 1,9,27,83,150 Sarbecovirus Rhinolophus sinicus Rhinolophidae Nobecovirus 1,12,17,23,30,31,38,40,43,82,83,100- Rhinacovirus 109,145,147,152,159,163,173,174 Sarbecovirus Rhinolophus stheno Rhinolophidae Rhinacovirus 29,152,161 Sarbecovirus Rhinolophus thomasi Rhinolophidae Rhinacovirus 17,163 Sarbecovirus Rhinolophus trifoliatus Rhinolophidae 18,163 Rhinonicteris aurantia Rhinonycteridae Hibecovirus 33 Triaenops afer Rhinonycteridae Setracovirus 1,3,32,142 Triaenops menamena Rhinonycteridae 3 Triaenops persicus Rhinonycteridae Merbecovirus 1,6,142 Nobecovirus Setracovirus Rhinopoma hardwickii Rhinopomatidae Nobecovirus 10,163 Sarbecovirus Bauerus dubiaquercus Vespertilionidae 1 Chalinolobus gouldii Vespertilionidae 110 Chalinolobus morio Vespertilionidae 110 Corynorhinus townsendii Vespertilionidae 154 Eptesicus fuscus Vespertilionidae 56,111-113,149 Eptesicus isabellinus Vespertilionidae Merbecovirus 42 Eptesicus nilssonii Vespertilionidae Merbecovirus 114 Eptesicus serotinus Vespertilionidae Merbecovirus 8,92,98,115,116,171 Glauconycteris poensis Pteropodidae 163 Glauconycteris variegata Pteropodidae Nobecovirus 163 Falsistrellus mackenziei Vespertilionidae 110 Hypsugo alaschanicus Vespertilionidae 140,171 Hypsugo pulveratus Vespertilionidae Merbecovirus 101,159 Hypsugo savii Vespertilionidae Merbecovirus 42,94,117 Ia io Vespertilionidae Merbecovirus 1,118,145 Kerivoula hardwickii Vespertilionidae 163 Kerivoula pellucida Vespertilionidae 163 Kerivoula titania Vespertilionidae 14 Murina cyclotis Vespertilionidae 152 Murina leucogaster Vespertilionidae 17,23 Murina recondita Vespertilionidae 14 Myotis adversus Vespertilionidae 174 Myotis aurascens Vespertilionidae 171 Bat species Bat family Key coronavirus Reference subgenera Myotis bechsteinii Vespertilionidae 119,120 Myotis blythii Vespertilionidae 42,89,115 (includes Myotis oxygnathus) Myotis bombinus Vespertilionidae 140 Myotis brandtii Vespertilionidae 114 Myotis californicus Vespertilionidae 1 Myotis capaccinii Vespertilionidae 8 Myotis chinensis Vespertilionidae 145,174 Myotis dasycneme Vespertilionidae 116,120,121,167 Myotis daubentonii Vespertilionidae Merbecovirus 1,8,23,29,31,42,86,89,114,116,120-122,163,167 Rhinacovirus Myotis davidii Vespertilionidae 17 Myotis emarginatus Vespertilionidae 41,90 Myotis evotis Vespertilionidae 113 Myotis fimbriatus Vespertilionidae 14,98,163 Myotis formosus Vespertilionidae 14 (formerly Myotis flavus) Myotis horsfieldii Vespertilionidae Nobecovirus 1,27,145,163 Myotis ikonnikovi Vespertilionidae Merbecovirus 171 Myotis laniger Vespertilionidae Rhinacovirus 152,163 Myotis longipes Vespertilionidae 1,174 Myotis lucifugus Vespertilionidae 113,123,124,125 Myotis macrodactylus Vespertilionidae 140,171 Myotis macropus Vespertilionidae 33,126 Myotis muricola Vespertilionidae 152 Myotis myotis Vespertilionidae 1,8,42,86,89,127,157 Myotis nattereri Vespertilionidae 8,41,86,89,116,119,122 Myotis nigricans Vespertilionidae 51 Myotis occultus Vespertilionidae 111 Myotis pequinius Vespertilionidae Merbecovirus 98 Myotis petax Vespertilionidae 140,171 Myotis pilosus Vespertilionidae Merbecovirus 1,31,38,43,46,98,145,163,174 (formerly Myotis ricketti) Rhinacovirus Myotis punicus Vespertilionidae 8 Myotis riparius Vespertilionidae 51 Myotis siligorensis Vespertilionidae Merbecovirus 17,163,174 Rhinacovirus Myotis velifer Vespertilionidae 1,56 Myotis volans Vespertilionidae 113 Myotis welwitschii Vespertilionidae 163 Neoromicia capensis Vespertilionidae Merbecovirus 7,128,129 Neoromicia cf. zuluensis Vespertilionidae Merbecovirus 130 Neoromicia somalica Vespertilionidae Nobecovirus 163 Nyctalus lasiopterus Vespertilionidae 42 Nyctalus leisleri Vespertilionidae 45 Nyctalus noctula Vespertilionidae Merbecovirus 94,121,157 Nyctalus plancyi Vespertilionidae 1,31 Bat species Bat family Key coronavirus Reference subgenera (includes Nyctalus velutinus) Nyctophilus geoffroyi Vespertilionidae 110 Nyctophilus gouldi Vespertilionidae 110 Perimyotis subflavus Vespertilionidae 131 Pipistrellus abramus Vespertilionidae Merbecovirus 31,38,43,92,101,118,132,145,171,174 Nobecovirus Sarbecovirus Pipistrellus cf. hesperidus Vespertilionidae Merbecovirus 6,133 Pipistrellus coromandra Vespertilionidae Merbecovirus 1,27,163 Pipistrellus hesperidus Vespertilionidae Merbecovirus 1,163 Pipistrellus inexspectatus Vespertilionidae 172 Pipistrellus kuhlii Vespertilionidae Merbecovirus 10,42,78,89,94,117,134,168 (includes Pipistrellus Nobecovirus deserti) Pipistrellus nathusii Vespertilionidae Merbecovirus 59,119,120 Pipistrellus pipistrellus Vespertilionidae Merbecovirus 1,41,43,59,89,118,121,135,157,166 Pipistrellus pygmaeus Vespertilionidae Merbecovirus 59,86,116,119,120,167 Pipistrellus tenuis Vespertilionidae Merbecovirus 118 (formerly Pipistrellus minus) Plecotus auritus Vespertilionidae Merbecovirus 57,89 Sarbecovirus Plecotus taivanus Vespertilionidae 14 Scotophilus dinganii Vespertilionidae Nobecovirus 1,32,142,172 Scotophilus heathii Vespertilionidae Nobecovirus 9,26,163,174 Scotophilus kuhlii Vespertilionidae Nobecovirus 1,9,14,27,43,97,136,137,145,147,163,174 Scotophilus leucogaster Vespertilionidae Nobecovirus 1,172 Scotophilus nux Vespertilionidae 1,163,172 Submyotodon latirostris Vespertilionidae 14 Tylonycteris pachypus Vespertilionidae Merbecovirus 1,31,38,43,46,101,118,132,145,155,159,163,174 Rhinacovirus Tylonycteris robustula Vespertilionidae Rhinacovirus 101,174 Vespadelus baverstocki Vespertilionidae 110 Vespadelus pumilus Vespertilionidae 33 Vespadelus regulus 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Study type and Number of studies Overview What we can learn Advantages Caveats description Experimental Bat cell lines: 29 Bat cell experiments • Characterization of • Ability to test Koch's • Relies on existing viral Experimental infection Live bats: 6 • Target cells: brain, newly detected postulates using isolates; cannot of individual bats or bat embryo, intestine, viruses different strains and isolate new cell lines, or other viral kidney, lung • Bat species bat species pathogens manipulations in a • Tested viruses: susceptibility to • Causal inference • No ecological controlled environment multiple bat SARS- infection and dose- • Controlled context; impossible related CoVs, response environment to accurately BatCoV HKU4, relationships • Rapid technological replicate BatCoV HKU9, • Magnitude, quality, advances make environmental HCoV-229E, HCoV- and kinetics of diagnostic tools conditions NL63, MERS-CoV, immune responses to affordable • Lab conditions may PEDV, Ro-BatCoV pathogens, and • Relatively rapid data not effectively mimic GCCDC1, SADS- mechanisms of viral acquisition the environmental CoV, SARS-CoV, control or tolerance conditions that drive SARS-CoV-2, • Disease pathogenesis infections in reservoir Scotophilus bat CoV (or lack thereof) hosts 512, TGEV • Individual and within- • Challenging and Live bat experiments host infection, expensive to house • Tested hosts and disease, and and breed colonies of viruses: Artibeus immunological bats jamaicensis (MERS- processes, especially • Often requires CoV), Eptesicus those required for biosafety level 3 or 4 fuscus (SARS-CoV- dynamic modeling facilities and 2), Myotis lucifugus (e.g., infectious specialized training (Myl-CoV), periods, acute vs. • A bat is not a bat, and Rousettus latent infections, a virus is not a virus: leschenaultii waning immunity, species-specific (BatCoV HKU9), etc.) responses to Rousettus • Tissue tropism and infection make it aegyptiacus (bat routes of virus difficult to generalize SARSr-CoV WIV1, excretion and across species or bat SARS-CoV-2) transmission families Study type and Number of studies Overview What we can learn Advantages Caveats description • Receptor binding • In vitro studies miss efficiency in bats and differences in cell other potential hosts recruitment and • Facilitative or localization or cell- antagonistic cell interaction interactions between • Immortalized cells coinfecting viruses behave differently • Virus surface survival from primary cells or and sensitivity to heat cells in an in vivo or desiccation context • Development of • Fundamental model systems, knowledge of bat laboratory protocols, immune systems and and screening tools basic tools for for the field probing bat immune • Spillover potential to responses are lacking other/novel hosts • Experiments are usually time-limited (e.g., limited ability to study immune function senescence, viral recrudescence, etc.) Longitudinal 14 • Countries: Australia, • Some spatial and • Ability to identify and • May not be truly Repeated sampling of China, Denmark, temporal dynamics of isolate novel longitudinal: without individuals, single Germany, Malaysia, pathogens in pathogens known recapture of populations, or multiple Singapore, South populations, and • May have ability to individuals, repeated populations over time; Korea, Thailand maybe in individuals repeatedly collect longitudinal ideally, this occurs in • Serially sampled • Spatiotemporal covariate data or monitoring at a closed populations with species: Eonycteris patterns of infection track life-histories of geographic location known individual life- spelaea, Hipposideros (e.g., travelling individuals may instead histories cervinus, Myotis waves) • More power to represent multiple daubentonii, Myotis • Transmission rates exclude time- cross-sectional macropus, Myotis and dynamics, using invariant differences surveys of the myotis, Pteropus lylei, carefully collected between individuals, population Study type and Number of studies Overview What we can learn Advantages Caveats description Rhinolophus sinicus, age-prevalence and populations, or • Expensive, time- Rousettus age-seroprevalence environments consuming, and leschenaultii data • Identification of logistically • Variation in temporal trends (e.g., challenging; slow prevalence/seropreva seasonality) data acquisition lence with host traits • Potential for • Effective or environmental forecasting and implementation covariates prediction requires a strong • Parameters of the • Intervention analysis ecological disease process in • Relationship between understanding of the individuals and time-series variables study system and populations required collection of data to for dynamic modeling determine sampling (e.g., seasonality, frequency and maybe transmission duration rates, life-history • May be temporally traits) biased; sampling at • Some dynamics of co- regular intervals may circulating viruses consistently detect or • Interventions that consistently miss viral might reduce shedding prevalence or • May be spatially magnitude of an biased; difficult to epizootic or enzootic sample spatially replicated populations • Determining disease dynamics is difficult: requires consistent recapture of individuals, longitudinal sampling that exceeds pathogen infectious period, nonlethal Study type and Number of studies Overview What we can learn Advantages Caveats description pathogen detection, and moderate prevalence • Large sample sizes, spatially replicated populations, and short sampling intervals are needed to understand environmental drivers, and individual and population-level variation in viral shedding • Relationships that exist for groups may not apply to individuals (ecological fallacy, e.g., virus x detected in all population subgroups sampled in Habitat A; therefore, all individuals or other population subgroups in Habitat A must also carry virus x. Cross-sectional 14 • Genetic variation of • Relatively fast and • No ability to detect (intra-species) strains within host inexpensive seasonality or other Sampling of a bat population(s) • Sampling of isolated temporal trends population or • Spatial distribution of populations can help • No causal inference population subgroup(s) strains within host distinguish between • Large amounts of at a specific timepoint population(s) population-level data are required to • Some differences pathogen persistence account for variation between and spatiotemporally irregular transmission Study type and Number of studies Overview What we can learn Advantages Caveats description demographic stages • Can sample among individuals or (dependent on populations populations sampling time-point) adaptively in • Effective • Possible to integrate response to spillover implementation with longitudinal • Ability to isolate requires a strong studies of same pathogens ecological species • Some ability to detect understanding of the • Natural routes of spatial variation or study system excretion statistically analyze • May be temporally differences. biased: sampling during peaks or troughs in population prevalence will over- or underestimate geographic variation in prevalence or genetic diversity • May be spatially biased: at one timepoint, different population subgroups may have peaks or troughs in prevalence • Ecological fallacy (as in longitudinal studies) Cross-sectional 123 • Sampled countries: • Identity of potential • Rapid detection of • Same caveats as (inter-species) 69 reservoir hosts viruses in multiple intra-species cross- Sampling of bat • Sampled bat families: • Potential exchange of species sectional studies assemblages or a 18 strains between hosts • Ability to isolate • Often low sample subset of a bat • Positive bat families: • Host and geographic pathogens sizes for assemblage (>1 species) 14 factors that impact • Some ability to detect opportunistically at a specific timepoint • Sampled bat species: viral diversity species-level sampled species 543 differences • Species bias: research effort may Study type and Number of studies Overview What we can learn Advantages Caveats description • Positive bat species: • Relatively fast and inadvertently skew 238 inexpensive importance of a particular species as a reservoir or spillover host • Ecological fallacy (as in longitudinal and intra-species cross- sectional studies) Multi-pathogen 36 • Viral species diversity, • Can be combined • Same caveats as detection abundance, and with next-generation longitudinal or cross- Detection of multiple community dynamics sequencing to sectional studies, pathogens (virus • Some information identify viral depending on design families, strains, or about periods of communities • May be difficult to other parasite taxa) potential spillover risk • May require little to distinguish between using metagenomic for newly detected no fieldwork if facilitative or sequencing or other viruses not yet known samples are already antagonistic targeted methods on to be zoonotic available interactions between samples collected • Coinfection and some • Can be relatively coinfecting viruses or during cross-sectional insight into inexpensive with viruses synchronously or longitudinal sampling interactive effects of rapid data acquisition shed from a bat at the individual- or viruses on hosts (design dependent) population; requires population-level large sample sizes combined with simulation or experimental studies • Drivers of multi-viral infection or shedding may be difficult to detect (e.g., may be driven by facilitative interaction between known or undetected coinfecting viruses, interactions with host physiology/immunity, Study type and Number of studies Overview What we can learn Advantages Caveats description and/or a response to optimal environmental conditions) • Biased detection: high titers of one virus in a sample may reduce assay sensitivity to other viruses • No causal inference • Co-detection of pathogens in pooled or population-level samples may reflect coinfection or contribution of multiple bats to the collected sample Sequencing only 29 • Comparative • Requires little • No ecological or Viral sequencing on genomics background physiological context samples collected • Mutation and knowledge of study • No causal inference during longitudinal or evolutionary rates system cross-sectional • Virus discovery • Relatively sampling; little • Effective population inexpensive; rapid collection of data on size and genetic data acquisition other covariates diversity of virus • May require little to within or across no fieldwork if subpopulations samples are already • Some information on available viral dynamics may be possible (e.g., through phylodynamics)