In this study, we have gathered evidence of bats being in close evolutionary and ecological relationship with several genera of mammalian PVs. The investigation of viral host associations can be a lengthy and controversial process that depends on targeted and ecologically informed sampling21,22,23,24,25,26,27,28,29. We believe that we have assembled the largest and most diversified single-sample set for investigating viruses in mammalian hosts. We focused on bats and rodents because of their unique properties among the mammals in terms of large social group sizes, intense social interaction and high population densities. Further criteria included high spatial mobility in the case of bats, and high population turnover in the case of rodents1,2,4,30. However, our data were not limited to these taxa, as an essential part of our analysis involved database-derived PV from a large range of other mammals as well as birds. The evolutionary distance between the analysed host taxa considerably exceeded that of either the bats or the rodents studied. It should be mentioned that PV entries in GenBank with and without our new data were not over-emphasising chiropteran or rodent hosts. As shown in Supplementary Fig. S6, the majority of PV entries were from primates, birds, carnivores and ungulates. Even after addition of our novel data to GenBank data sets, the number of PV sequences from bats was just even with that from ungulates.

Like almost all PVs currently contained in GenBank, our novel viruses were identified by RT–PCR and sequencing. PCR primers have an inherent bias due to their sequence specificity. We have applied a large range of published and own primer sets to compensate for this bias. Some of these have been thoroughly validated on clinical panels from a large range of hosts, confirming high sensitivity over a large genetic range of viruses31. More recent studies have suggested to improve the detection of novel viruses by hypothesis-free approaches such as random cDNA amplification from serum followed by deep sequencing32. Inclusion of such an approach in our study indeed revealed novel viral sequences that should be investigated further, but did not yield any PV. Low serum concentrations of PV RNA may have prevented detection, reminding us that hypothesis-free virus testing may not be sufficiently sensitive to enable comprehensive detection of viral flora32,33.

ICTV currently lists 36 accepted PV species. Gene databases comprise more viruses, many of which are only partially sequenced and not classified into defined species (Supplementary Tables S6 and S7). Although 83 clearly distinct PV taxa could be discriminated in our data set based on phylogeny, a classification criterion using a distance threshold comparable to that between HeV and NiV (7.0–7.5% in the L-gene fragment used) identified 66 independent novel taxonomic entities on the rank of tentative species to be represented in our data. Nevertheless, the number of novel taxa added to the PV phylogeny is unlikely to cause a bias toward bat- and rodent viruses in subsequent analyses of host associations. This is foremostly because reference sequences in these analyses were selected to maximise the patristic distance within each genus of PV (Fig. 8a). The algorithm underlying the parsimony-based ancestral state reconstruction only counts host switches once per resolved phylogenetic root point, irrespective of the number of depending leaves carrying identical traits. In general, probabilistic approaches such as ML- and Bayesian methods are considered more powerful than parsimony models for the reconstruction of character states evolving along phylogenetic branches34. However, this is mainly because probabilistic models take branch lengths into account, whereas parsimony-based methods only consider tree topology34. For the particular task of reconstructing viral host associations, we favour parsimony approaches out of theoretical considerations. First, we had to assume that host transition happens rarely and is unlikely to take place in a bidirectional manner, because of the fitness valley effect that will prevent host changes to be reversed easily4,10. In particular, viruses conquering a new host will leave behind population immunity in their original AR, preventing re-introduction, and making back and forward transitions (as well as host switching as a whole) a rare process4,10. Another argument was the uncertainty of deeper branch lengths in viral phylogenies35. More recent studies on non-retroviral RNA viruses invading mammalian germlines demonstrated tremendous discrepancies between apparent evolutionary rates of extant RNA viruses versus those of phylogenetic stem lineages36,37,38,39. Rate differences in deep branches will have great influence on probabilistic models, but will not affect parsimony assumptions. Even though recent findings of viral germline fossils comply with the idea that ancient PV may have existed in mammals36,37,38,39, such data are so far unavailable for PV, and we have not attempted in our analysis to determine the evolutionary origins of PV. Such an analysis would require a different approach to sampling, as viruses from taxa other than mammals and birds are currently underrepresented in databases. To attenuate the contribution of deep phylogenetic nodes, we have limited our analysis to an estimation of trait switches along trees, rather than inferring host associations for deep phylogenetic nodes. With this limitation in mind, we can conclude that bats have most often been the donors of those viruses currently encountered in other mammals.

In spite of our preference for parsimony-based analysis, we have challenged this result by repetition in a probabilistic approach, testing the influence of alternative hypotheses by either restricting the trait change model or imposing fossilised ancestral state assumptions40. These analyses confirmed the results of the parsimony model and formally excluded other host hypotheses within the available data set. However, it should be mentioned that ancestral state analyses have not been systematically applied to the theoretical problem of viral host switching before. Many open questions remain, including the essential issue of developing significance criteria in host hypothesis testing. Only if complex data sets like ours become available for several other virus taxa, it will be possible to approach this major theoretical task. The current limitation, however, is in the sparse and incomplete biological sampling of habitat.

Beyond phylogenetic analysis, we have identified traits of the natural history of infection that suggest a specific connection between PV and bats. The epidemiology of a morbilli-related virus was fundamentally different from that of measles virus in humans, or that of Rinderpest in cattle. Human measles is the prototype of viruses depending on steady transmission in sufficiently large social groups, potentially absent from isolated and remote populations41. Black41 has defined this pattern of pathogen prevalence as the 'introduced disease' pattern, based on observations in isolated human tribes. Strikingly, in bats, the morbilli-related virus was excreted by adult animals at similar rates as by young animals, which is very untypical of morbilliviruses in other mammals. This pattern of prevalence was classified by Black41 as 'endemic—high incidence, low morbidity', as exemplified in humans by hepatitis B virus or herpes viruses. Some researchers have argued that bats in general might deal with viral infections differently than other mammals42. However, we have recently described a variance of viral persistence patterns in bats that is congruent with observations in other mammals, with a typical 'introduced disease' pattern for astro- and coronaviruses, although a bat adenovirus in the same group of animals showed Black's 'endemic' pattern41,43. Adenoviruses provide a good template to explain the shedding pattern of the morbilli-related virus found in the present study, as they are known to persist in tissue and to be shed without signs beyond the acute phase of disease—a property determining the ability of viruses to persist in small populations41,44. Accordingly, the morbilli-related virus in our study was detected in a species of bats forming small- to medium-sized social groups (M. myotis), possibly requiring long-term excretion for virus maintenance on group level. This is rather untypical for morbilliviruses in other mammals that depend heavily on efficient transmission and sufficient group size to be maintained30,41,45. In this light, the difference in organ association between bats and rodents was quite interesting. Although PVs in rodents were associated with the kidney, favouring excretion, their highest concentrations and prevalences in bats were seen in the spleen. Although we have no further direct proof, this matches the concept that bat-borne PV might not as much depend on highly efficient transmission, but might routinely employ mechanisms of persistence to follow Black's 'endemic' pattern of prevalence41. This anomality might indeed identify bats as AR of these viruses. Moreover, the morbilli-related bat virus was detected in Europe, but also in an unrelated species forming rather small social groups in sub-Saharan Africa. Even though we have not been able to conduct longitudinal investigations of excretion in other PV genera, detection in groups without social connection as well as re-detection in subsequent years was seen also for rubula- and henipaviruses in this study. Detection of these viruses was not associated with changes in serum chemistry parameters, suggesting symptomless infection despite virus replication in internal organs, which may be regarded as typical for a virus in its natural host context that is not dependent on efficient horizontal transmission4,7,46.

Beyond virus evolution and ecology, these data might have important implications for public health. HeV and NiV may be of African descent and have highly diversified relatives in Africa. These viruses might be associated with unrecognised disease, given the tremendous number of unresolved cases of encephalitis often ascribed to malaria in Africa47. Observed patterns of viral loads suggest that virus could be acquired during slaughtering of bats for alimentary purposes48, but possibly also through contact with ubiquitous bat faeces (Supplementary Tables S3 and S8). It is for this same reason that the significance of Henipavirus-related agents in widely distributed bats in America deserves urgent further investigation. Moreover, the finding of agents serologically related to eradicable viruses, such as mumps, distemper and measles virus, is highly relevant in assessing perspectives and consequences of virus eradication19,49,50,51. Clearly, the bats investigated in this study carried viruses that were only similar but not identical to those agents endemic in humans or livestock. These new data therefore emphasise the importance of investigating possible transmission chains, as exemplified by the case of severe acute respiratory syndrome, in which an agent derived from bats was probably passed to humans by intermediate hosts such as carnivores52. In the case of the mumps-related bat virus, a direct antigenetic relatedness between human and bat viruses has been confirmed, and the close genetic proximity between both viruses suggests that even cross-neutralisation might be possible. In light of the still narrow representation of genetic diversity of bats covered in this study (ca. 7.5% of bat species), further research might reveal further bat-borne PV in close relationship to known pathogens of humans and livestock. If antigenic overlap exists, this could become relevant for virus eradication concepts. Relevant antigenic overlap would be defined by proof of cross-neutralisation between reservoir-borne and human or livestock pathogens. In this latter case, elimination of circulating virus and the subsequent cessation of vaccination might leave humans or livestock susceptible for reservoir-borne, antigenetically related viruses.

Although these data identify a potential reservoir of important mammalian viruses, we can only begin to understand their true significance by functional investigation. Knowledge on the genetic range of pathogens carried by speciose small mammals may enable early recognition of zoonotic epidemics and rapid decision-making in the public health sector53,54. However, much more (and different) work needs to be done to actually assess and ameliorate zoonotic risks. The most relevant provision in this field is that epidemic risks emanating from wildlife virus reservoirs should trigger wildlife conservation rather than interference with wild animal populations2.