The ongoing, unprecedented Ebola virus disease epidemic in Western Africa highlights the urgent need to uncover the biological and ecological factors that underlie the distribution, evolution, and emergence of filoviruses. While a full answer to this question will require the integration of knowledge across multiple levels of biological organization, from genes to populations to ecosystems, previous work has shown that studies of molecular interactions between viruses and their host cells can contribute important pieces to this puzzle. The essential interactions between viruses and their entry receptors provide particularly cogent examples. A switch in receptor binding from the feline to the canine ortholog of the transferrin receptor drove the emergence of a new virus, canine parvovirus, and fueled a global disease pandemic in dogs (Allison et al., 2014). Analyses of interactions of SARS-like coronaviruses with their receptor ACE2 have helped to trace the emergence of SARS coronavirus from bats to humans, and its use of civets as intermediate amplifying hosts (Demogines et al., 2012; Ge et al., 2013; Ren et al., 2008).

In this study, we show that interactions between filoviruses and their entry receptor NPC1 can influence the cellular susceptibility of bats to infection. This observation is especially striking in light of previous findings that filoviruses could efficiently infect a broad range of mammalian cells, including some derived from bats (Kuhn, 2008; Kuhl et al., 2011). Indeed, this prior work and the results of experimental infection studies in rodents and bats have led to the hypothesis that interactions between viral components and those of the host innate and adaptive immune systems constitute the primary molecular variables influencing filovirus host range in nature (Ebihara et al., 2006; Volchkov et al., 2000).

Here, we propose that NPC1 is also a genetic determinant of filovirus susceptibility in bats. The essential nature of NPC1 for infection in cells derived from mammals of multiple species, including bats (Figure 3), and for infection and in vivo pathogenesis in lethal EBOV infection mouse models argues against the existence of alternative filovirus entry receptors (Carette et al., 2011; Miller et al., 2012; Herbert et al., 2015). Therefore, strong reductions in the affinity of virus-NPC1 recognition are predicted to reduce or eliminate infection in whole bat hosts, as observed in NPC1-deficient mice (Carette et al., 2011; Herbert et al., 2015), barring viral mutation to enhance this affinity. It is conceivable that even modest defects or delays in viral multiplication through such a mechanism could help determine host range by accelerating viral immune clearance, as recently observed in NPC1-heterozygous mice (Herbert et al., 2015), or by synergizing with other host-virus barriers. The highly virus- and host species-specific nature of the virus-receptor mismatch uncovered in this study warrants the determination of more bat NPC1 sequences for inclusion in genetic analyses (see below), and a more comprehensive phenotypic examination of virus-bat pairs. Such studies maydiscover additional interesting bat-filovirus dynamics, including incompatibilities between filoviruses and NPC1 or other proviral/antiviral host factors. Such discoveries have potential implications for our understanding of the molecular basis of filovirus infection, virulence, and host range.

We found that a single amino acid change, at residue 502, in the African straw-colored fruit bat ortholog of NPC1 (EhNPC1) greatly diminished the susceptibility of cells from multiple tissues and individuals to EBOV. These migratory pteropodids are widely distributed across sub-Saharan Africa (Figure 1A), roost in large colonies near human settlements, and host other RNA viruses with zoonotic potential (Baker et al., 2013; Peel et al., 2013). Moreover, they are extensively hunted for bushmeat in Western Africa (Kamins et al., 2011), making them ideal candidates to transmit viruses directly to humans. Unfortunately, there is little information currently available on the susceptibility of African straw-colored fruit bats to EBOV or their potential role as filovirus hosts. Serologic surveys have found some evidence for exposure to one or more filovirus; however, neither infectious virus nor coding-complete or full viral genomes—the gold standards—have been successfully obtained from these bats, indicating they may only have been exposed to filoviruses, rather than being productively infected (reviewed in [Wahl-Jensen et al., 2013; Olival and Hayman, 2014]). While more extensive wildlife sampling and, if feasible, experimental infections of African straw-colored fruit bats will be required to clarify this picture, we can extrapolate to several possible scenarios. First, these bats are fully resistant to EBOV, and therefore cannot be the source of this virus in the 2013–present EBOV disease outbreak in Western Africa or the 2014 outbreak in Middle Africa. Second, because African straw-colored fruit bat cells do remain weakly susceptible to EBOV (Figure 3C), it is conceivable that they support EBOV replication at low levels. Indeed, this is one hallmark of a sustaining viral reservoir. Third, the filoviruses circulating in these bats, whether EBOV or otherwise, bear one or more GP mutations (e.g., V141A) that circumvent the infection barrier imposed by EhNPC1. Assessing this last hypothesis and understanding the nature of the selection pressures that drive GP evolution in vivo will require the isolation of ebolavirus GP sequences from bats—there are none currently available.

Although these results suggest that African straw-colored fruit bats are selectively refractory to EBOV, our genetic findings indicate that this is not merely a special relationship between one host and one virus. Rather, we used a diverse set of bat NPC1 sequences, only one of which is from African straw-colored fruit bats, to show that a number of codons, including residue 502, have evolved under recurrent positive selection. This is a process in which resistant NPC1 variants are ‘serially replaced’ in response to compensating viral mutations that restore susceptibility. We provide evidence that the filovirus GP interaction surface in the second luminal domain of NPC1, domain C, is a hotspot for such positive selection (Figure 5). By contrast, the vast majority of codons in mammalian NPC1 have evolved under purifying selection. We propose that this pattern of selection is the signature of a long-term genetic conflict between filoviruses and NPC1 in bats, superimposed over the normal evolutionary signature of a housekeeping gene with a critical role in cellular cholesterol trafficking. Similar signatures of recurrent positive selection have been identified in other housekeeping genes that encode viral receptors, including the transferrin receptor (Kaelber, et al., 2012; Demogines et al., 2013) (TfR; receptor for New World arenaviruses [Radoshitzky et al., 2007], the betaretrovirus murine mammary tumor virus [Ross et al., 2002], and parvoviruses [Parker et al., 2001]), bat angiotensin-converting enzyme-2 (Demogines et al., 2012) (ACE2; receptor for SARS-like coronaviruses [Li et al., 2003]), and mammalian dipeptidyl peptidase-4 (Cui et al., 2013) (DPP4; receptor for MERS-like coronaviruses [Raj et al., 2013]). In these cases as well, the preponderance of positively-selected residues localize to virus-receptor interfaces. Interestingly, the sequence polymorphism at NPC1 residue 502 did not impair cholesterol clearance from lysosomes (Figure 3), and none of the residues under positive selection were found to be mutated in Niemann-Pick type C disease patients (Runz et al., 2008; Vanier and Millat, 2003). Thus, despite being constrained by its housekeeping function, NPC1 appears to retains a sizeable sequence space accessible to adaptive mutation.

It is tempting to speculate that sequence variation at residue 141 (Figure 6) and potentially other positions in the receptor-binding site of filovirus glycoproteins represents the other half of the genetic arms race, shaped by selective pressure to utilize restrictive NPC1 receptors. Although more data, especially filovirus sequences from bats, are needed, our findings raise the tantalizing possibility that filoviruses, including those yet undiscovered, are each adapted to specific bat hosts, with co-evolved virus-receptor interactions constituting one potential biological barrier to interspecies viral transmission. Alternatively, it is conceivable that repeated contacts between unknown (non-bat) reservoir hosts carrying specific filoviruses, and bats of particular species, have driven positive selection in bat NPC1 to limit infection (and selection of filoviruses with compensating sequence changes in GP). In this scenario, detection of anti-filovirus antibodies or filovirus genome-derived oligonucleotides may reflect a type of spillover event from the actual filovirus reservoir hosts into bats.

Our hypothesis that NPC1 in bats has been genetically sculpted by filoviruses (and vice versa) presupposes not only a long-term coevolutionary relationship, but also one in which these viruses have imposed selective pressure on bats to limit or eliminate infection. The discovery of filovirus NP- and VP35-related endogenous viral elements (EVEs) in bat genomes is consistent with such a long-term relationship (Taylor et al., 2010; 2011; Katzourakis and Gifford, 2010). To further investigate the deeper origins of filoviruses in bats, we screened all available bat genomes for filovirus-related EVEs. We obtained evidence for synteny between a filovirus nucleoprotein (NP)-like EVE in the genome of the big brown bat (Eptesicus fuscus) and those previously identified in three, more distantly-related, myotis bats (Figure 7 and Supplementary file 5) (Taylor et al., 2011). This new discovery strongly suggests that all four EVEs resulted from a single insertion event prior to the divergence of the Myotis and Eptesicus genera, ≈25 million years ago (Miller-Butterworth et al., 2007). Therefore, bats may have been exposed to filovirus-like agents for far longer than previously recognized (≈13 million years ago [Taylor et al., 2011]).

Figure 7 Download asset Open asset Orthologous endogenous viral elements (EVEs) derived from filovirus nucleoprotein (NP) genes indicate that filoviruses have infected bats for at least 25 million years. The time-calibrated phylogeny shown to the left is based on estimates obtained in Miller-Butterworth et al., 2007. The schematic to the right shows the orthologous EVEs and empty insertion sites as they occur in each bat genome. Also see Supplementary file 5. https://doi.org/10.7554/eLife.11785.016

Available experimental exposure studies, although limited in number and scope, suggest that some filoviruses isolated from humans can replicate in bats without causing substantial host pathology (e.g., MARV and RAVV in Egyptian rousettes [Amman et al., 2015; Jones et al., 2015; Paweska et al., 2012]). These observations therefore prompt a key question: what is the origin and nature of the selective pressure that has driven accelerated NPC1 evolution in bats? Our scant understanding admits a number of possibilities. First, it is conceivable that some filoviruses do indeed replicate in a manner that is deleterious to their specific bat hosts—we may simply not have identified the viruses and hosts in question. Indeed, the filovirus LLOV, discovered in Schreibers's long-fingered bat carcasses in Spain and Portugal, may exemplify this possibility (Negredo et al., 2011). Alternatively, in some cases (e.g., ebolaviruses and Egyptian rousettes), the human viral isolates used in challenge studies may differ from these bat isolates in important respects due to human adaptation (human EBOV, BDBV, TAFV, RESTV, and SUDV isolates do not infect Egyptian rousettes [Jones et al., 2015]). Second, filoviruses may have been more virulent in bats in the past. Thus, the positive selection signatures observed in bat NPC1, which cannot be accurately dated, may represent fixed alleles that are the consequence of a selective process driven by ancient filoviruses with properties distinct from their modern counterparts. Indeed, the lack of virulence observed in some bats may reflect a détente that was shaped by precisely these historic genetic conflicts between filoviruses and bats. Third, we cannot rule out the (unlikely) possibility that the evolution of NPC1 in bats was driven by an entirely different infectious agent that also utilizes (or utilized) NPC1 to multiply in its hosts. Regardless of the mechanisms that genetically shaped NPC1, we propose that polymorphisms in this gene nevertheless impose host barriers that impede the colonization and spread of present-day filoviruses in bats in Africa and elsewhere. Our findings set the stage for broader explorations of species-specificity in filovirus interactions with proviral and antiviral host factors, with an eye to uncovering new molecular arms races between filoviruses and bats and new genetic determinants of filovirus host range and host switching.