Brief Communication https://doi.org/10.1038/s41564-018-0227-2

The discovery of Bombali adds further support for bats as hosts of

Tracey Goldstein 1,14*, Simon J. Anthony2,3,4,14*, Aiah Gbakima5, Brian H. Bird1, James Bangura5, Alexandre Tremeau-Bravard1, Manjunatha N. Belaganahalli 1, Heather L. Wells 2, Jasjeet K. Dhanota 1, Eliza Liang2,4, Michael Grodus2, Rohit K. Jangra6, Veronica A. DeJesus6, Gorka Lasso7, Brett R. Smith1, Amara Jambai8, Brima O. Kamara9, Sorie Kamara10, William Bangura11, Corina Monagin1,12, Sagi Shapira7,13, Christine K. Johnson1, Karen Saylors12, Edward M. Rubin12, Kartik Chandran6, W. Ian Lipkin2,3 and Jonna A. K. Mazet1

Here we describe the complete genome of a new , also positive using a separate ebolavirus ‘genus-level’ cPCR assay. Bombali virus (BOMV) detected in free-tailed bats in Sierra The resulting 187-bp fragment showed 83% nucleotide identity Leone (little free-tailed (Chaerephon pumilus) and Angolan to known ebolaviruses. All samples collected from dogs, cats and free-tailed (Mops condylurus)). The bats were found roost- rodents were negative when both assays were used. Given the 2013 ing inside houses, indicating the potential for human trans- virus disease outbreak, we also screened all samples for EBOV mission. We show that the viral glycoprotein can mediate using specific real-time PCR (rtPCR); however, all samples, includ- entry into human cells. However, further studies are required ing those from bats, were negative. to investigate whether exposure has actually occurred or if All bats (n =​ 244) were barcoded to confirm the species BOMV is pathogenic in humans. (Supplementary Table 1). Of the four positive bats, three were iden- Ebolaviruses (family: ) are non-segmented, negative- tified as little free-tailed bats (Chaerephon pumilus) based on 98% sense, single-stranded RNA . Five species have been sequence identity in the MT-Cytb gene and 99% in the MT-CO1 described to date, for which the prototypic viruses are Zaire virus gene. The fourth bat was identified as an Angolan free-tailed bat (EBOV), Bundibugyo virus (BDBV), Sudan virus (SUDV), Taï (Mops condylurus) based on 98% identity in the MT-Cytb gene and Forest virus (TAFV) and (RESTV)1. With the exception 99% in the MT-CO1 gene. These bats co-roost and are widely dis- of RESTV, all have been associated with severe disease in humans. tributed across Western and sub-Saharan Africa (Supplementary EBOV was the first ebolavirus described and since 1976 more than Figure 1). The four positive bats were adult females sampled between 25 outbreaks have been recognized2. The most significant outbreak the 21 and 28 May 2016 at three different sites within 20 km of occurred in , and Liberia in 2013–2016 where each other in the Bombali District (Supplementary Figure 1). They an estimated 28,000 humans were infected and 11,325 died3. were sampled inside human dwellings in small villages, where ani- Despite more than 40 years of research and continued outbreaks, mals (poultry, goats, sheep) and crops (fruit, vegetables, oil trees) the reservoirs of EBOV and the other ebolaviruses remain unknown. were raised for local consumption and sale (Supplementary Table 2). Current evidence points to bats4–9, although failure to isolate a virus Using unbiased high-throughput sequencing, 98% of the genome or recover a complete genome means that no ebolavirus has been was recovered from the oral swab of the Angolan free-tailed bat conclusively linked to any particular bat species. Therefore, we ini- with an average depth of 12×.​ Using virome capture sequencing tiated a survey in Sierra Leone to identify hosts of EBOV as well as (VirCapSeq), 42% of the genome was recovered with an average any additional filoviruses that might be circulating in wildlife. depth of 5×.​ Gene walking using PCR and Sanger sequencing was Between March and September 2016, 1,278 samples were col- used to obtain a second genome from the rectal swab of a little free- lected from 535 animals (244 bats, 46 rodents, 240 dogs, 5 cats) tailed bat. The termini for both sequences were then verified using from 20 locations in Sierra Leone (Supplementary Figure 1). rapid amplification of cDNA ends (RACE) to generate two com- Three oral and two rectal swabs from four insectivorous bats were plete BOMV genomes (GenBank accession numbers: MF319185 positive using a broadly reactive filovirus ‘family-level’ consensus and MF319186). The two genomes share 99.1% sequence identity PCR (cPCR) assay (4/244, Supplementary Table 1). The resulting to each other. 680-bp fragment showed 75% nucleotide identity to other known Phylogenetic analyses showed that BOMV is sufficiently dis- ebolaviruses. Rectal swabs for two of the four positive bats were tinct to represent the prototypic strain of a new species within the

1One Health Institute & Karen C. Drayer Wildlife Health Center, School of Veterinary Medicine, University of California, Davis, CA, USA. 2Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, USA. 3Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY, USA. 4EcoHealth Alliance, New York, NY, USA. 5Metabiota, Inc. Sierra Leone, Freetown, Sierra Leone. 6Department of and Immunology, Albert Einstein College of Medicine, New York, NY, USA. 7Department of Systems Biology, Irving Cancer Research Center, Columbia University, New York, NY, USA. 8Ministry of Health and Sanitation, Freetown, Sierra Leone. 9Ministry of Health and Sanitation, Makeni, Sierra Leone. 10Livestock and Veterinary Services Division, Ministry of Agriculture, Forestry and Food Security, Freetown, Sierra Leone. 11Forestry and Wildlife Division, Ministry of Agriculture, Forestry and Food Security, Freetown, Sierra Leone. 12Metabiota, Inc., San Francisco, CA, USA. 13Department of Microbiology & Immunology, Columbia University, New York, NY, USA. 14These authors contributed equally: Tracey Goldstein, Simon J. Anthony. *e-mail: [email protected]; [email protected]

Nature Microbiology | www.nature.com/naturemicrobiology Brief Communication NATure MicrOBiOLOgy a b NP EBOV/H.sap/COD/76 VP35 EBOV/H.sap/COD/76 ω = 0.1708 EBOV/H.sap/COD/95 ω = 0.1540 EBOV/H.sap/COD/95 EBOV/H.sap/COD/07 EBOV/H.sap/LBR/14 EBOV/H.sap/GIN/14 EBOV/H.sap/GIN/14 EBOV/H.sap/LBR/14 EBOV/H.sap/SLE/15 1 1 EBOV/H.sap/SLE/15 EBOV/H.sap/COD/07 BDBV/H.sap/UGA/07 BDBV/H.sap/UGA/07 1 1 1 1 TAFV/H.sap/CIV/94 TAFV/H.sap/CIV/94 BOMV/M.con/SLE/16 BOMV/M.con/SLE/16 1 1 BOMV/C.pum/SLE/16 BOMV/C.pum/SLE/16 SUDV/H.sap/UGA/00 SUDV/H.sap/UGA/00 1 0.98 1 0.94 RESTV/M.fas/USA/89 RESTV/M.fas/USA/89 1 LLOV/M.sch/ESP/03 1 LLOV/M.sch/ESP/03 MARV/H.sap/COD/99 MARV/H.sap/COD/99 1 1 MARV/R.aeg/UGA/09 MARV/R.aeg/UGA/09 0.5 0.4 c VP40 EBOV/H.sap/COD/76 d GP EBOV/H.sap/COD/76 EBOV/H.sap/COD/95 EBOV/H.sap/COD/95 ω = 0.1096 ω = 0.2409 EBOV/H.sap/GIN/14 EBOV/H.sap/COD/07 EBOV/H.sap/LBR/14 EBOV/H.sap/LBR/14 EBOV/H.sap/SLE/15 EBOV/H.sap/GIN/14 1 EBOV/H.sap/COD/07 0.95 EBOV/H.sap/SLE/15 BDBV/H.sap/UGA/07 BDBV/H.sap/UGA/07 1 1 TAFV/H.sap/CIV/94 1 TAFV/H.sap/CIV/94 BOMV/M.con/SLE/16 BOMV/M.con/SLE/16 1 1 BOMV/C.pum/SLE/16 BOMV/C.pum/SLE/16 SUDV/H.sap/UGA/00 SUDV/H.sap/UGA/00 1 1 1 1 RESTV/M.fas/USA/89 RESTV/M.fas/USA/89 1 LLOV/M.sch/ESP/03 1 LLOV/M.sch/ESP/03 MARV/H.sap/COD/99 1MARV/H.sap/COD/99 1 MARV/R.aeg/UGA/09 MARV/R.aeg/UGA/09 0.5 0.4 e VP30 EBOV/H.sap/COD/76 f VP24 EBOV/H.sap/COD/76 EBOV/H.sap/COD/95 EBOV/H.sap/COD/95 ω = 0.1503 ω = 0.0874 EBOV/H.sap/GIN/14 EBOV/H.sap/LBR/14 EBOV/H.sap/SLE/15 EBOV/H.sap/GIN/14 EBOV/H.sap/LBR/14 EBOV/H.sap/SLE/15 1 EBOV/H.sap/COD/07 1 EBOV/H.sap/COD/07 BDBV/H.sap/UGA/07 BDBV/H.sap/UGA/07 0.99 1 1 TAFV/H.sap/CIV/94 0.98 TAFV/H.sap/CIV/94 0.73 SUDV/H.sap/UGA/00 0.64 BOMV/M.con/SLE/16 1 RESTV/M.fas/USA/89 BOMV/C.pum/SLE/16 BOMV/M.con/SLE/16 1 RESTV/M.fas/USA/89 1 BOMV/C.pum/SLE/16 1 SUDV/H.sap/UGA/00 1 LLOV/M.sch/ESP/03 1 LLOV/M.sch/ESP/03 MARV/H.sap/COD/99 MARV/H.sap/COD/99 1 1 MARV/R.aeg/UGA/09 MARV/R.aeg/UGA/09 0.3 0.2 g L EBOV/H.sap/COD/76 h Full genome EBOV/H.sap/COD/76 EBOV/H.sap/COD/95 EBOV/H.sap/COD/95 = 0.1206 ω EBOV/H.sap/COD/07 EBOV/H.sap/COD/07 EBOV/H.sap/LBR/14 EBOV/H.sap/LBR/14 EBOV/H.sap/GIN/14 EBOV/H.sap/GIN/14 1 1 EBOV/H.sap/SLE/15 EBOV/H.sap/SLE/15 BDBV/H.sap/UGA/07 BDBV/H.sap/UGA/07 1 1 1 1 TAFV/H.sap/CIV/94 TAFV/H.sap/CIV/94 BOMV/M.con/SLE/16 1 1 BOMV/M.con/SLE/16 BOMV/C.pum/SLE/16 BOMV/C.pum/SLE/16 SUDV/H.sap/UGA/00 SUDV/H.sap/UGA/00 1 1 1 1 RESTV/M.fas/USA/89 RESTV/M.fas/USA/89 1 LLOV/M.sch/ESP/03 1 LLOV/M.sch/ESP/03 MARV/H.sap/COD/99 MARV/H.sap/COD/99 1 1 MARV/R.aeg/UGA/09 MARV/R.aeg/UGA/09 0.3 0.4

Fig. 1 | Phylogenetic tree comparing the relationship of BOMV to other known filoviruses. a–h, Each protein is presented separately (a–g) and as a complete genome (h). The values of ω​ were generated using SLAC analysis in Datamonkey based on 15 filovirus sequences (ω​ <​ 1 indicates purifying selection, ω​ > 1​ indicates positive selection). GenBank accession numbers for the reference sequences are included in the ‘Data availability’ section.

Ebolavirus genus (Fig. 1; Supplementary Figure 2). We suggest the variability were identified throughout the genome (Supplementary species should be named Bombali ebolavirus to reflect the loca- Figure 4). No evidence of recombination was observed. Selection tion of first detection, which is consistent with the naming of other analysis indicated that all genes were undergoing purifying ebolavirus species. Assessment using NCBI’s PAirwise Sequence selection; however, several individual residues showed evidence of Comparison (PASC) tool supports this new species assignment; positive selection (Supplementary Table 3). it also meets all the criteria for a novel virus species as suggested A BOMV-specific rtPCR assay was used to rescreen all samples by Bào et al.10 (Supplementary Figure 3). Overall, the virus showed and to quantify the of positive samples. This assay detected 55–59% nucleotide identity (64–72% amino acid identity) to other down to 10 genome copies with 91% efficiency and did not cross- ebolaviruses, though areas of high sequence conservation and high react with virus (genus , family Filoviridae),

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a b 8

) 7

–1 Cell clone: BOMV GP 6 U2OS–WT M (K): r IU ml NPC1–KO – cDNA 160 10 5 125 GP1 NPC1– KO + cDNA Viral infectivit y 90 (log 4

3 rVSV–BOMV GP rVSV–GP: BOMV EBOVVSV c 79 88 111 118 140 155 170

EBOV D V P S A T K R W G F R N L E I K K P D G S H K V S G T G P C A G D F A F H K E V I Y SUDV D I P S A T K R W G F R N L E I K K P D G S H K A Q G T G P C P G D Y A F H K D V I Y RESTV D V P S A T K R W G F R N L E I K K S D G S H K V Q G T G P C P G D L A F H K N V I Y BDBV D V P T A T K R W G F R N L D I K K A D G S H K V S G T G P C P E G Y A F H K E I I Y TAFV D V P T A T K R W G F R N L A I K K V D G S H K V S G T G P C P G G L A F H K E I I Y BOMV D V P S A T K R W G F R N L E I K K P D G S H K V S G T G S C E S G F A F H K E I I Y d

Fig. 2 | BOMV GP-mediated entry and infection is NPC1-dependent. a, BOMV GP1,2 is incorporated into rVSV particles. Pelleted rVSV–BOMV GP particles (equivalent to 18,000 infectious units on U2OS cells) were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting with an antiserum specific for ebolavirus GP1 residues 86–97 (EBOV GP1,2 numbering). Western blots were repeated twice, each at three dilutions of rVSV–BOMV GP (Supplementary Figure 5). b, Infectivity of rVSVs bearing BOMV, EBOV or VSV GPs on wild-type (WT) or NPC1 knockout (KO) U2OS cells complemented with or without human NPC1 cDNA (average ±​ s.d.; n =​ 15 from 5 independent experiments for WT and NPC1 KO cDNA cells; n =​ 6 from two independent experiments for NPC1 KO +​ cDNA cells; the dotted orange line indicates the limit of detection for the assay). c, GP1 alignment of the known human-infecting ebolaviruses (EBOV, SUDV, RESTV, BDBV, TAFV) and BOMV. Displayed regions pertain to the GP1 interface based on the GP1-human NPC1 crystal structure (PDB: 5F1B). Conserved residues are shown in blue; viral-specific residues in yellow. Squared positions correspond to residues whose side chain heavy atoms are within 5 Å of any heavy atom in the human NPC1 receptor. d, Left panel: atomic representation of the interaction between the human NPC1 (red) and the EBOV GP1 protein (blue) (PDB: 5F1B). Middle panel: close-up view of the interface. Right panel: close-up view of the modelled interface between the human NPC1 crystal structure (red) and the BOMV GP1 atomic model (blue). Displayed viral residues (in yellow) correspond to interfacial positions with different amino acids in the BOMV GP1 protein. Displayed residues on the human NPC1 (in white) correspond to residues with side chain heavy atoms within 5 Å of residues 146 and/or 148 in the EBOV or BOMV GP1.

Lloviu virus (genus Cuevavirus, family Filoviridae) or other known Free-tailed bats have been previously implicated as hosts of ebolaviruses (BDBV, EBOV, RESTV, SUDV, TAFV). Viral load in the ebolaviruses. Both little and Angolan free-tailed bats were shown four positive animals varied from 10,000 to 4 genome copies per μ​l. to survive experimental infection with EBOV (human Kikwit A rectal swab from one additional little free-tailed bat was found to variant11), while Angolan free-tailed bats were suggested as the be weakly positive with approximately three genome copies per μ​l. source of the 2013 Ebola virus disease outbreak in Western Africa3. Given that little and Angolan free-tailed bats are insectivorous, Angolan free-tailed bats were also shown to have antibodies we considered the possibility that insects or other arthropods against EBOV or a related virus5. The discovery of BOMV further could be the source of this virus. However, sequences of arthropod supports their role as hosts of ebolaviruses, although additional mitochondrial DNA were only obtained from one of the positive surveillance will be required to determine if BOMV is distributed samples—the oral swab of the Angolan free-tailed bat. Sequences throughout their range and whether these bats sustain BOMV of two different arthropods were detected: an unidentified butterfly transmission over time (that is, whether they are true reservoirs within the Papillionoidea and a hexapod within the Fujientomidae. of BOMV). BOMV load in this specimen was approximately 2,800 genome Given that BOMV was found in close proximity to humans, copies. By comparison, a rectal swab from a little free-tailed bat we tested whether the BOMV envelope glycoprotein GP1,2 could had an estimated 10,000 genome copies but no arthropod DNA. mediate virus entry into human cells. We generated a recombinant Therefore, despite previous suggestions that insects may be reser- vesicular stomatitis virus (rVSV) encoding the BOMV GP gene voir hosts or vectors of ebolaviruses11,12, we found no correlation (Fig. 2, panel A), and showed that the rVSV–BOMV GP was infec- between the presence of insect DNA and BOMV. These data suggest tious in human osteosarcoma (U2OS) cells (Fig. 2, panel B). These that BOMV was not present merely as a component of the bat diet. data indicate that BOMV GP1,2 is fully competent to mediate viral

Nature Microbiology | www.nature.com/naturemicrobiology Brief Communication NATure MicrOBiOLOgy entry. Entry and infection of rVSV–BOMV GP was also completely thiocyanate were shipped to the One Health Institute laboratory, University of dependent on Niemann-Pick C1 protein (NPC1; Fig. 2, panel B), California, Davis for analysis under Public Health Service permit no. 2016-06-092. Bat host species identification was confirmed by DNA barcoding of the providing additional evidence that NPC1 is a universal receptor for MT-Cytb and MT-CO1 mitochondrial genes28. The presence of invertebrate 13,14 filoviruses . Sequence analysis showed that BOMV GP1,2 shares DNA in BOMV-positive samples was examined by PCR for a fragment of the 92% of the known NPC1-interacting residues found in other ebo- MT-CO1 gene29 (up to 48 clones sequenced from each). laviruses, with only two unique mutations identified at the binding interface, P146S and A148E (Fig. 2, panel C)15–18. The correspond- Viral discovery and sequencing. Total RNA was extracted using Direct-zol RNA ing NPC1 residues found within 5 Å of P146S and A148E were con- columns (Zymo Research) and cDNA prepared using SuperScript III (Invitrogen). Samples were screened using three assays: (1) a nested filovirus ‘family-level’ cPCR served between humans and free-tailed bats (both have D502 and targeting a 680-bp fragment of the filovirus L gene; (2) an Ebolavirus ‘genus-level’ V505). Neither of these mutations were predicted to interfere with cPCR targeting a 187-bp fragment of the NP gene30; and (3) an rtPCR specific for GP–NPC1 recognition to block binding (Fig. 2, panel D); this was the EBOV virus, targeting the L gene31. Primer sequences for the cPCR filovirus supported by our experimental data. assay were: round 1: Filo-MOD-FWD: TITTYTCHVTICAAAAICAYTGGG, FiloL.conR: ACCATCATRTTRCTIGGRAAKGCTTT; round 2: Filo- We acknowledge that binding is not the only determinant of host MOD-FWD: TITTYTCHVTICAAAAICAYTGGG, Filo-MOD-RVS: susceptibility; however, it represents the first critical step in spillover. GCYTCISMIAIIGTTTGIACATT. To quantify the BOMV load, a Further, even if BOMV is able to establish a productive infection, it quantitative rtPCR was designed. Primers and probe sequences were: is not known whether the virus is virulent in humans since RESTV Filo_UCD_qFor: TCTCGACGAAGGTCATTAGCGA; Filo_UCD_qRev: can also infect human cells but does not cause disease19,20. Data on TTGCTCTGGTACTCGCTTGGT; and Filo_UCD_probe: FAM- TGCTGGGATGCTGTCTTTGAGCCT-BHQ. pro-inflammatory cytokine expression in human macrophages Libraries for genome sequencing were generated with the KAPA Hyper Prep or the degree to which BOMV antagonizes the human interferon kit (Roche)32 and with VirCapSeq for vertebrate viruses (VirCapSeq-VERT33) and response could help to clarify the pathogenic potential of this virus. sequenced on the Miseq platform (Illumina). Contigs and unique singletons were Certain key motifs in BOMV 35 (VP35) (interferon assembled as described previously32. A second genome was generated by PCR induction) and VP24 (interferon signalling)19,21–23 are more similar walking using gene-specific primers. The termini were amplified using RACE, adopting anchor- and virus-specific primers. Host NPC1 sequences were generated to EBOV, while others are more similar to RESTV (Supplementary by mapping unassembled singletons onto a reference NPC1 gene. Figure 4). Thus, predictions of pathogenicity in humans cannot be made from the sequence alone. Phylogenetic analyses. Sequences were edited using Geneious (version 9.1.7; While the pathogenic potential of BOMV is unknown, our data https://www.geneious.com/) and aligned with CLUSTALW (https://www.genome. on cell entry suggest that the virus could infect humans. Evidence of jp/tools-bin/clustalw). Bayesian coalescent phylogenetic analysis was implemented ebolavirus-reactive antibodies in humans before the 2013 outbreak24 using BEAST (http://beast.community/tree_priors). Nucleotide substitution models were chosen using jModelTest (https://github.com/ddarriba/jmodeltest2) and a suggests that an ebolavirus was already circulating in humans in this Yule process speciation model. Each analysis was run for 1,000,000 generations. area. We suggest that it is unlikely that a virulent pathogen such as Maximum clade credibility trees were generated using the TreeAnnotator program EBOV would circulate in humans without causing disease. Given in BEAST and edited using FigTree. Alignments and trees were created separately also the cross-reactivity between ebolaviruses (Supplementary for each gene and a concatenation was used for the complete genome. Sequences 10 Table 4, Supplementary Figure 6), and that BOMV was discov- were also analysed using the PASC tool to classify sequences taxonomically . The nucleotide alignment of the ebolavirus genomes was screened for recombination ered in bats inside houses, it is possible that BOMV or some other using the seven algorithms in the Recombination Detection Program (version 4.87; potentially non-pathogenic ebolavirus has already spilled over. http://web.cbio.uct.ac.za/~darren/rdp.html). The ebolavirus nucleotide alignment Serosurveys of humans in contact with little and Angolan free-tailed was analysed for evidence of selection using the SLAC, FEL, MEME and FUBAR bats would help to confirm whether exposure has occurred. algorithms, executed in Datamonkey (http://datamonkey.org/) and with the M7 Our study contributes to a better understanding of the diversity and M8 codon models in codeml (Phylogenetic Analysis by Maximum Likelihood (PAML) package; http://abacus.gene.ucl.ac.uk/software/paml.html). The codon and ecology of ebolaviruses. First, our data provide strong evidence models in PAML were implemented using both a gene-specific tree for each gene and that bats serve as hosts for ebolaviruses and that additional unknown a species-level tree (the concatenated alignment tree). Model fit was compared using 2 ebolaviruses may exist in wildlife. Identifying these viruses and a likelihood ratio test (χ d.f. = 2). To visualize the variation in selective pressure across testing their capacity for human infection would greatly enhance the genome, the empirical Bayes posterior mean ω​ ±​ credible interval was plotted for our understanding of ‘pre-emergent’ viral diversity. Second, it sug- each codon position and colour-coded according to the posterior probability of ω​ >​ 1. gests that insectivorous bats play an important role in the ecology BOMV GP1,2 interaction with human NPC1. Wild-type (WT) and NPC1 of ebolaviruses. To date, surveys have tended to focus on fruit bats. knockout U2OS cells complemented with human NPC1 cDNA were cultured as While they seem to be important hosts4,5,25,26, we support the previ- described previously34. Rescue of the rVSVs bearing EBOV GP1,2 and VSV G ous suggestion by Marí Saéz et al.2 that future surveillance should be have also been described previously35. The U2OS human carcinoma cell line was expanded to include insectivorous bats. obtained from ATCC and authenticated at the source. The U2OS knockout cell line was produced by CRISPR–Cas9 engineering and sequenced to confirm the Finally, we stress that our study is not meant to create alarm or deletion of the NPC1 allele. All cell lines were routinely tested and were negative incite the retaliatory culling of bats. While bats have been impli- for Mycoplasma. cated as reservoirs for a number of other infectious pathogens, they Sequence encoding the full-length BOMV GP1,2 from the Angolan free- are also important insectivores, pollinators and seed dispersers. tailed bat was cloned between MluI and NotI restriction sites into the plasmid 35 Previous studies have shown that killing or disturbing bats in their VSV vector to replace the VSV G open reading frame . The resulting plasmid was used to rescue the rVSV–BOMV GP virus using the plasmid-based natural habitat does not reduce the risk of transmission; rather, it infectious VSV rescue system on 293FT cells as described previously36,37. The can increase the number of susceptible bats and enhance disease 293T human embryonic kidney fibroblast cell line was obtained from ATCC transmission27. While BOMV has the potential to infect human and authenticated at the source. The rescued virus was expanded on Vero cells cells, there is currently no evidence that the virus causes disease. and the BOMV GP1,2 sequence was verified by rtPCR followed by Sanger Nonetheless, local community engagement is ongoing to explain the sequencing. Incorporation of BOMV GP1,2 into the VSV particles was detected by immunoblotting using a rabbit antiserum specific for ebolavirus GP1 residues current state of understanding regarding BOMV. 86–97 (EBOV GP numbering)38,39. Monolayers of WT, NPC1 knockout or NPC1 knockout U2OS cells Methods complemented with human NPC1 cDNA U2OS cells were infected with Animal sampling.. Oral and rectal swabs, and whole blood when possible, were serial log dilutions of rVSVs expressing enhanced green fluorescent protein collected into guanidinium thiocyanate, frozen in liquid nitrogen and stored at (eGFP) and bearing the EBOV, BOMV or VSV glycoproteins for 1 h at 37 °C. −​80 °C until analysis. All animal sampling activities were conducted with Ammonium chloride at a final concentration of 20 mM was added at 1 h post- permission from Te Ministry of Agriculture, Forestry and Food Security and infection to prevent subsequent rounds of infection. Infections were enumerated under the Institutional Animal Care and Use Committee at the University of by counting eGFP-positive cells at 12–14 h post-infection and expressed as California, Davis (protocol number: 16048). Inactivated samples in guanidinium infectious units per ml.

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Protein structure modelling. A sequence alignment between EBOV GP1,2 and 7. Yuan, J. et al. Serological evidence of ebolavirus infection in bats, China. BOMV GP1,2 was carried out with T-coffee (https://www.ebi.ac.uk/Tools/msa/ Virol. J. 9, 236 (2012). tcoffee/). Interfacial residues were identified using the crystal structure of the 8. Negredo., A. et al. Discovery of an ebolavirus-like flovirus in Europe. EBOV GP1,2 protein bound to human NPC118. BLAST was used for template PLoS Pathog. 7, e1002304 (2011). search and alignment, while NEST (http://honig.c2b2.columbia.edu/nest/) was 9. Jayme, S. I. et al. Molecular evidence of Ebola Reston virus infection in used to model the structure of the BOMV GP1,240. A non-redundant set of Philippine bats. Virol. J. 12, 107 (2015). sequences was assembled, corresponding to the proteins in the NCBI PDB, using a 10. Bào, Y. et al. Implementation of objective PASC-derived taxon demarcation sequence identity cut-off of 1.0 with CD-HIT43(http://weizhongli-lab.org/cd-hit/). criteria for ofcial classifcation of floviruses. Viruses 9, 106 (2017). A single iteration of BLAST was run against this data set and the template and 11. Swanepoel, R. et al. Experimental inoculation of plants and animals with alignment with the lowest e-value was selected (PDB: 5FHC; e-value: 1.4 e−139). Ebola virus. Emerg. Infect. Dis. 2, 321–325 (1996). The interaction of the human NPC1 protein with BOMV GP1,2 was assessed 12. Leendertz, S. A. J. Testing new hypotheses regarding ebolavirus reservoirs. using a structural alignment of the GP1,2 atomic model to the crystallized human Viruses 8, 30 (2016). NPC1–EBOV GP1,2 protein complex40 with the SKA program (http://honig.c2b2. 13. Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter columbia.edu/ska). Niemann-Pick C1. Nature 477, 340–343 (2011). 14. Côté, M. et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential Peptide enzyme-linked immunosorbent assay (ELISA) assay. We designed for Ebola virus infection. Nature 477, 344–348 (2011). and synthesized a series of peptides with increasing specificity for BOMV GP1, 15. Miller, E. H. et al. Ebola virus entry requires the host-programmed including one BOMV peptide with high sequence similarity with the other recognition of an intracellular receptor. EMBO J. 31, 1947–1960 (2012). ebolaviruses (GP-100); one that shares sequence similarity with some, but not all, 16. Ng, M. et al. Filovirus receptor NPC1 contributes to species-specifc patterns ebolaviruses (GP-270); and one BOMV peptide that shows no sequence homology of ebolavirus susceptibility in bats. eLife 4, e11785 (2015). with the other ebolaviruses (GP-471, Supplementary Figure 5). Our rationale was 17. Bornholdt, Z. A. et al. Host-primed Ebola virus GP exposes a hydrophobic to demonstrate decreasing cross-reactivity as a function of sequence variation. NPC1 receptor-binding pocket, revealing a target for broadly neutralizing Peptides with high sequence similarity for EBOV (GP-313) and TAFV (GP-378) antibodies. mBio 7, e02154-15 (2016). were designed to demonstrate specificity of peptides with known sequence 18. Wang, H. et al. Ebola viral glycoprotein bound to its endosomal receptor homology to other ebolaviruses. ELISA was performed as described by King Niemann-Pick C1. Cell 164, 258–268 (2016). 43 et al. with slight modifications. We coated plates overnight with each peptide 19. Pappalardo, M. et al. Conserved diferences in protein sequence determine 1 1 (6 μ​g ml− ) or recombinant EBOV glycoprotein (0.5 μ​g ml− ; IBT Bioservices), the human pathogenicity of Ebolaviruses. Sci. Rep. 6, 23743 (2016). blocked (1% bovine serum albumin) and used 100 μ​l primary rabbit polyclonal 20. Miranda, M. 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Ebola virus contains an immunosuppressive-like domain similar to oncogenic retroviruses. FEBS Lett. 305, 181–184 (1992). Reporting Summary. Further information on research design is available in the 24. Schoepp, R. J., Rossi, C. A., Khan, S. H., Goba, A. & Fair, J. N. Nature Research Reporting Summary linked to this article. Undiagnosed acute viral febrile illnesses, Sierra Leone. Emerg. Infect. Dis. 20, 1176–1182 (2014). Data availability. All sequences obtained or utilized in this study are available in 25. Towner, J. S. et al. infection detected in a common African GenBank. Accession numbers are as follows: BOMV/M.con/SLE/16 (MF319185); bat. PLoS ONE 2, e764 (2007). BOMV/C.pum/SLE/16 (MF319186); EBOV/H.sap/COD/76 (KC242801); 26. Yang, X. L. et al. Genetically diverse floviruses in Rousettus and Eonycteris EBOV/H.sap/COD/95 (KR867676); EBOV/H.sap/COD/07 (KC242786); spp. bats, China, 2009 and 2015. Emerg. Infect. Dis. 23, 482–486 (2017). EBOV/H.sap/GIN/14 (KT765131); EBOV/H.sap/LBR/14 (KR075003); EBOV/H. 27. Amman, B. R. et al. Marburgvirus resurgence in Kitaka Mine bat sap/SLE/15 (KT357856); BDBV/H.sap/UGA/07 (KU182911); TAFV/H.sap/ population afer extermination attempts, Uganda. Emerg. Infect. Dis. 20, CIV/94 (KU182910); SUDV/H.sap/UGA/00 (KR063670); RESTV/M.fas/USA/89 1761–1764 (2014). (AF522874); LLOV/M.sch/ESP/03 (JF828358); MARV/H.sap/COD/99 (JX458851); 28. Townzen, J. S., Brower, A. V., & Judd, D. D. Identifcation of mosquito MARV/R.aeg/UGA/09 (JX458854); Bat filovirus/Rousettus/CHN/09/Bat2202 bloodmeals using mitochondrial cytochrome oxidase subunit I and (KX371882); Bat filovirus/Rousettus/CHN/09/Bat2188 (KX371878); Bat filovirus/ cytochrome b gene sequences. Med. Vet. Entomol. 22, 386–393 (2008). Rousettus/CHN/15/Bat9447-1 (KX371887); Bat filovirus/Rousettus/CHN/09/ 29. Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. DNA Bat2196 (KX371880); Bat filovirus/Rousettus/CHN/09/Bat2187 (KX371877); primers for amplifcation of mitochondrial cytochrome c oxidase Bat filovirus/Rousettus/CHN/09/Bat2199 (KX371881); Bat filovirus/Rousettus/ subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. CHN/15/Bat9447-2 (KX371888); Bat filovirus/Rousettus/CHN/15/Bat9434 3, 294–299 (1994). (KX371883); Bat filovirus/Rousettus/CHN/15/Bat9445 (KX371886); Bat filovirus/ 30. Towner, J. S. et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse Rousettus/CHN/15/Bat9447-3 (KX371889); Bat filovirus/E.spe/CHN/15/Bat9442 transcription-PCR in an outbreak setting and assessment of patient viral load (KX371884); Bat filovirus/Rousettus/CHN/15/Bat9435 (KX371885); Bat filovirus/ as a predictor of outcome. J. Virol. 78, 4330–4341 (2004). Rousettus/CHN/09/Bat2190 (KX371879); Bat filovirus/Rousettus/CHN/09/ 31. Jääskeläinen, A. J. et al. Development and evaluation of a real-time Bat2180 (KX371875); Bat filovirus/Rousettus/CHN/15/Bat9447-4 (KX371890); EBOV-L-RT-qPCR for detection of . J. Clin. Virol. 67, Bat filovirus/Rousettus/CHN/09/Bat2176 (KX371874); Bat filovirus/Rousettus/ 56–58 (2015). CHN/09/Bat2181 (KX371876); Bat filovirus/R.les/CHN/13/BtFV-WD04 32. Anthony, S. J. et al. Further evidence for bats as the evolutionary source of (KP233864); and Bat filovirus/E.spe/CHN/09/Bat2162 (KX371873). Middle East respiratory syndrome coronavirus. mBio 8, e00373-17 (2017). 33. Briese, T. et al. Virome capture sequencing enables sensitive viral diagnosis Received: 20 June 2018; Accepted: 25 July 2018; and comprehensive virome analysis. mBio 6, e01491-15 (2015). Published: xx xx xxxx 34. Spence, J. S., Krause, T. B., Mittler, E., Jangra, R. K. & Chandran, K. Direct visualization of Ebola virus fusion triggering in the endocytic pathway. mBio References 7, e01857-15 (2016). 1. Burk, R. et al. Neglected floviruses. 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39. Ng, M. et al. Cell entry by a novel European flovirus requires host Author contributions endosomal cysteine proteases and Niemann-Pick C1. 468–470, T.G. helped design the study, collected, analysed and interpreted the data, helped with 637–646 (2014). the literature search and with writing the manuscript. S.J.A. helped design the study, 40. Petrey, D. et al. Using multiple structure alignments, fast model building, and collected, analysed and interpreted the data and helped with writing the manuscript. energetic analysis in fold recognition and homology modeling. Proteins 53, A.G. supervised the in-country activities, including obtaining permissions, and helped S430–S435 (2003). with sample and data collection. B.H.B. helped design the study, analysed the data and 41. Li, W., Jaroszewski, L. & Godzik, A. Clustering of highly homologous helped with the literature search. J.B. performed and supervised all aspects of field sequences to reduce the size of large protein databases. Bioinformatics 17, activities and helped with sample and data collection. A.T-B. and M.N.B. collected and 282–283 (2001). analysed data. H.W. collected, analysed and interpreted data and helped with writing the 42. King, D. P. et al. Humoral immune responses to phocine herpesvirus-1 in manuscript. J.K.D. collected the data. E.L. and M.G. collected, analysed and interpreted Pacifc harbor seals (Phoca vitulina richardsii) during an outbreak of clinical data and helped with writing the manuscript. R.K.J. collected and analysed the data disease. Vet. Microbiol. 80, 1–8 (2001). and helped with writing the manuscript. V.A.D. collected and analysed the data. G.L. collected, analysed and interpreted the data and helped with writing the manuscript. Acknowledgements B.R.S. collected the data. A.J., B.O.K., S.K. and W.B. provided project permissions and logistical support. C.M. helped design the study and collected the data. S.S. analysed and We thank the government of Sierra Leone for permission to conduct this work; the interpreted the data and helped with writing the manuscript. C.K-J. helped design the Sierra Leone district and community stakeholders for their support and for allowing us study. K.S. oversaw the project. E.M.R. oversaw the project and analysed the data. K.C. to perform sampling in their districts and communities; the Bombali Ministry of Health collected and analysed the data and helped with writing the manuscript. W.I.L. analysed and Sanitation and Ministry of Agriculture district officers, field teams and regional lead and interpreted the data and helped with writing the manuscript. J.A.K.M. oversaw and including M. LeBreton, F. Jean Louis, K. Kargbo, L.A.M. Kenny, V. Lungay, W. Robert, designed the project and helped with writing the manuscript. E. Amara, D. Kargbo, V. Merewhether-Thompson, M. Kanu, E. Lavallie, A. Bangura, M. Turay, F.V. Bairoh, M. Sinnah and S. Yonda for performing sample collection; Yongai Saah Bona for administrative and logistic support; laboratory staff for assistance with Competing interests processing the samples, including M. Coomber and O. Kanu (University of Makeni) The authors declare no competing interests. and V. Ontiveros (UC Davis); T. O’Rourke, D. O’ Rourke (Metabiota) and D. Greig (UC Davis) for assistance with data entry, B. Lee for bioinformatics assistance and J. Morrison Additional information and A. Rasmussen for technical guidance (Columbia University); N. Randhawa for map Supplementary information is available for this paper at https://doi.org/10.1038/ graphics (UC Davis); and W. Karesh and J. Epstein (EcoHealth Alliance) for global input s41564-018-0227-2. into study design. This study was made possible by the generous support of the American Reprints and permissions information is available at www.nature.com/reprints. people through the United States Agency for International Development (USAID) Emerging Pandemic Threats PREDICT project (cooperative agreement number GHN- Correspondence and requests for materials should be addressed to T.G. are S.J.A. A-OO-09-00010-00) and by support from the National Institutes of Health (GM030518, Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in S10OD012351, S10OD021764 and GM109018-05). published maps and institutional affiliations.

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Corresponding author(s): Tracey Goldstein

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1 ` Software nature research | life sciences reporting summary Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this ClustalW, BEAST, jModelTest, TreeAnnotator, FigTree, Recombination Detection Program study. (RDP version 4.87), PASC tool, Geneious, SLAC, FEL, MEME, and FUBAR algorithms executed in datamonkey, the M7 and M8 codon models in codeml (PAML package), CD-HIT, SKA program, PRINSEQ software (v 0.20.2), Bowtie2 mapper (v 2.0.6), MIRA assembler (v4.0)

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` Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of No restrictions unique materials or if these materials are only available for distribution by a third party. 9. Antibodies Describe the antibodies used and how they were validated Anti EBOV, TAFV, SUDV, BUDV antibodies were purchased from IBT Bioservices, eEnzyme, for use in the system under study (i.e. assay and species). Sino Biological Inc., Alpha Diagnostic and R&D systems and validated by the company for use in ELISA platforms. Checkerboard dilutions were performed in our lab to determine the appropriate dilution to use for our ELISA assays and negative controls were included to determine if they cross reacted with non-ebolavirus antigens. Likewise, Rabbit antiserum specific for ebolavirus GP1 was purchased from Santa Cruz Biotechnology and used in immmunoblotting to detect BOMV GP1 VSV particles, used per manufacturer instructions. NPC1 domain C was detected by a horseradish-conjugated anti-Flag antibody (Sigma-Aldrich), using ultra-TMB substrate (Thermo Scientific). This is a standard antibody used to detect Flag tags and is well validated by the company. All appropriate negative controls were used to confirm specific binding.

All commercial vendors provide physical quality control and data sheets for all antibodies. 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. All commercially-available cell lines and primary cells are obtained from American Type Culture Collection (ATCC, subsidiary of BEI Resources) or Lonza Group, and are validated at their source. These include Vero African grivet kidney cells, U2OS human carcinoma cells and 293T human embryonic kidney fibroblast cells. This work also involves the generation of a cell line (U2OS-KO) genetically-modified by CRISPR/Cas9 engineering. All engineered cell lines are sequenced to genetically define each allele of the target gene. Further, functional knockout of the target gene and lack of major off-target effects are validated by genetic complementation, by transducing them with retroviruses expressing NPC1 gene variants.

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Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines November 2017 11. Description of research animals Provide all relevant details on animals and/or Oral and rectal swabs, and whole blood when possible, were humanely collected (capture animal-derived materials used in the study. and release) from live captured animals under permits provided by The Ministry of Agriculture, Forestry and Food Security and under the Institutional Animal Care and Use Committee at the University of California, Davis (protocol number: 16048).

2 Policy information about studies involving human research participants nature research | life sciences reporting summary 12. Description of human research participants Describe the covariate-relevant population Not applicable characteristics of the human research participants. November 2017

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