Detection of Bombali Virus in a Mops condylurus Bat in Kyela, Tanzania

Bombali virus (BOMV) is a novel Orthoebolavirus that has been detected in free-tailed bats in Sierra Leone, Guinea, Kenya, and Mozambique. We screened our collection of 349 free-tailed bat lungs collected in Côte d’Ivoire and Tanzania for BOMV RNA and tested 228 bat blood samples for BOMV antibodies. We did not detect BOMV-specific antibodies but found BOMV RNA in a Mops condylurus bat from Tanzania, marking the first detection of an ebolavirus in this country. Our findings further expand the geographic range of BOMV and support M. condylurus’ role as a natural BOMV host.


Introduction
Ebola virus disease (EVD) is a severe disease with high case-fatality rates (25-90%) that sporadically occurs in Sub-Saharan Africa [1].EVD outbreaks are caused by zoonotic spillovers of ebolaviruses from unknown animal reservoirs, and exceptionally, by resurgence from human EVD survivors [2,3].Different bat species have been implicated as ebolavirus reservoir hosts by molecular, serological, and circumstantial findings, but conclusive evidence remains elusive [4][5][6].
In 2018, Bombali virus (BOMV), a novel Orthoebolavirus, was discovered in two freetailed bat species (Chaerephon pumilus and Mops condylurus) from Bombali District, Sierra Leone [7].Since then, the virus has also been detected in M. condylurus from Guinea, Kenya, and Mozambique [8][9][10].A follow-up study in Kenya showed that BOMV was present in bats caught at the initial sampling site after one year [11].Histopathological examination revealed no differences between PCR-positive and PCR-negative lung tissue, suggesting that BOMV infections in bats are mild [12].The repeated and geographically widespread detections of asymptomatic BOMV infections point to M. condylurus and potentially other free-tailed bats as natural BOMV hosts.
These findings prompted us to screen our collection of free-tailed bat specimens sampled in Tanzania and Côte d'Ivoire between 2013 and 2017 for BOMV RNA and antibodies.Here, we report the detection of BOMV RNA in an M. condylurus bat from southern Tanzania.This finding extends the geographic range of BOMV and lends further support to the role of M. condylurus as its natural host.

Bat Sampling
Our collection comprised 349 free-tailed bats sampled for different studies in Tanzania and Côte d'Ivoire between 2013 and 2017 (Figure 1 and Table S1).
Viruses 2024, 16, x FOR PEER REVIEW 2 of 15 suggesting that BOMV infections in bats are mild [12].The repeated and geographically widespread detections of asymptomatic BOMV infections point to M. condylurus and potentially other free-tailed bats as natural BOMV hosts.These findings prompted us to screen our collection of free-tailed bat specimens sampled in Tanzania and Côte d'Ivoire between 2013 and 2017 for BOMV RNA and antibodies.Here, we report the detection of BOMV RNA in an M. condylurus bat from southern Tanzania.This finding extends the geographic range of BOMV and lends further support to the role of M. condylurus as its natural host.

Bat Sampling
Our collection comprised 349 free-tailed bats sampled for different studies in Tanzania and Côte d'Ivoire between 2013 and 2017 (Figure 1 and Table S1).Bats were caught with 12 m mist nets or self-made funnel traps.The captured bats were anesthetized using 10 mg/kg ketamine and 2 mg/kg xylazine and euthanized by exsanguination by cardiac puncture.Blood and organ samples were collected following strict biosafety protocols, snap-frozen in liquid nitrogen, and placed at −80 °C for longterm storage.All steps were performed by trained personnel under veterinary supervision and with permission by the relevant local authorities.In Tanzania, the study was conducted under the Clearance Certificate for Conducting Medical Research in Tanzania NIMR/HQ/R.8a/Vol.IX/2468.In Côte d'Ivoire, the ethics approval for sampling and analyzing small mammal organs was obtained from the National Commission of Ethics (CNER) under the number 033/MSLS/CNER-dkm.Bats were caught with 12 m mist nets or self-made funnel traps.The captured bats were anesthetized using 10 mg/kg ketamine and 2 mg/kg xylazine and euthanized by exsanguination by cardiac puncture.Blood and organ samples were collected following strict biosafety protocols, snap-frozen in liquid nitrogen, and placed at −80 • C for long-term storage.All steps were performed by trained personnel under veterinary supervision and with permission by the relevant local authorities.In Tanzania, the study was conducted under the Clearance Certificate for Conducting Medical Research in Tanzania NIMR/HQ/R.8a/Vol.IX/2468.In Côte d'Ivoire, the ethics approval for sampling and analyzing small mammal organs was obtained from the National Commission of Ethics (CNER) under the number 033/MSLS/CNER-dkm.

Taxonomic Assignment
In the field, all bats were identified at least to the family level.To confirm and refine the taxonomic assignment, we performed a PCR targeting an ~800 bp fragment of the cytochrome B (CytB) gene (Table A1) and sequenced the PCR products using Sanger's method.The resulting sequences were matched against the nucleotide collection of the National Center for Biotechnology Information (NCBI) using the basic local alignment search tool (BLASTn) [13].
Because not all sequences could be assigned to the species level using BLASTn, we performed a species delimitation analysis using a Bayesian Poisson tree process (bPTP) model [14].Briefly, we selected CytB sequences generated in this study with a length ≥ 700 bp and ran cdhit to remove identical sequences [15].We included Myotis daubentoni (AB106589) as an outgroup and aligned the CytB sequences with MAFFT implemented in Geneious Prime 2020.2.3 (https://www.geneious.com,accessed on 31 May 2021).The resulting alignment was used to build a maximum likelihood (ML) phylogenetic tree using IQ-TREE version 2.1.2with automatic model selection [16].TPM2 + F + G4 was selected as the best-fitting model.Branch robustness was assessed with Shimodaira-Hasegawa-like approximate likelihood ratio tests (SH-like aLRTs) with 100,000 replicates [17].The ML tree was uploaded to the bPTP web server (https://species.h-its.org/,accessed on 31 May 2021) for species delimitation analyses [14].We ran the bPTP model with 500,000 Markov chain Monte Carlo (MCMC) generations and checked for convergence.

Molecular Screening
For BOMV screening, we selected lung tissue based on previous studies indicating a lung tropism for BOMV and other viruses from the Filoviridae family in bats [8,9,11,18].Extraction and cDNA synthesis were performed in two different laboratories, i.e., at the Robert Koch Institute, Berlin, and at the Institute of Virology of the Charité, Berlin.Therefore, different extraction and cDNA synthesis kits were used.At the Robert Koch Institute, we extracted full nucleic acids from lung tissue using the QIAamp ® Viral RNA Kit and generated cDNA using SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, MA, USA) and random hexamer primers (Roche, Basel, Switzerland).At the Charité, we extracted RNA using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and converted it to cDNA using M-MLV Reverse Transcriptase (Invitrogen) with random hexamer primers (Roche).
We used an in-house semi-nested PCR assay targeting a separate region of the L gene of members of the Filoviridae family to confirm a positive result from the BOMVspecific qPCR (Table A1).The semi-nested PCR assay was designed at the Institute of Virology of the Charité based on the RefSeq genomes of Tai Forest ebolavirus (NC_014372), BDBV (NC_014373), EBOV (NC_002549), SUDV (NC_006432), RESTV (NC_004161), the batinfecting Lloviu cuevavirus (NC_016144), Marburg marburgvirus isolate Musoke (MARV; NC_001608), and Marburg marburgvirus isolate Ravn (NC_024781), as well as a batderived filoviral sequence (KP233864).Briefly, for the first round of PCR, we added 5 µL of extracted RNA to a 20 µL master mix containing 1.1 µL of molecular-grade H 2 O, 12.5 µL of 2× Reaction Mix, 0.4 µL of MgSO4 (50 mM), 1µL of Bovine serum albumin, 2 µL of each of primers PanFiloVMC_F2 (10 µM) and PanFiloVMC_R1(10 µM), and 1 µL of SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen).After an initial reverse transcription for 20 min at 48 • C, we applied a touch-down approach with 50 cycles of 15 s denaturation at 98 • C, 20 s annealing starting at 60 • C and decreasing by 1 • C per cycle for the first 10 cycles, and 45 s elongation at 72 • C, followed by a final elongation for 2 min at 72 • C. We used 1 µL of the first-round PCR product in a 24 µL master mix consisting of 18.15 µL of H 2 O, 2.5 µL of 10× PCR buffer, 0.5 µL of dNTP mix (10 mM each), 1.25 µL of MgCl 2 (50 mM), 0.75 µL of each of primers PanFiloVMC_F3 (10 µM) and PanFiloVMC_R1(10 µM), and 0.1 µL of Platinum™ Taq DNA Polymerase (Invitrogen).The cycling protocol was identical to the first-round PCR without the reverse transcription step.The sensitivity of the assay was assessed by serial dilution of two photometrically quantified in vitro transcribed RNAs of EBOV and MARV.The detection limit of the assay was 11.5 RNA copies/µL and 31.0 copies/µL, respectively.PCR products from the BOMVspecific qPCR and the semi-nested Filoviridae PCR were sequenced using Sanger's method.

High-Throughput Sequencing
To generate additional genetic data, we sequenced the positive specimen on Illumina platforms with and without prior enrichment by in-solution hybridization capture.
For shotgun sequencing, we prepared a library using the KAPA RNA Hyper Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.Up to 100 ng RNAs were fragmented for 6 min at 85 • C and amplified for eight PCR cycles.The resulting libraries were quantified using the Qubit dsDNA HS Kit (Thermo Fisher Scientific, Waltham, MA, USA) and visualized using the High-Sensitivity D1000 Kit on an Agilent 4200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA).The library was paired-end sequenced on an Illumina NextSeq with 150 cycles.
For target enrichment, we first performed DNase treatment on 30 µL of nucleic acid extract using the TURBO DNA-free™ Kit (Invitrogen), followed by clean-up with the RNA Clean and Concentrator-5 Kit (Zymo Research, Irvine, CA, USA), cDNA synthesis using the SuperScript™ IV First-Strand Synthesis System (Invitrogen) and second-strand synthesis with the NEBNext ® Ultra™ II Non-Directional RNA Second-Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA).We fragmented 47.6 ng double-stranded cDNA with a Covaris S220 Focused ultrasonicator to generate 400 bp fragments and built a dualindexed Illumina library using NEBNext ® Ultra™ II DNA Library Prep Kit from Illumina ® (New England Biolabs).The library was quantified using the KAPA Library Quantification Kit (Roche).Briefly, the 125 ng library was used as input for in-solution hybridization capture using the myBaits Hybridization Capture Kit (Daicel Arbor Biosciences, Ann Arbor, MI, USA) with custom RNA baits designed to cover genomes of different Filoviridae (Table A2).We performed two rounds of hybridization capture at 65 • C for 24 h.After each round, the capture product was amplified using the KAPA HiFi HotStart ReadyMix (Roche) with primers targeting the Illumina adapters and quantified using the KAPA Library Quantification Kit (Roche).The final product was sequenced on an Illumina MiniSeq platform using the MiniSeq High Output Reagent Kit for 300 cycles (Illumina).

Phylogenetic Analyses
To assess the relationship between the generated sequences and known BOMV strains, we performed maximum likelihood and Bayesian phylogenetic analysis.We assembled four different datasets for phylogenetic analysis: For ingroup analysis, we used all published complete and partial BOMV sequences together with all Sanger and Illumina sequences generated in this study (set1), or with only Sanger sequences (set2).For outgroup analysis, we added the reference genome of EBOV (NC_002549.1) to both datasets and only used coding sequences to allow for correct alignment (set3 and set4).
Sequences were aligned with MAFFT implemented in Geneious Prime.The resulting alignments were used to build maximum likelihood (ML) phylogenetic trees using IQ-TREE version 2.1.2with automatic model selection [16].Branch robustness was assessed with Shimodaira-Hasegawa-like approximate likelihood ratio tests (SH-like aLRTs) with 100,000 replicates [17].For ingroup analyses (set1 and set2), TIM + F was selected as the best-fitting model.
To assess the placement of the root, we performed outgroup analyses (set3 and set4), with GTR + F + I as the best-fitting model.To confirm the tree topology, we performed Bayesian phylogenetic analyses on set1 and set2 in BEAST v1.10.4. [22].For this, we built a model using BEAUti v1.10.4 assuming a strict molecular clock, constant population size with a lognormal distribution, and GTR with empirical base frequencies as the substitution model.We assessed the temporal signal in set1 and set2 using the Bayesian evaluation of temporal signal (BETS) test in BEAST with a generalized stepping-stone marginal likelihood estimation using 50 stepping stones in 50,000 generations [23].A (log) Bayes factor of 1 for both set1 and set2 (Table A3) suggested that sampling dates covered an insufficient time span for crossing the phylodynamic threshold [24], and therefore tip dates were not used for calibration.Two independent MCMCs were run for 10,000,000 states, and the MCMC trace files were checked in Tracer v1.7.2 (24).Log and tree files were combined in LogCombiner v1.10.4,and TreeAnnotator 1.10.4 was used to summarize the posterior distributions of trees as maximum clade credibility (MCC) trees [22].

Virus Isolation
We attempted virus isolation in cell culture from lung, kidney, and blood samples of the BOMV-positive bat (performed under BSL-4 conditions).Vero cells and different M. condylurus cells, i.e., immortalized kidney cells (MoKi-Immortalized), primary lung cells (MoLu Prim), and primary brain cells (MoBra Prim) [25], were inoculated with tissue homogenate and checked for cytopathic effect every 48 h over ten days before being passaged (total of two passages).After each passage, a sample was taken and tested by BOMV-specific qPCR [7].

Taxonomic Assignment
BLASTn analyses of the CytB sequences clearly identified 83 bats as belonging to the species M. condylurus.The other sequences either produced similarly good hits to several species within the genus Chaerephon (n = 157) or did not yield any highly similar hits (n = 109) (Tables 1 and S1).The bPTP species delimitation analysis estimated four to nine species in our dataset (mean = 4.24), of which three species-including the outgroup-were supported by high posterior probability values (>0.95).The two well-supported free-tailed bat species covered the M. condylurus sequences (species 1) and the cluster of sequences without highly similar BLASTn hits (species 2) (Figure A1).Based on morphological traits and geographical distribution, species 2 was assigned as Chaerephon cf.major.A third cluster containing sequences highly similar to Chaerephon sp. received a slightly lower posterior probability of 0.85 (species 3) (Figure A1).According to the closest BLASTn hits, species 3 was assigned as Chaerephon pumilus/leucogaster group.Based on their very low posterior probability values (<0.13), we considered the remaining putative species as unlikely.The individuals excluded from phylogenetic and species delimitation analyses (i.e., <700 bp) could all be assigned to species 1-3 based on their sequence with the exception of CIV919, which could be assigned to the genus Chaerephon but did not cluster with the C. pumilus/leucogaster group (Table 1).

Molecular BOMV Testing and Sequencing
Using a BOMV-specific qPCR [7], 1/349 lung extracts were repeatedly weakly positive in one replicate (CT = 36.74and 40.27, <10 copies/µL) (Table 1).The same extract was also positive in an in-house semi-nested PCR assay targeting the L gene of different filoviruses.
The positive sample originated from an M. condylurus bat (TZ154) from Kyela District in southern Tanzania.All other available sample types for TZ154 (i.e., liver, spleen, kidney, intestines, blood, and throat swab) were negative in the BOMV qPCR assay.
The Sanger sequencing of the PCR products produced two fragments of the L gene: a 106 nucleotides (nt) fragment for the BOMV qPCR (accession: PP175521), which differed from the positive control in two sites, and a 385 nt fragment for the semi-nested PCR (accession: PP175522) (Figure A2).High-throughput sequencing without prior enrichment (i.e., shotgun sequencing) did not produce any reads that could be mapped to the BOMV reference.After enrichment by hybridization capture, 70,059 (2.57%) reads were mapped to the reference, resulting in 19 unique reads after the removal of duplicate reads.This produced three additional short BOMV fragments of 102 nt (NP gene), 148 nt (L gene), and 154 nt (L gene) (accession: SUB14147099) albeit at low sequencing depth (3×) (Table 2 and Figure A2).We were unable to generate a complete BOMV genome, likely because of the very low viral copy numbers and the poor quality of the tissue, which had already been thawed twice before BOMV testing.Sites covered 3× 0 404

Phylogenetic Analyses
In all analyses, TZ154 formed a well-supported cluster with the Kenyan BOMV strains (Figures 2 and A3-A5).Outgroup analyses placed the root between the West African and East African BOMV strains but with low statistical support (Figure A4).The Bayesian phylogenetic tree confirmed this tree topology and separated BOMV strains in a West African and an East African clade (again with low statistical support) (Figures 2 and A5).
Viruses 2024, 16, x FOR PEER REVIEW 7 of 15 reference.After enrichment by hybridization capture, 70,059 (2.57%) reads were mapped to the reference, resulting in 19 unique reads after the removal of duplicate reads.This produced three additional short BOMV fragments of 102 nt (NP gene), 148 nt (L gene), and 154 nt (L gene) (accession: SUB14147099) albeit at low sequencing depth (3×) (Table 2 and Figure A2).We were unable to generate a complete BOMV genome, likely because of the very low viral copy numbers and the poor quality of the tissue, which had already been thawed twice before BOMV testing.

Phylogenetic Analyses
In all analyses, TZ154 formed a well-supported cluster with the Kenyan BOMV strains (Figures 2 and A3-A5).Outgroup analyses placed the root between the West African and East African BOMV strains but with low statistical support (Figure A4).The Bayesian phylogenetic tree confirmed this tree topology and separated BOMV strains in a West African and an East African clade (again with low statistical support) (Figures 2 and A5).Bayesian phylogenetic tree of BOMV sequences (set1).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and all sequences generated from TZ154.Leaves are colored according to country and labeled with strain name or accession number.Posterior probability (PP) is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.

Virus Isolation and Serology
No BOMV replication was detected in any of the used cell types and passages.The lack of success in virus culture attempts can likely be attributed to the low viral copy numbers and the poor quality of the tissue, which had already been thawed twice before BOMV testing.
While 3/228 bat blood samples were reactive to one or two ebolavirus proteins, none reacted to multiple proteins of the same ebolavirus and none to BOMV GP, including TZ154 (Tables 1 and S1).
Figure 2. Bayesian phylogenetic tree of BOMV sequences (set1).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and all sequences generated from TZ154.Leaves are colored according to country and labeled with strain name or accession number.Posterior probability (PP) is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.

Virus Isolation and Serology
No BOMV replication was detected in any of the used cell types and passages.The lack of success in virus culture attempts can likely be attributed to the low viral copy numbers and the poor quality of the tissue, which had already been thawed twice before BOMV testing.
While 3/228 bat blood samples were reactive to one or two ebolavirus proteins, none reacted to multiple proteins of the same ebolavirus and none to BOMV GP, including TZ154 (Tables 1 and S1).

Discussion
In this study, we tested samples of 349 free-tailed bats captured in Côte d'Ivoire and Tanzania for BOMV, including 83 M. condylurus bats.We screened lung samples of all

Figure 1 .
Figure 1.Map of bat sampling sites.The pie chart size corresponds to the number of individuals sampled at each site (log scale); M. condylurus in red and other free-tailed bats in blue.M. condylurus distribution is marked in dark gray.

Figure 1 .
Figure 1.Map of bat sampling sites.The pie chart size corresponds to the number of individuals sampled at each site (log scale); M. condylurus in red and other free-tailed bats in blue.M. condylurus distribution is marked in dark gray.

Figure 2 .
Figure2.Bayesian phylogenetic tree of BOMV sequences (set1).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and all sequences generated from TZ154.Leaves are colored according to country and labeled with strain name or accession number.Posterior probability (PP) is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.
(i.e., all individuals in the cdhit clusters that were used for phylogenetic analysis) are indicated next to the black bars.Posterior probabilities are indicated over the branch leading to the species' clades.Putative species with very low posterior probability values (<0.13) are not shown.Clades are collapsed and colored according to country.

Figure A1 .
Figure A1.ML tree of CytB sequences.Rooted based on the outgroup M. daubentoni.Branches w SH-aLRT branch support <90% are colored in gray.Putative species names and the number of in viduals (i.e., all individuals in the cdhit clusters that were used for phylogenetic analysis) are in cated next to the black bars.Posterior probabilities are indicated over the branch leading to the s cies' clades.Putative species with very low posterior probability values (<0.13) are not show Clades are collapsed and colored according to country.

Figure A2 .
Figure A2.Coverage plot of BOMV genome.The line plot shows coverage per genome position.T top part shows the structure of the BOMV genome.Genes are marked in green and labeled with abbreviated gene name.Sequences used in phylogenetic analyses are marked in blue (Illumina quences, minimum 3× coverage) and orange (Sanger sequences from PCR products).

Figure A2 .
Figure A2.Coverage plot of BOMV genome.The line plot shows coverage per genome position.The top part shows the structure of the BOMV genome.Genes are marked in green and labeled with the abbreviated gene name.Sequences used in phylogenetic analyses are marked in blue (Illumina sequences, minimum 3× coverage) and orange (Sanger sequences from PCR products).Viruses 2024, 16, x FOR PEER REVIEW 11 of 15

Figure A3 .
Figure A3.Unrooted ML tree of BOMV sequences (ingroup analysis).The sequences are colored according to country and labeled with strain name or accession number.Branch support was assessed with Shimodaira-Hasegawa-like approximate likelihood ratio tests with 100,000 replicates and is given next to the branches.Scale bar indicates substitutions per site: (A) tree built from set1 alignment including Sanger and Illumina sequences of TZ154; (B) tree built from set2 alignment including only Sanger sequences of TZ154.

Figure A3 .
Figure A3.Unrooted ML tree of BOMV sequences (ingroup analysis).The sequences are colored according to country and labeled with strain name or accession number.Branch support was assessed with Shimodaira-Hasegawa-like approximate likelihood ratio tests with 100,000 replicates and is given next to the branches.Scale bar indicates substitutions per site: (A) tree built from set1 alignment including Sanger and Illumina sequences of TZ154; (B) tree built from set2 alignment including only Sanger sequences of TZ154.

Figure A3 .
Figure A3.Unrooted ML tree of BOMV sequences (ingroup analysis).The sequences are colored according to country and labeled with strain name or accession number.Branch support was as sessed with Shimodaira-Hasegawa-like approximate likelihood ratio tests with 100,000 replicate and is given next to the branches.Scale bar indicates substitutions per site: (A) tree built from set alignment including Sanger and Illumina sequences of TZ154; (B) tree built from set2 alignmen including only Sanger sequences of TZ154.

Figure A4 . 15 Figure A4 .
Figure A4.Rooted ML tree of BOMV sequences (outgroup analysis) based on outgroup EBOV.The sequences are colored according to country and labeled with strain name or accession number.Branch support was assessed with Shimodaira-Hasegawa-like approximate likelihood ratio test with 100,000 replicates and is given next to the branches.Branches with SH-aLRT branch support <90% are colored in gray.Branch lengths are shown as equal: (A) tree built from set1 alignment including Sanger and Illumina sequences of TZ154; (B) tree built from set2 alignment including only Sanger sequences of TZ154.

Figure A5 .
Figure A5.Bayesian phylogenetic tree of BOMV sequences (set2).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and only Sanger sequences generated from TZ154 (set2).Leaves are colored according to country and labeled with strain name or accession number.The posterior probability is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.

Figure A5 .
Figure A5.Bayesian phylogenetic tree of BOMV sequences (set2).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and only Sanger sequences generated from TZ154 (set2).Leaves are colored according to country and labeled with strain name or accession number.The posterior probability is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.

Figure A5 .
Figure A5.Bayesian phylogenetic tree of BOMV sequences (set2).Maximum clade credibility tree summarized from BEAST analysis using all published partial and complete BOMV sequences and only Sanger sequences generated from TZ154 (set2).Leaves are colored according to country and labeled with strain name or accession number.The posterior probability is given beside nodes (PP < 0.95 is marked in gray).Scale bar indicates substitutions per site.

Figure A6 .
Figure A6.Luminex titration curves for TZ154.Shown are median fluorescence intensities (MFI) per 100 beads measured for different dilutions (1:200, 1:400, 1:1000, and 1:2000) of the blood sample for different ebolavirus proteins.Different ebolavirus species are displayed in different colors, different proteins are indicated with different symbols.GP_EBOVk refers to the glycoprotein from the EBOV strain from Kissidougou/Makona (2014) in Guinea, and GP_EBOVm refers to the strain from Mayinga (1976) in DRC.

Table 1 .
Overview of sampling information and laboratory results.Positive test results are marked in bold.

Table 2 .
High-throughput sequencing results on Illumina platforms.

Table 2 .
High-throughput sequencing results on Illumina platforms.

Table A3 .
Bayesian evaluation of temporal signal (BETS).Comparing log marginal likelihood estimates (MLEs) of a tip-dated model and a model with isochronous sampling are shown.