Bat capture and processing

All of the work described herein was performed as a collaborative effort between Njala University, the Centers for Disease Control and Prevention (CDC), the University of Makeni, and the University of California, Davis (UCD) USAID-PREDICT. All animal sampling was performed with permission from the Ministry of Agriculture, Forestry, and Food Security, with approval of both the Institutional Animal Care and Use Committees (IACUC) at the University of California, Davis (protocol number: 16048) and the CDC (protocol number: 2943AMMMULX). The chiropteran taxonomy used in this manuscript follows that of Wilson and Reeder (2005)44. Bats were captured at four sites in the Sierra Leone districts of Moyamba (Kasewe Cave), Kailahun (Tailu Village), Koinadugu (Kakoya Cave), and Kono (Koema Cave; Fig. 1). Bats at Kasewe Cave and Tailu Village were captured using mist nets placed at locations around the cave or in a suitable habitat with natural flyways and corridors. Captured bats were placed in breathable cotton bags and transported to a site where they were processed via complete necropsy45. Captured bats were humanely euthanized under anesthesia whereupon a cardiac blood sample was obtained. Two oral secretion samples were taken using synthetic poly-tipped swabs (Fisher Scientific, Grand Island, NY, USA or Puritan, Guilford, ME, USA), of which one was placed in virucidal lysis buffer (MagMax; Life Technologies) for PCR analysis and the other in viral transport media for virus isolation. Full necropsies were performed, and visceral tissues (liver, spleen, heart, lung, kidney, salivary gland, axillary lymph node) were collected and either flash frozen in liquid nitrogen for storage or placed in virucidal lysis buffer for inactivation and PCR analysis. When handling bats, all personnel wore appropriate personal protective equipment that included disposable gowns, double gloves including bite gloves (if necessary), face shield and respiratory protection.

Bats at Kakoya and Koema Caves were also captured with mist nets and placed in cotton bags until sampling11. Samples were collected in duplicate using non-destructive techniques (venipuncture, oral, and rectal swabs) and placed into viral transport media, frozen at −80 °C or into virucidal lysis buffer (Trizol; Roche Diagnostics) for inactivation and PCR analysis. Bat species field identifications were confirmed using mtDNA barcoding of the cytochrome b and cytochrome oxidase subunit 1 mitochondrial genes46.

Statistical analyses

All statistical analyses were done using SPSS Statistics v25 (IBM Corp, Armonk, NY). Measurements were taken on distinct samples. The ecological data collected at the trapping sites were analyzed for age and sex bias using two-sided Pearson’s chi-squared tests.

Viral detection and sequencing

Tissue and oral swab samples from bats collected at Kasewe Cave and Tailu Village were analyzed in country at Njala University by quantitative reverse transcriptase PCR (qRT-PCR) targeting marburgvirus protein 40 (VP40—MBGConTaqM F-1: GGA CCA CTG CTG GCC ATA TC, MBGConTaqM R-3Rev-1: GAG AAC ATl TCG GCA GGA AG; Probes: MBG5313-Prb: 56-FAM-ATC CTA AAC-ZEN-AGG CTT GTC TTC TCT GGG ACT T-3IABkFQ, MBG5313-PrbRav: 56-FAM-ATC CTG AAT-ZEN-AAG CTC GTC TTC TCT GGG ACT T-3IABkFQ). Total RNA was extracted from tissue and oral swabs. RNA from tissues, approximately 100 mg of tissue was placed in 2 mL grinding vials (OPS Diagnostics, Lebanon, NJ, USA) with 1 mL of MagMax lysis buffer concentrate (Ambion MagMAX, Applied Biosystems, Foster City, CA, USA) and homogenized. 125 µL of tissue lysate was then mixed with 75 µL of 100% isopropanol; for oral swab RNA, 500 µL of swab eluate in lysis buffer was extracted on the MagMax Express-96 deep-well magnetic particle processor using pre-loaded protocols. A commercially available eukaryotic 18S rRNA primer/probe assay (Applied Biosystems, Grand Island, NY, USA) was used according to manufacturer’s instructions for an extraction control.

Sequences were obtained using products generated from RT-PCR for MARV VP35 (Round 1: MBGVP35-F1: GCTTACTTAAATGAGCATGG, MBGVP35-R1: AGIGCCCGIGTTTCACC; Round 2: MBGVP35-F3: CAAATCTTTCAGCTAAGG, MBGVP35-R2: TCAGATGAATAIACACAIACCCA) and NP amplicons (Round 1: MBG704F1+: GTAAAYTTGGTGACAGGTCATG, MBG1248R1-: TCTCGTTTCTGGCTGAGG; Round 2: MBG719F2+: GGTCATGATGCCTATGACAGTATCAT; MBG1230R2-: ACGGCIAGTGTCTGACTGTGTG)5,18. For the first round of PCR, 10 µL of extracted RNA was used. The second round of PCR used 7 µL of the first round PCR reaction. PCR amplicons were analyzed on 2% E-gel. PCR reactions were purified using Ampure beads and quantitated using Qbit dsDNA HS Assay Kit on a Qubit 3.0 fluorimeter. The MinION platform was used with R9 flow cells (FLO-MIN107) and the 1D Barcoded ligation sequencing kit (1D SQK-LSK108 with the EXP-NBD103 Native Barcoding kit 1D) for sequencing of amplicons. Sequencing libraries were constructed and performed according to manufacturer’s instructions. Base calling was performed using ONT Albacore sequencing pipeline software (version 2.1.10). Reads were mapped to the reference genome by means of bwa mem using the -x ont2d flag. Reads were then converted to BAM format using SAMtools view. SAMtools was then used to sort the aligned BAM files. Consensus sequences were obtained using alignment-base consensus generation in Geneious (version 11.1.4). Each consensus sequence was manually reviewed and edited for base calling errors (specifically in homopolymer regions). Primer sequences used to generate the amplicon were manually trimmed.

Bats captured at Kakoya and Koema Caves were processed at the University of Makeni. Samples were then shipped to UC Davis where total RNA was extracted using Direct-Zol RNA columns (Zymo Research Corp), and cDNA prepared using Superscript III (Invitrogen). Samples were analyzed using a nested filovirus family level consensus PCR (cPCR) targeting a 680 bp fragment of the filovirus L gene11 (Round 1: Filo-MOD-FWD: TITTYTCHVTICAAAAICAYTGGG, FiloL.conR: ACCATCATRTTRCTIGGRAAKGCTTT; Round 2: Filo-MOD-FWD: TITTYTCHVTICAAAAICAYTGGG, Filo-MOD-RVS: GCYTCISMIAIIGTTTGIACATT), an Ebolavirus genus level cPCR targeting a 187 bp fragment of the NP gene47 (Round 1: SudZaiNP1(+): GAGACAACGGAAGCTAATGC, SudZaiNP1(−): AACGGAAGATCACCATCATG; Round 2: SudZaiNP2(+): GGTCAGTTTCTATCCTTTGC, SudZaiNP2(−): CATGTGTCCAACTGATTGCC), a RT-PCR specific for Ebola virus (EBOV) virus targeting the L-gene48 (EBOV FWD:AACTGATTTAGAGAAATACAATCTTGC, EBOV RVS: AATGCATCCAATTAAAAACATTC, Probe 1: FAM-ATTGCAACCGTTGCTATGGT-MGB, Probe 2: FAM-TAGAATATTGTAACCGTTGCT-MGB) and a RT-PCR specific for the BOMV virus, targeting the L-gene11 (Filo_UCD_qFor: TCTCGACGAAGGTCATTAGCGA, Filo_UCD_qRev: TTGCTCTGGTACTCGCTTGGT, Filo_UCD_probe: FAM-TGCTGGGATGCTGTCTTTGAGCCT-BHQ). Samples were analyzed for MARV using qRT-PCR targeting the VP35 gene. Bands of the expected size were excised from 1% agarose and purified using the Qiaquick kit (Qiagen Inc.). Purified PCR products were cloned (pCR4-TOPO vector; Invitrogen Corp.) and sequenced (ABI 3730 Capillary Electrophoresis Genetic Analyzer; Applied Biosystems, Inc., Foster City, CA). Libraries for genome sequencing were generated with the Kapa Hyper Library kit (Kapabiosystems, Roche)32 and with VirCapSeq-VERT33, and sequenced on the Illumina Miseq platform.

For each of the qRT-PCR positive bats from Kasewe, RNA was also purified using Tri-pure reagent (Invitrogen) and the Direct-Zol RNA Miniprep Plus kit (Zymo Research). NP and VP35 PCR products were obtained and sequenced on the SeqStudio Genetic Analyzer (Thermofisher Scientific).

RNA from the virus isolates was obtained and purified as described above and then used to perform whole genome sequencing on the MiSeq (Illumina). DNA sequencing libraries were constructed using the NEBNext rRNA Depletion kit (Human/Mouse/Rat) #E6310 and NEBNext Ultra II RNA Library Prep Kit for Illumina” #E7335. Indexed libraries generated from pooled liver and spleen (Liv/Spl) of bats 960 and 968, as well as axillary lymph node (LN) from bat 960 were pooled and run on the same flowcell using the MiSeq v2 reagent kit. Libraries from bat 1000 Liv/Spl were run on a separate flowcell.

Phylogenetic analysis

Sequences from the NP and VP35 genes were concatenated and a multiple sequence alignment was generated using the Clustal Omega program49. A maximum likelihood phylogeny was constructed using the best-fit nucleotide substitution model (GTR+G) in PhyML 3.050,51.

The reads from the virus isolates were mapped to the Angola 1379c reference strain (GenBank No.: DQ447653), and a consensus sequence was obtained for each using Geneious v 11.1.2. The consensus sequences were then aligned with all other MARV full-length sequences using the Clustal Omega program. A maximum likelihood phylogeny was constructed as described above using the best-fit nucleotide substitution model (GTR+I+G).

Virus isolation and immunofluorescence assay

All virus isolations were performed at the CDC under biosafety level 4 conditions. Tissue homogenates were placed into 500 µL DMEM/fungizone/penstrep (100 units/mL penicillin; 100 µg/mL streptomycin; 2.50 µg/mL amphotericin B; Life Technologies) with 2% fetal calf serum14. The entire eluate was used to inoculate Vero-E6 cells (American Type Culture Collection, CRL-1586; mycoplasma-free) in 25 cm2 flasks for 1 h at 37 °C and 5% CO 2 . Maintenance media (DMEM containing 2% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin) was then added to cultures; cells were monitored for 14 days with a media change on day 7.

All cultures were tested by immunofluorescence assay for MARV antigen at 7 and 14 DPI14. Immunofluorescence assay spot slides prepared from inoculated Vero E6 cells were fixed in acetone and then gamma-irradiated. After being incubated with a 1:100 dilution of rabbit anti-MARV polyclonal (in-house) or normal rabbit serum (negative control; in-house) for 30 min at 37 °C, rinsed two times with 1× PBS for 10 min, incubated with a 1:40 dilution of goat anti-rabbit fluorescein isothiocyanate (Capel-ICN Pharmaceuticals, Aurora, OH, USA) for 30 min at 37 °C, rinsed with 1× PBS for 7 min, stained with Eriochrome Black T (in-house) for 7 min and rinsed with 1× PBS for 7 min, the slides were observed under a fluorescence microscope.

Serology

Serum samples were tested at the CDC for the presence of MARV-specific IgG antibodies by indirect ELISA52. Wells of 96-well ELISA plates were coated (100 µL) with a 1:2000 dilution of MARV antigen lysate (in-house) and corresponding wells were coated with an equivalent dilution of uninfected control lysate (in-house). After incubation overnight at 4 °C, the plates were washed with PBS containing 0.1% Tween-20 (PBS-T) and 100 µL of serum diluent (PBS containing 5% skim milk and 0.1% tween-20) was added to each well of the plate. After 10 min, 33 µL of a 21:521 dilution of gamma-irradiated bat serum pre-diluted in masterplate diluent (PBS containing 5% skim milk powder, 0.5% tween-20 and 1% thimerosal) was added to the first well of the plate and four-fold serial dilutions were performed. Final bat serum concentrations were 1:100, 1:400, 1:1600, and 1:6400. Following a 1 h incubation at 37 °C, the plates were washed with PBS-T and 100 µL of a 1:11,000 dilution of goat anti-bat IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX, USA) in serum diluent was added to the plates. The manufacturer notes that this antibody reacts specifically with bat IgG and with light chains common to other immunoglobulins. After incubation for 1 h at 37 °C, the plates were washed with PBS-T, 100 µL of the Two-Component ABTS Peroxidase System (KPL, Gaithersburg, MD, USA) was added, and the plates were allowed to incubate for 30 min at 37 °C. The plates were then read on a microplate spectrophotometer set at 410 nm. The optical density (OD) values of each four-fold serial dilution were visually inspected to ensure linearity. To negate non-specific background reactivity, adjusted OD values were calculated by subtracting the ODs at each four-fold dilution of wells coated with uninfected control antigen lysate from ODs at corresponding wells coated with MARV antigen lysate. The adjusted sum OD value was determined by summing the adjusted OD values at each four-fold serial dilution. A conservative threshold for MARV seropositivity of 0.92 was applied52.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.