Article DOI: https://doi.org/10.3201/eid2505.

181666

Bombali Ebola Virus in Mops condylurus

Bat, Kenya
Appendix

Methods

Bats were captured in February 2016 and May 2018 as part of an ongoing virus screening
project in the Taita Hills area of rural Kenya; all M. condylurus were captured in 2018. We
employed mist and hand netting, and structured trapping site selection to focus on habitat and
species diversity and minimize the number of individuals collected from any 1 species or site.
Captured bats were placed into individual cotton bags, and processed at the University of
Helsinki Taita Research Station. Species identifications were made in the field using keys (1).
Non-conservation priority bat species (classified as least concern by the IUCN) were euthanized
via cervical dislocation to collect blood, lung, liver, spleen, kidney, intestine and brain samples,
as well as urine, feces, and ectoparasites when possible. Dissections were performed in a
sheltered outside area, using personal protective equipment, including FFP3 facemasks, latex
gloves, and safety gowns. Bat tissues were placed into separate marked tubes with RNAlater
(Sigma, https://www.sigmaaldrich.com), stored at 20°C, and later sent on dry ice to Helsinki,
Finland.

At the University of Helsinki, under enhanced BSL-3 conditions, bat tissue samples were
treated with Tripure (Roche, http://www.roche.com) to inactivate any potential hazardous agents
before RNA extractions (Tripure method) and screening by a pan-filovirus RT-qPCR (2).
Filovirus screening was initially conducted as a precaution, to facilitate screening for other
viruses under less-strict biosafety conditions. The pan-filo RT-qPCR has been tested to detect
Zaire EBOV, Bundibugyo, Sudan, Taï Forest, and Reston ebolavirus, in addition to Marburg
virus (MARV) and Ravn Virus RNA. EBOV and MARV RNA were used as positive controls (in
vitro RNAs) (3). Following the identification of a positive individual, with particularly high viral
loads in the lung, lung samples from all bats were also screened with a Bombali virus–specific

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real-time RT-PCR (4). All tissue, excreta, and ectoparasite samples were screened from the
positive individual (Appendix Table 1), and viral loads determined by RT-qPCR with an in vitro
transcribed RNA serving as the quantification standard. A full list of each bat species captured
and screened is provided in Appendix Table 2.

Prior to whole-genome sequencing, RT-PCR positive samples were treated with DNase I
(Thermo Fisher, http://www.thermofisher.com), and purified with Agencourt RNA Clean XP
magnetic beads (Beckman Life Sciences, https://www.beckman.com). Ribosomal RNA was
removed using a NEBNext rRNA depletion kit (New England BioLabs, https://www.neb.com),
according to the manufacturer’s protocol. The sequencing library was prepared using a NEBNext
Ultra II RNA library prep kit (New England BioLabs). Libraries were quantified using a
NEBNext Library Quant kit for Illumina (New England BioLabs). Pooled libraries were then
sequenced on a MiSeq platform (Illumina, https://www.illumina.com) using a MiSeq v3 reagent
kit with 300 bp paired-end reads. Raw sequence reads were trimmed and low-quality (quality
score <15) and short (<36 nt) sequences were removed using Trimmomatic (5). Thereafter, de
novo assembly was conducted using MegaHit (6). Open reading frames were sought using
MetaGeneAnnotator (7), followed by taxonomic annotation using SANSparallel (8). We
confirmed bat species identity of the positive individual by retrieving cytochrome-b sequences
from the NGS reads (GenBank accession no. MK330941).

The phylogenetic tree was constructed using the Bayesian Markov chain Monte Carlo
(MCMC) method, implemented in Mr Bayes version 3.2 (9) using a GTR-G-I model of
substitution with 2 independent runs and 4 chains per run. The analysis was run for 5 million
states and sampled every 5,000 steps. The average standard deviation of split frequencies was
0.000732.

Febrile patients seeking care at 3 health facilities in the Taita Hills (Wundanyi, Mwatate,
and Voi) were recruited into the study by clinicians. A questionnaire was used to capture socio-
demographic data and pertinent history, including a tickbox question regarding contact with bats
at home or work. Based on the criterion of exposure to bats, a total of 81 patients (2.9–83.4 years
of age; average, 38.8 years) were selected for analysis of filovirus RNA. Samples were collected
within 5 days of the onset of fever. No patients reported bleeding. Reported symptoms included,
in addition to fever; myalgia (54/81), joint pain (45/81), rash (9/81), diarrhea (8/81), vomiting

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(7/81), headache (6/81) and cough (4/81). Serum samples were stored at the University of
Helsinki Taita Research station at 20°C for <3 weeks, and then transported on ice to a central
laboratory at the University of Nairobi where they were stored at 80°C and later shipped on dry
ice to Helsinki. Nucleic acids were extracted from 100µL of serum and eluted to 50µL using the
QIAamp Viral RNA Mini Kit (QIAGEN, https://www.qiagen.com) according to manufacturer’s
instructions. Pan-filovirus RT-qPCR was then conducted as described above, as well as Bombali
virus–specific RT-PCR (4).

Human serum samples were analyzed for Ebola virus–specific IgG antibodies using an
immunofluorescence assay (IFA) based on a recombinant Zaire ebolavirus VP-40 with a similar
IFA protocol as described before (10), and demonstrated within the EbolaMoDRAED EU-IMI
project to react with Zaire ebolavirus patient serum. Bombali virus VP40 protein is 75%–78%
similar to that of other ebolaviruses, which have been demonstrated to cross-react within the
genus (11). As antigen, we used acetone-fixed Vero E6 cells transfected with the pCAGGS-
Ebola VP40 construct (Zaire ebolavirus, isolate Ebola virus/ H.sapiens-wt/SLE/2014/Makona-
G3856.1 sequence, GenBank KM233113.1), and as controls, cells transfected with the empty
vector. Patient serum samples were diluted 1:60 in PBS and incubated for 1 h at 37°C.
Fluorescein isothiocyanate–conjugated anti-human IgG (Jackson ImmunoResearch,
https://www.jacksonimmuno.com) was diluted 1:30 in PBS, and incubated for 30 min at 37°C.
Unbound antibodies and anti-human IgG were washed 3 times with PBS and then once with
distilled water. The slides were covered with mounting medium and coverslips, and read using a
×20 objective of fluorescence microscope Olympus IX71 (Olympus Corporation, www.olympus-
global.com).

Additional Results

Serologic analysis revealed antibodies against ebolavirus in the blood of the tissue-
positive bat (Appendix Figure), but antibodies were not present in blood from the other bats.
Note that bat blood samples (from RNA-negative individuals) were first heat inactivated under
enhanced BSL-3 conditions. To minimize exposure risk, the blood sample from the positive bat
was sent to the Public Health Agency of Sweden and screened under BSL-4 conditions. To
detect bat antibodies in blood samples, Vero E6 cells transfected as above to produce ZEBOV

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VP40, or at Public Health Agency of Sweden, infected with Zaire ebolavirus, were used in IFA
according to a previously described protocol (12). Blood samples were diluted to 1:20 in PBS
before incubation. Detection was done with goat anti–bat antibody Ig (Bethyl Laboratories,
https://www.bethyl.com) at 1:1,000, followed by donkey anti–goat cyanin 2 (Cy2)-labeled Ig
(Dianova, https://www.dianova.com) at 1:100. Slide staining and analysis were conducted as
described above.

References

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Appendix Table 1. Viral loads from Mops condylurus bat that tested positive for Bombali Ebola virus.
Sample Ct value Copy number/500 ng total RNA
Mouth swab† 24.00 Not applicable
Spleen 32.76 414
Liver 33.95 181
Intestine 32.76 413
Heart 29.82 3,173
Feces 29.14 5,121
Lung 16.74 27,950,000
Kidney Negative 0
Urine Negative 0
Fleas Negative 0
*Viral loads for each sample type were estimated using a standard curve based
on in vitro transcribed and quantified RNA.
†Mouth swab has no copy number because it was screened in a BSL-4
laboratory in Sweden using a different protocol and without the standard curve.

Appendix Table 2. Bat species screened for filoviruses, Kenya*

Species 2016 2018
Mops condylurus 0 16
Chaerephon pumilus 4 7
Cardioderma cor 36 20
Chaerephon chapini 1 0
Epomophorus wahlbergi 19 23
Glauconycteris argentata 1 0
Hipposideros caffer 2 1
Lavia frons 0 1
Lissonycteris angolensis 10 0
Myotis tricolor 1 0
Neoromicia nana 0 3
Nycticeinops schlieffeni 3 1
Nycteris thebaica 0 1
Rhinolophus clivosus 0 2
Rhinolophus landeri 1 2

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Species 2016 2018
Rousettus aegyptiacus 17 13
Scotoecus hirundo 34 4
Scotophilus dinganii 4 12
Rousettus lanosus 0 1
Pipistrellus sp. 20 0
Neoromicia sp. 0 1
Miniopterus sp. 6 0
Hypsugo sp. 4 0
*Bats were captured from the Taita Hills area in 2016 and 2018. All bat lung
samples were screened for filovirus RNA via a new pan-filovirus reverse
transcription qualitative PCR (2) and a Bombali virus–specific real-time reverse
transcription PCR (4).

Appendix Figure. Detection of Ebola virus–specific antibodies in Bat B241 (the BOMV RNA positive
individual) using an immunofluorescence assay based on Zaire ebolavirus (ZEBOV)–infected, acetone-
fixed Vero E6 cells. The slides contain ZEBOV-infected and noninfected control cells. A) 4,6-diamidino-2-
phenylindole (DAPI) staining for cell nuclei. B) Staining with rabbit anti–ZEBOV-GP showing ZEBOV-
infected cells. C) Staining with bat B241 serum at a dilution of 1:200, demonstrating specific granular
staining of ZEBOV-infected cells. D) A merge of stains demonstrating that the antibody response of bat
B241 is Ebola virus genus cross-reactive, but targeting other viral proteins than the ZEBOV GP.

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