Investigating the Circulation of Ebola Viruses in Bats during the Ebola Virus Disease Outbreaks in the Equateur and North Kivu Provinces of the Democratic Republic of Congo from 2018

Abstract

With 12 of the 31 outbreaks, the Democratic Republic of Congo (DRC) is highly affected by Ebolavirus disease (EVD). To better understand the role of bats in the ecology of Ebola viruses, we conducted surveys in bats during two recent EVD outbreaks and in two areas with previous outbreaks. Dried blood spots were tested for antibodies to ebolaviruses and oral and rectal swabs were screened for the presence of filovirus using a broadly reactive semi-nested RT-PCR. Between 2018 and 2020, 892 (88.6%) frugivorous and 115 (11.4%) insectivorous bats were collected. Overall, 11/925 (1.2%) to 100/925 (10.8%) bats showed antibodies to at least one Ebolavirus antigen depending on the positivity criteria. Antibodies were detected in fruit bats from the four sites and from species previously documented to harbor Ebola antibodies or RNA. We tested for the first time a large number of bats during ongoing EVD outbreaks in DRC, but no viral RNA was detected in the 676 sampled bats. Our study illustrates the difficulty to document the role of bats as a source of Ebolaviruses as they might clear quickly the virus. Given the increasing frequency of EVD outbreaks, more studies on the animal reservoir are urgently needed.

Dynamics of Antibodies to Ebolaviruses in an Eidolon helvum Bat Colony in Cameroon

“…The species identification recorded in the field was molecularly confirmed on a subset of bats using DNA extracted from DBS as previously described [ 11 , 34 ]. Briefly, a fragment of approximately 800 base pairs (bp) of the mitochondrial cytochrome b (cytb) was amplified using primers adapted from Irwin and colleagues [ 34 , 35 ]. Amplicons were sequenced using the Sanger technique.…”

“…Dried blood spots (DBS) were tested with a Luminex-based serological assay adapted for bats as previously described [ 11 , 34 , 37 ]. The assay included nine recombinant proteins, i.e., glycoprotein (GP), nucleoprotein (NP), or viral protein 40 (VP40) for three Ebolavirus species that circulate in Africa (Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV)).…”

“…The assay included nine recombinant proteins, i.e., glycoprotein (GP), nucleoprotein (NP), or viral protein 40 (VP40) for three Ebolavirus species that circulate in Africa (Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV)). Whole blood from DBS was reconstituted as previously described [ 11 , 34 , 37 ]. Plasma was reconstituted from one DBS spot in 1 mL of incubation buffer, consisting of phosphate-buffered saline (PBS) containing 0.75 mol/L NaCl, 1%(wt/vol) bovine serum albumin (Sigma Aldrich, St. Quentin Fallavier, France), 5% (vol/vol) fetal bovine serum (Gibco-Invitrogen, Cergy Pontoise, France), and 0.2% (vol/vol) Tween-20 (Sigma-Aldrich, Hohenbrunn, Germany).…”

“…Secondly, we used more stringent conditions and determined consensus cut-offs as the mean of cut-offs obtained using three previously described statistical methods used in the absence of well documented positive controls, i.e., change point analysis method and fitted univariate binomial and exponential distributions at a 0.001 risk for error [11,34]. The cut-offs used were those described by Lacroix and colleagues [34] and were calculated on a total of 8741 bats from Guinea, DRC, and Cameroon, including the Eidolon helvum colony reported in this study (Supplementary Table S1) [34]. Analyses were performed with R version 4.0.2 software (https://www.r-project.org/, last accessed on 30 November 2021).…”

“…cDNA was prepared using the Reverse Transcription System kit with random primers (Promega, Madison, WI, USA), following the manufacturer's instructions. PCR screening was performed by a semi-nested PCR targeting a 630 bp fragment of the L gene using degenerated primers that detect a wide diversity of filoviruses, as previously described, i.e., Filo-MOD-FWD: TITTYTCHVTICAAAAICAYTGGG and FiloL.conR: ACCATCATRTTRCTIGGRAAKGCTTT in round 1 and Filo-MOD-FWD: TITTYTCHVTI-CAAAAICAYTGGG, and Filo-MOD-RVS: GCYTCISMIAIIGTTTGIACATT in round 2 [34] Briefly, cDNA was amplified using the GoTaq Hot Start Master Mix PCR kit (Promega, Madison, WI, USA) as follows for first and second PCR rounds: 10 cycles of 92 • C for 20 s, 50 • C for 30 s with −0.5 • C/cycle and 72 • C for 1 min, 35 cycles of 92 • C for 20 s, 50 • C for 30 s and 72 • C for 1 min. Additionally, samples with bands of the expected size were also tested using a previously described Ebola Zaire specific semi-nested PCR assay targeting a 184 bp fragment on the VP35 gene [38].…”