Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011–2016

To determine whether fruit bats in Singapore have been exposed to filoviruses, we screened 409 serum samples from bats of 3 species by using a multiplex assay that detects antibodies against filoviruses. Positive samples reacted with glycoproteins from Bundibugyo, Ebola, and Sudan viruses, indicating filovirus circulation among bats in Southeast Asia.


The Study
During 2011-2016, we collected serum from bats of the 3 aforementioned species in Singapore and screened samples for evidence of exposure to filoviruses. Samples were collected with permission from the National University of Singapore Institutional Animal Care and Use Committee (B01/12) and the National Parks Board (NP/RP11-011-3a). We diluted venous blood 1:10 in phosphate-buffered saline and then centrifuged, recovered, and heat-inactivated the serum at 56°C for 30 minutes and stored it at −80°C.
We developed a Bio-Plex (Bio-Rad, Hercules, CA, USA) bead-based multiplex assay that simultaneously probes serum for immunoglobulins specific to the viral envelope glycoproteins (GPs) from representative strains of all described Ebolavirus and Marburgvirus species ( Table  1). A human FreeStyle 293-F stable cell-line expression system was used to produce the Ebolavirus and Marburgvirus spp. GPs as a soluble GP consisting of the entire ectodomain, sGP (1,2) , which retains a native-like oligomeric conformation, as described previously with modifications (10). In brief, each GP (1,2) coding sequence was truncated at the C-terminus to remove the predicted transmembrane domain and cytoplasmic tail, then appended with the GCN trimerization peptide sequence (10) together with a factor Xa protease cleave site and a Twin-Strep-tag sequence (IBA Lifesciences, Göttingen, Germany). The sGP (1,2) proteins were produced in serum-free conditions and purified by Strep-Tactin XT technology (IBA Lifesciences). The Twin-Strep-tag was removed by factor Xa enzymatic cleavage; factor Xa was removed by Xarrest Agarose (Merck Millipore, Billerica, MA, USA); sGP (1,2) was purified further by S-200 size exclusion chromatography, concentrated, and stored frozen. These sGP (1,2) s were coupled to carboxylated beads (Bio-Rad). Screening was performed on a Bio-Rad Bio-Plex 200.
To further determine the cross-reactivity of positive samples and to corroborate Bio-Plex assay results for a selected number of samples, we performed Western blot (WB) assays ( Figure 2). The filovirus GP (1,2) is a trimer of heterodimeric GP 1 and GP 2 subunits. The trimeric-like sGP (1,2) is the antigen in the multiplex Bio-Plex assay, whereas linearized monomeric sGP 1 and sGP 2 subunits are the antigens in WBs. Reduced and denatured EBOV or BDBV unconjugated sGP (1,2) was loaded on 8% sodium dodecyl sulfate-polyacrylamide electrophoresis gels, transferred to a polyvinylidene difluoride membrane, and probed with 1:100 dilutions of positive and negative bat serum, as previously determined by the Bio-Plex assay. All 3 E. spelaea bat samples and 2 of 3 C. brachyotis bat samples that were Bio-Plex positive were also positive by WB and displayed reactivity with EBOV and BDBV GP 1 and GP 2 antigens; no P. lucasi bat samples positive by Bio-Plex were positive by WB.

Conclusions
We present evidence of antibodies specific to filoviruses antigenically related to Ebolavirus spp. in 3 species of fruit bats widely distributed throughout Southeast Asia. We detected seroreactivity with Ebolavirus spp. but not Marburgvirus spp. GP. Despite the close relatedness of the viruses, we detected samples reacting with only SUDV, not RESTV, GP. This finding contrasts  with previous reports of bat serum cross-reactivity with RESTV nucleoprotein (5,7,14). Possible explanations include 1) the fact that our customized Bio-Plex assay is based on conformational sGP (1,2) , which can differentiate antibody specificity better than the more sequence conserved nucleoprotein, and 2) the lack of evidence of RESTV GP positivity with Cynopterus and Eonycteris bat serum samples, which is in line with previous findings (both species were negative while only Rousettus amplexicaudatus bats were positive) (7). E. spelaea bats were previously predicted to be filovirus hosts (15), and sequences of novel filoviruses have been discovered in E. spelaea bat populations in Yunnan, China (14). Our data provide additional empirical evidence that populations of C. brachyotis, E. spelaea, and P. lucasi bats in Southeast Asia are hosts of filoviruses, which seem antigenically more closely related to EBOV, BDBV, and SUDV than to RESTV. Examination of cross-reactivity of positive samples from E. spelaea, C. brachyotis, and P. lucasi bats revealed no clear patterns of preferential reactivity with EBOV, BDBV, or SUDV GP. Factors that might contribute to the lack of P. lucasi positivity by WB include sensitivity differences between Bio-Plex and WB assays paired with the change in sGP (1,2) conformation. Two Bio-Plex EBOV-positive samples (E. spelaea samples 0805149 and 011603) reacted with EBOV sGP 2 and BDBV sGP 1 in the WB. Bio-Plex and WB data strongly suggest the presence of yet-undetected batborne filoviruses, which are antigenically related to but distinct from BDBV, EBOV, and SUDV circulating in local bat populations. Reasons why these filoviruses have remained undetected include their inability to cross the species barrier, the rarity of spillovers into humans or domestic animals, or the fact that spillover events cause mild or no disease. We suggest that a yet-undescribed diversity of filoviruses exists in Southeast Asia bat populations, a hypothesis supported by the recent identification of filovirus sequences in E. spelaea and R. leschenaulti bats in China (14,16 Figure 2.
isolation, will help elucidate the characteristics of filoviruses endemic to Asia and identify bat species that function as maintenance populations and reservoirs.