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Emerging Infectious Diseases logoLink to Emerging Infectious Diseases
. 2018 Jan;24(1):122–126. doi: 10.3201/eid2401.170401

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

Eric D Laing 1,2,3,4,5, Ian H Mendenhall 1,2,3,4,5, Martin Linster 1,2,3,4,5, Dolyce H W Low 1,2,3,4,5, Yihui Chen 1,2,3,4,5, Lianying Yan 1,2,3,4,5, Spencer L Sterling 1,2,3,4,5, Sophie Borthwick 1,2,3,4,5, Erica Sena Neves 1,2,3,4,5, Julia S L Lim 1,2,3,4,5, Maggie Skiles 1,2,3,4,5, Benjamin P Y-H Lee 1,2,3,4,5, Lin-Fa Wang 1,2,3,4,5, Christopher C Broder 1,2,3,4,5, Gavin J D Smith 1,2,3,4,5,
PMCID: PMC5749470  PMID: 29260678

Abstract

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.

Keywords: virus surveillance, serology, Southeast Asia, Ebola virus, Bundibugyo virus, Sudan virus, virus envelope glycoprotein, viruses, filoviruses, fruit bats, Singapore


The genus Ebolavirus comprises 5 virus species: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), Taï Forest ebolavirus (TAFV), and Reston ebolavirus (RESTV). The genus Marburgvirus comprises 1 species, Marburg marburgvirus, which includes 2 closely related virus strains: Marburg virus (MARV) and Ravn virus (RAVV). Viruses within the Ebolavirus and Marburgvirus genera are zoonotic; EBOV was the causative agent of the 2014–2016 Ebola virus disease epidemic in West Africa (1). Rousettus bats in Africa have been identified as Marburgvirus hosts (2), and viral nucleic acid and serologic evidence suggests that bats are also natural hosts of Ebolavirus spp. (3). Yet it remains unclear which species are the definitive reservoirs of filoviruses.

Ecologic models of Ebolavirus and Marburgvirus geographic distribution and habitat ranges of potential reservoir bat species suggest that both groups are distributed throughout Asia (3,4). Serologic evidence of filoviruses in frugivorous bats in Bangladesh, China, and the Philippines has been reported (57), and RESTV nucleic acid was detected in an insectivorous bat in the Philippines, where RESTV is considered endemic (8). We examined pteropodid bats of 3 species: Cynopterus brachyotis, Eonycteris spelaea, and Penthetor lucasi, which are widely distributed across Southeast Asia and share ecologic niches (9).

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.

Table 1. Ebolavirus and Marburgvirus species soluble envelope glycoproteins conjugated Bio-Plex beads used in multiplex assay to detect antibodies against filoviruses*.

Virus Isolation host/location Bio-Plex bead no. NCBI accession no.
Ebola virus/H.sapiens/COD/1976/Yambuku-Mayinga Human/DRC 33 NC_002549.1
Bundibugyo virus/H. sapiens/UGA/2007 Human/Uganda 64 FJ217161.1
Taï Forest virus/H. sapiens/COV/1994/Pauleoula-CI Human/Côte d'Ivoire 57 NC_014372
Sudan virus/H. sapiens/UGA/2000/Gulu-808892 Human/Uganda 77 NC_006432.1
Reston virus/M. fascicularis/USA/1989/Pennsylvania Macaque/USA 85 AF522874.1
Reston virus/S. domesticus/PHL/2008/Reston08-A Swine/Philippines 72 FJ621583.1
Marburg virus/H. sapiens/KEN/1980/Musoke Human/Kenya 37 Z12132 S55429
Marburg virus/H. sapiens/AGO/2005/Ang0126 Human/Angola 28 DQ447656.1
Ravn virus/H. sapiens/KEN/1987/Kitum cave-810040 Human/Kenya 49 NC_024781.1

*Bio-Plex manufactured by Bio-Rad (Hercules, CA, USA). DRC, Democratic Republic of the Congo; NCBI, National Center for Biotechnology Information.

In the absence of confirmed filovirus-negative bat serum, we used methods developed by Peel et al. to establish a median fluorescence intensity (MFI) cutoff value (11). We confirmed a cutoff value of 200 MFI (Technical Appendix), as was previously used for Eidolon helvum bat serum in a Bio-Plex serologic assay (12). We screened 409 samples with our Ebolavirus and Marburgvirus spp. sGP(1,2) Bio-Plex assay modified from that described by Bossart et al. (13). Samples were diluted 1:100 and tested in duplicate; the sGP(1,2)-coupled beads were mixed with individual samples; and a 1:1 combination of recombinant biotinylated-protein A/protein G (1:500) (Pierce, Rockford, IL, USA) was added to the wells, followed by addition of streptavidin-phycoerythrin (1:1,000) (Bio-Rad) and determination of MFI.

Samples were positive for 17 (9.1%) of 186 E. spelaea, 13 (8.5%) of 153 C. brachyotis, and 3 (4.3%) of 70 P. lucasi bats (Figure 1). Positive samples reacted with EBOV, BDBV, SUDV, or TAFV sGP(1,2). However, no samples were positive for RESTV, MARV, or RAVV sGP(1,2). We further examined positive samples to determine cross-reactivity between the Ebolavirus spp. sGP(1,2) (Table 2). Twelve (71%) samples from E. spelaea bats cross-reacted with >2 Ebolavirus spp. sGP(1,2) (BDBV, EBOV, SUDV, or TAFV). In contrast, 8 (62%) C. brachyotis and 2 (66%) P. lucasi samples were positive for only 1 sGP(1,2) (BDBV or SUDV).

Figure 1.

Figure 1

Mean fluorescence intensity (MFI) values obtained from Bio-Plex assay (Bio-Rad, Hercules, CA, USA) screening of individual serum samples from bats of 3 species with soluble filovirus glycoproteins. Dashed line indicates the cutoff value at 200 MFI. 1, Zaire ebolavirus; 2, Bundibugyo ebolavirus; 3, Taï Forest ebolavirus; 4, Sudan ebolavirus; 5, Reston ebolavirus–monkey; 6, Reston ebolavirus–pig; 7, Marburg virus–Musoke; 8, Marburg virus–Angola; 9, Ravn virus.

Table 2. Bio-Plex median fluorescence intensity values for bat serum positive for >1 filovirus antigen*.

Bat species, ID EBOV BDBV TAFV SUDV RESTVm RESTVp MARV(Mus) MARV(Ang) RAVV
Eonycteris spelaea, n = 186
0805149† 738 124 68 40 44 22 23 21 24
080814 86 318 105 258 26 12 17 16 20
082154 143 161 113 214 35 41 21 31 39
052313 284 408 177 285 89 72 29 23 30
052335 203 191 124 219 42 21 38 38 24
052339 357 306 141 293 54 31 26 26 42
071839 330 299 164 480 65 44 28 33 45
071842 446 327 202 362 65 49 42 38 57
110733 126 416 166 95 58 42 34 42 58
011603† 1151 130 91 69 36 32 51 35 39
011616 252 294 168 175 32 49 47 29 50
011656 306 386 204 394 89 73 18 39 37
012309† 579 659 315 69 35 31 27 33 35
021303 478 431 188 450 52 37 24 30 47
111903 469 384 276 113 52 57 37 69 54
111907 285 336 213 158 39 36 29 50 30
042722
260
262
174
167
75
31
54
24
42
Cynopterus brachyotis, n = 153
051253 121 133 59 242 40 41 19 25 68
0516613 146 293 127 73 47 36 25 29 22
0516632 138 139 86 356 35 25 28 34 34
0726122† 119 501 100 60 40 46 25 19 29
1103241 84 141 128 241 50 47 66 38 34
100903 148 201 71 108 42 33 18 16 36
100914 74 228 70 55 39 38 30 27 26
100925 166 304 109 116 43 18 33 30 28
021357 201 299 179 264 65 44 25 55 47
050804 242 276 140 124 41 30 34 33 44
050818 383 374 198 332 60 55 29 26 68
040807† 297 597 194 192 40 38 122 95 32
042701†
339
547
222
417
60
78
54
25
62
Penthetor lucasi, n = 70
062590† 34 496 93 39 36 18 23 17 23
070409† 95 238 129 89 62 27 34 36 37
112112† 251 352 148 235 51 29 23 23 29

*Bio-Plex manufactured by Bio-Rad (Hercules, CA, USA). Boldface indicates positive results. BDBV, Bundibugyo virus; EBOV, Ebola virus; ID, specimen identification number; MARV(Mus), Marburg virus–Musoke; MARV(Ang), Marburg virus–Angola; RESTVm, Reston virus–monkey; RESTVp, Reston virus–pig; SUDV, Sudan virus; RAVV, Ravn virus; TAFV, Taï Forest virus. 
†Sample screened by Western blot and shown in Figure 2.

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 GP1 and GP2 subunits. The trimeric-like sGP(1,2) is the antigen in the multiplex Bio-Plex assay, whereas linearized monomeric sGP1 and sGP2 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 GP1 and GP2 antigens; no P. lucasi bat samples positive by Bio-Plex were positive by WB.

Figure 2.

Figure 2

Western blot results of individual bat serum samples probed against Zaire ebolavirus and Bundibugyo ebolavirus glycoproteins 1 and 2 (GP1, GP2). Boldface indicates positivity by Western blot and underlining indicates positivity by Bio-Plex (Bio-Rad, Hercules, CA, USA). 1, soluble GP1 and GP2 blotted with control anti–Ebola virus nonhuman primate polyclonal serum that demonstrates cross-reactivity with Bundibugyo ebolavirus soluble GP. Other numbers along baseline correspond to the following sample identifiers, also used in Table 2: 2, 0805149; 3, 012309; 4, 011603; 5, 0116048; 6, 0719036; 7, 1128015; 8, 0726122; 9, 042701; 10, 040807; 11, 0512540; 12, 1009010; 13, 0408029; 14, 070409; 15, 112112; 16, 062590; 17, 0228004; 18, 0919025; 19, 0625095. BDBV, Bundibugyo virus; EBOV, Ebola virus.

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 sGP2 and BDBV sGP1 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). Comprehensive surveillance including serology and detection of viral nucleic acid, along with virus isolation, will help elucidate the characteristics of filoviruses endemic to Asia and identify bat species that function as maintenance populations and reservoirs.

Technical Appendix

Median fluorescence intensity cutoff value determination and results for filoviruses in serum from fruit bats, Singapore, 2011–2016.

17-0401-Techapp-s1.pdf (507.7KB, pdf)

Acknowledgment

We thank Alison J. Peel for assistance with determination of the median fluorescence intensity cutoff and statistical advice.

This study was supported by the Duke-National University of Singapore Signature Research Program funded by the Agency of Science, Technology and Research, and the Ministry of Health, Singapore, and by grants from National University of Singapore–Global Asia Institute (NIHA-2011-1-005), the National Medical Research Council (NMRC/BNIG/2005/2013), the Ministry of Health (CDPHRG/0006/2014) in Singapore, and the US Department of Defense, Defense Threat Reduction Agency. C.C.B., E.D.L., L.Y., and S.L.S. were supported by funding from the Biological Defense Research Directorate of the Naval Medical Research Center. E.D.L. was also supported by the National Science Foundation, an East Asia and Pacific Summer Institutes Fellowship award (1515304), with collaborative support from the National University of Singapore.

Biography

Dr. Laing is a postdoctoral fellow at the Uniformed Services University and performed this work while a National Science Foundation EAPSI fellow at Duke-National University of Singapore Medical School. His research focuses on biosurveillance, batborne viruses, and antiviral immunity.

Footnotes

Suggested citation for this article: Laing ED, Mendenhall IH, Linster M, Low DHW, Chen Y, Yan L, et al. Serologic evidence of fruit bat exposure to filoviruses, Singapore, 2011–2016. Emerg Infect Dis. 2018 Jan [date cited]. https://doi.org/10.3201/eid2401.170401

1

These authors contributed equally to this article.

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Supplementary Materials

Technical Appendix

Median fluorescence intensity cutoff value determination and results for filoviruses in serum from fruit bats, Singapore, 2011–2016.

17-0401-Techapp-s1.pdf (507.7KB, pdf)

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