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

The genus Ebolavirns comprises 5 virus species: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), Tai' 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 (5-7), 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[degrees]C for 30 minutes and stored it at -80[degrees]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, s[GP.sub.(1,2)], which retains a native-like oligomeric conformation, as described previously with modifications (10). In brief, each [GP.sub.(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, Gottingen, Germany). The s[GP.sub.(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); s[GP.sub.(1,2)] was purified further by S-200 size exclusion chromatography, concentrated, and stored frozen. These sGP(1 gs were coupled to carboxylated beads (Bio-Rad). Screening was performed on a Bio-Rad Bio-Plex 200.

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 (online Technical Appendix,, as was previously used for Eidolon helvum bat serum in a Bio-Plex serologic assay (12). We screened 409 samples with our Ebolavirns and Marburgvirus spp. s[GP.sub.(1,2)] Bio-Plex assay modified from that described by Bossart et al. (13). Samples were diluted 1:100 and tested in duplicate; the s[GP.sub.(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 s[GP.sub.(1,2)]. However, no samples were positive for RESTV, MARV, or RAVV s[GP.sub.(1,2)]. We further examined positive samples to determine cross-reactivity between the Ebolavirus spp. s[GP.sub.(1,2)] (Table 2). Twelve (71%) samples from E. spelaea bats cross-reacted with [greater than or equal to]2 Ebolavirus spp. s[GP.sub.(1,2)] (BDBV, EBOV, SUDV, or TAFV). In contrast, 8 (62%) C. brachyotis and 2 (66%) P. lucasi samples were positive for only 1 s[GP.sub.(1,2)] (BDBV or SUDV).

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.sub.(1,2)] is a trimer of heterodimeric [GP.sub.1] and [GP.sub.2] subunits. The trimeric-like s[GP.sub.(1,2)] is the antigen in the multiplex Bio-Plex assay, whereas linearized monomeric s[GP.sub.1] and s[GP.sub.2] subunits are the antigens in WBs. Reduced and denatured EBOV or BDBV unconjugated s[GP.sub.(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.sub.1] and [GP.sub.2] antigens; no P. lucasi bat samples positive by BioPlex were positive by WB.


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 s[GP.sub.(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 s[GP.sub.(1,2)] conformation. Two Bio-Plex EBOV-positive samples (E. spelaea samples 0805149 and 011603) reacted with EBOV s[GP.sub.2] 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 (14,15). 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.


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.

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.


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(2.) Towner JS, Amman BR, Sealy TK, Carroll SA, Comer JA, Kemp A, et al. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats. PLoS Pathog. 2009;5:e1000536.

(3.) Olival KJ, Hayman DT. Filoviruses in bats: current knowledge and future directions. Viruses. 2014;6:1759-88. 10.3390/v6041759

(4.) Peterson AT, Bauer JT, Mills JN. Ecologic and geographic distribution of filovirus disease. Emerg Infect Dis. 2004;10:40-7.

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(6.) Yuan J, Zhang Y, Li J, Zhang Y, Wang LF, Shi Z. Serological evidence of ebolavirus infection in bats, China. Virol J. 2012;9:236.

(7.) Taniguchi S, Watanabe S, Masangkay JS, Omatsu T, Ikegami T, Alviola P, et al. Reston Ebolavirus antibodies in bats, the Philippines. Emerg Infect Dis. 2011;17:1559-60.

(8.) Jayme SI, Field HE, de Jong C, Olival KJ, Marsh G, Tagtag AM, et al. Molecular evidence of Ebola Reston virus infection in Philippine bats. Virol J. 2015;12:107. s12985-015-0331-3

(9.) Mendenhall IH, Borthwick S, Neves ES, Low D, Linster M, Liang B, et al. Identification of a lineage D betacoronavirus in cave nectar bats (Eonycteris spelaea) in Singapore and an overview of lineage D reservoir ecology in SE Asian bats. Transbound Emerg Dis. 2016.

(10.) Chan YP, Yan L, Feng YR, Broder CC. Preparation of recombinant viral glycoproteins for novel and therapeutic antibody discovery. Methods Mol Biol. 2009;525:31-58, xiii.

(11.) Peel AJ, McKinley TJ, Baker KS, Barr JA, Crameri G, Hayman DT, et al. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats. J Virol Methods. 2013;193:295-303.

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(13.) Bossart KN, McEachern JA, Hickey AC, Choudhry V, Dimitrov DS, Eaton BT, et al. Neutralization assays for differential henipavirus serology using Bio-Plex protein array systems. J Virol Methods. 2007;142:29-40.

(14.) Yang XL, Zhang YZ, Jiang RD, Guo H, Zhang W, Li B, et al. Genetically diverse filoviruses in Rousettus and Eonycteris spp. bats, China, 2009 and 2015. Emerg Infect Dis. 2017;23:482-6.

(15.) Han BA, Schmidt JP, Alexander LW, Bowden SE, Hayman DT, Drake JM. Undiscovered bat hosts of filoviruses. PLoS Negl Trop Dis. 2016;10:e0004815.

(16.) He B, Feng Y, Zhang H, Xu L, Yang W, Zhang Y, et al. Filovirus RNA in fruit bats, China. Emerg Infect Dis. 2015;21:1675-7.

Address for correspondence: Gavin J. D. Smith, Programme in Emerging Infectious Diseases, Duke-National University Singapore Medical School, 8 College Rd, Singapore 169857, Singapore; email:

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Eric D. Laing, [1] Ian H. Mendenhall, [1] Martin Linster, Dolyce H. W. Low, Yihui Chen, Lianying Yan, Spencer L. Sterling, Sophie Borthwick, Erica Sena Neves, Julia S. L. Lim, Maggie Skiles, Benjamin P. Y. -H. Lee, Lin-Fa Wang, Christopher C. Broder, Gavin J. D. Smith

Author affiliations: Uniformed Services University, Bethesda, Maryland, USA (E.D. Laing, L. Yan, S.L. Sterling, C.C. Broder); Duke-National University of Singapore Medical School, Singapore, Singapore (I.H. Mendenhall, M. Linster, D.H.W. Low, Y. Chen, S. Borthwick, E.S. Neves, J.S.L. Lim, L.-F. Wang, G.J.D. Smith); North Carolina State University, Raleigh, North Carolina, USA (M. Skiles); National Parks Board, Singapore (B.P.Y-H. Lee); Duke University, Durham, North Carolina, USA (L.-F. Wang, G.J.D. Smith)


[1] These authors contributed equally to this article.

Caption: 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, Tat Forest ebolavirus; 4, Sudan ebolavirus; 5, Reston ebolavirus-monkey; 6, Reston ebolavirus-pig; 7, Marburg virus-Musoke; 8, Marburg virus-Angola; 9, Ravn virus.

Caption: Figure 2. Western blot results of individual bat serum samples probed against Zaire ebolavirus and Bundibugyo ebolavirus glycoproteins 1 and 2 ([GP.sub.1], [GP.sub.2]). Boldface indicates positivity by Western blot and underlining indicates positivity by Bio-Plex (Bio-Rad, Hercules, CA, USA). 1, soluble [GP.sub.1] and [GP.sub.2] blotted with control anti-Ebola virus nonhuman primate polyclonal serum that demonstrates crossreactivity 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.
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/      Bio-Plex
                                      location           bead no.

Ebola virus/H.sapiens/                Human/DRC             33

Bundibugyo virus/H.                 Human/Uganda            64

Tai Forest virus/H.              Human/Cote d'Ivoire        57

Sudan virus/H. sapiens/             Human/Uganda            77

Reston virus/M.                      Macaque/USA            85

Reston virus/S.                   Swine/Philippines         72

Marburg virus/H. sapiens/            Human/Kenya            37

Marburg virus/H. sapiens/           Human/Angola            28

Ravn virus/H. sapiens/               Human/Kenya            49
KEN/1987/Kitum cave-

Virus                               NCBI
                                accession no.

Ebola virus/H.sapiens/           NC_002549.1

Bundibugyo virus/H.               FJ217161.1

Tai Forest virus/H.               NC_014372

Sudan virus/H. sapiens/          NC_006432.1

Reston virus/M.                   AF522874.1

Reston virus/S.                   FJ621583.1

Marburg virus/H. sapiens/       Z12132 S55429

Marburg virus/H. sapiens/         DQ447656.1

Ravn virus/H. sapiens/           NC 024781.1
KEN/1987/Kitum cave-

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

Table 2. Bio-Plex median fluorescence intensity values for bat serum
samples positive for [greater than or equal to]1 filovirus antigen *

Bat species, ID            EBOV    BDBV   TAFV   SUDV   RESTVm   RESTVp

Eonycteris spelaea,
 n = 186
  0805149 ([dagger])       738#    124     68     40      44       22
  080814                    86     318#   105    258#     26       12
  082154                    143    161    113    214#     35       41
  052313                   284#    408#   177    285#     89       72
  052335                   203#    191    124    219#     42       21
  052339                   357#    306#   141    293#     54       31
  071839                   330#    299#   164    480#     65       44
  071842                   446#    327#   202#   362#     65       49
  110733                    126    416#   166     95      58       42
  011603 ([dagger])        1151#   130     91     69      36       32
  011616                   252#    294    168    175      32       49
  011656                   306#    386#   204#   394#     89       73
  012309 ([dagger])        579#    659#   315#    69      35       31
  021303                   478#    431#   188    450#     52       37
  111903                   469#    384#   276#   113      52       57
  111907                   285#    336#   213#   158      39       36
  042722                   260#    262#   174    167#     75       31

Cynopterus brachyotis,
 n = 153
  051253                    121    133     59    242#     40       41
  0516613                   146    293#   127     73      47       36
  0516632                   138    139     86    356#     35       25
  0726122 ([dagger])        119    501#   100     60      40       46
  1103241                   84     141    128    241#     50       47
  100903                    148    201#    71    108      42       33
  100914                    74     228#    70     55      39       38
  100925                    166    304#   109    116      43       18
  021357                   201#    299#   179    264#     65       44
  050804                   242#    276#   140    124      41       30
  050818                   383#    374#   198    332#     60       55
  040807 ([dagger])        297#    597#   194    192      40       38
  042701 ([dagger])        339#    547#   222#   417#     60       78

Penthetor lucasi, n = 70
  062590 ([dagger])         34     496#    93     39      36       18
  070409 ([dagger])         95     238#   129     89      62       27
  112112 ([dagger])        251#    352#   148    235#     51       29

Bat species, ID            MARV(Mus)    MARV(Ang)    RAVV

Eonycteris spelaea,
 n = 186
  0805149 ([dagger])           23           21        24
  080814                       17           16        20
  082154                       21           31        39
  052313                       29           23        30
  052335                       38           38        24
  052339                       26           26        42
  071839                       28           33        45
  071842                       42           38        57
  110733                       34           42        58
  011603 ([dagger])            51           35        39
  011616                       47           29        50
  011656                       18           39        37
  012309 ([dagger])            27           33        35
  021303                       24           30        47
  111903                       37           69        54
  111907                       29           50        30
  042722                       54           24        42

Cynopterus brachyotis,
 n = 153
  051253                       19           25        68
  0516613                      25           29        22
  0516632                      28           34        34
  0726122 ([dagger])           25           19        29
  1103241                      66           38        34
  100903                       18           16        36
  100914                       30           27        26
  100925                       33           30        28
  021357                       25           55        47
  050804                       34           33        44
  050818                       29           26        68
  040807 ([dagger])           122           95        32
  042701 ([dagger])            54           25        62

Penthetor lucasi, n = 70
  062590 ([dagger])            23           17        23
  070409 ([dagger])            34           36        37
  112112 ([dagger])            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-Angol a; RESTVm, Reston
virus-monkey; RESTVp, Reston virus-pig; SUDV, Sudan virus; RAVV,
Ravn virus; TAFV, Tai Forest virus.

([dagger]) Sample screened by Western blot and shown in Figure 2.

Note: Positive results indicated with #.
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Title Annotation:DISPATCHES
Author:Laing, Eric D.; Mendenhall, Ian H.; Linster, Martin; Low, Dolyce H.W.; Chen, Yihui; Yan, Lianying; S
Publication:Emerging Infectious Diseases
Geographic Code:90SOU
Date:Jan 1, 2018
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