Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011-2016.
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).
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, https://wwwnc.cdc.gov/Ero/article/24/1/170401-Techapp1.pdf), 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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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: email@example.com
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Eric D. Laing,  Ian H. Mendenhall,  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)
 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 COD/1976/Yambuku-Mayinga Bundibugyo virus/H. Human/Uganda 64 sapiens/UGA/2007 Tai Forest virus/H. Human/Cote d'Ivoire 57 sapiens/COV/1994/ Pauleoula-CI Sudan virus/H. sapiens/ Human/Uganda 77 UGA/2000/Gulu-808892 Reston virus/M. Macaque/USA 85 fascicularis/USA/1989/ Pennsylvania Reston virus/S. Swine/Philippines 72 domesticus/PHL/2008/ Reston08-A Marburg virus/H. sapiens/ Human/Kenya 37 KEN/1980/Musoke Marburg virus/H. sapiens/ Human/Angola 28 AGO/2005/Ang0126 Ravn virus/H. sapiens/ Human/Kenya 49 KEN/1987/Kitum cave- 810040 Virus NCBI accession no. Ebola virus/H.sapiens/ NC_002549.1 COD/1976/Yambuku-Mayinga Bundibugyo virus/H. FJ217161.1 sapiens/UGA/2007 Tai Forest virus/H. NC_014372 sapiens/COV/1994/ Pauleoula-CI Sudan virus/H. sapiens/ NC_006432.1 UGA/2000/Gulu-808892 Reston virus/M. AF522874.1 fascicularis/USA/1989/ Pennsylvania Reston virus/S. FJ621583.1 domesticus/PHL/2008/ Reston08-A Marburg virus/H. sapiens/ Z12132 S55429 KEN/1980/Musoke Marburg virus/H. sapiens/ DQ447656.1 AGO/2005/Ang0126 Ravn virus/H. sapiens/ NC 024781.1 KEN/1987/Kitum cave- 810040 * 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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|Author:||Laing, Eric D.; Mendenhall, Ian H.; Linster, Martin; Low, Dolyce H.W.; Chen, Yihui; Yan, Lianying; S|
|Publication:||Emerging Infectious Diseases|
|Date:||Jan 1, 2018|
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