Identification of GBV-D, a Novel GB-like Flavivirus from
Old World Frugivorous Bats (Pteropus giganteus) in
Bangladesh
Jonathan H. Epstein1, Phenix-Lan Quan2, Thomas Briese2*, Craig Street2, Omar Jabado2, Sean Conlan2,
Shahneaz Ali Khan1,3, Dawn Verdugo2, M. Jahangir Hossain4, Stephen K. Hutchison5, Michael Egholm5,
Stephen P. Luby4, Peter Daszak1*, W. Ian Lipkin2
1 Conservation Medicine Program, Wildlife Trust, New York, New York, United States of America, 2 Center for Infection and Immunity, Mailman School of Public Health,
Columbia University, New York, New York, United States of America, 3 Chittagong Veterinary & Animal Sciences University, Chittagong, Bangladesh, 4 Programme on
Infectious Disease and Vaccine Sciences, International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh, 5 454 Life Sciences, Branford,
Connecticut, United States of America

Abstract
Bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, SARS-like corona-, Marburg-, Ebola-, andastroviruses. In an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16
Pteropus giganteus bats from Faridpur, Bangladesh, using high-throughput pyrosequencing. Sequence analyses indicated thepresence of a previously undescribed virus that has approximately 50% identity at the amino acid level to GB virus A and C(GBV-A and -C). Viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. Infection was not
associated with evidence of hepatitis or hepatic dysfunction. Phylogenetic analysis indicates that this first GBV-like flavivirus
reported in bats constitutes a distinct species within the Flaviviridae family and is ancestral to the GBV-A and -C virus clades.
Citation: Epstein JH, Quan P-L, Briese T, Street C, Jabado O, et al. (2010) Identification of GBV-D, a Novel GB-like Flavivirus from Old World Frugivorous Bats
(Pteropus giganteus) in Bangladesh. PLoS Pathog 6(7): e1000972. doi:10.1371/journal.ppat.1000972
Editor: Colin Parrish, Cornell University, United States of America
Received January 11, 2010; Accepted May 27, 2010; Published July 1, 2010
Copyright: ! 2010 Epstein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by awards from the National Institutes of Health including K08AI067549, AI079231, AI57158 (Northeast Biodefense CenterLipkin), AI070411, the United States Agency for International Development (USAID) Emerging Pandemic Threats (EPT) Program, PREDICT project, under the termsof Cooperative Agreement Number GHN-A-OO-09-00010-00, the Department of Defense, the Rockefeller Foundation, and Google.org. The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: thomas.briese@columbia.edu (TB); daszak@wildlifetrust.org (PD)Introduction

contaminated with bat feces, urine or saliva overnight when bats suchas P. giganteus come to feed from the collecting pots [14,15]. Date palm
sap or other foods eaten by both bats and people, may also serve as avehicle for transmission of other bat-borne agents.
Several zoonotic flaviviruses, including Japanese encephalitis
virus, West Nile virus, and Kyasanur forest virus have been
identified in bats; however, to date, GB viruses have not [1]. GB
viruses A and C (GBV-A and -C) represent two recently identified
species that are currently unassigned members of the family
Flaviviridae [16]. GBV-A viruses have been described in New World
primates and are not known to infect humans [17–19], while GBVC (also known as Hepatitis G virus (HGV)) have frequently been
isolated from humans in many regions of the World, including India
and Bangladesh [19–23], and from wild chimpanzees (Pan troglodytes)
in Africa [24,25]. Here we describe discovery of a virus in the serumof healthy bats in Bangladesh, tentatively named GB virus D (GBVD), that is distantly related to GBV-A and -C and represents a newmember of the family Flaviviridae.

Bats (order Chiroptera), after rodents, comprise the most diverse
group of mammals with more than 1,100 species. They are present
on six continents, often have substantial habitat overlap withhumans [1] and harbor several zoonotic viruses causing significant
human morbidity and mortality, including Ebola- and Marburgvirus, Nipah virus (NiV), and SARS-like coronaviruses [2–5].
Proximity of bats to human populations may facilitate the zoonotic
transmission of viruses either through direct contact, via amplifying
domestic animal hosts, or through food-borne routes [6–8].
The current study was set up as part of a viral discovery effort totarget key wildlife reservoirs in emerging disease hotspots. Bangladesh
is a ‘hotspot’ for emerging zoonotic diseases [9], with a relatively highdiversity of wildlife that likely harbors new zoonotic pathogens, one ofthe densest human populations on the planet, and a high level ofconnectivity between people, domestic animals and wildlife. In
Bangladesh and India, frugivorous Pteropus giganteus bats have been
identified as a reservoir for NiV [10,11], which has been recognized
as the cause of several outbreaks of encephalitis [12–14]. Pteropus
giganteus bats are common throughout the Indian subcontinent, living
in close association with humans and feeding on cultivated fruit [14].
NiV transmission from bats to humans has been linked with theharvest and consumption of raw date palm sap, which becomes
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Materials and Methods
Ethics statement
Every effort was made to minimize bat stress and avoid injury
during capture, restraint, and sampling procedures. This study was
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Detection of a Novel Flavivirus in Bats

Huntsville, AL, USA) [26]. After RNase H treatment cDNA wasamplified by the polymerase chain reaction (PCR), applying a 9:1
mixture of the defined 17-mer primer sequence and the random
octamer-linked 17-mer primer sequence, respectively [27].
Products of .70 base pairs (bp) were selected by column purification (MinElute, Qiagen, Hilden, Germany) and ligated to
specific linkers for sequencing on the 454 Genome Sequencer
FLX (454 Life Sciences, Branford, CT, USA) without DNA
fragmentation [28,29]. Sequences were analyzed using software
applications implemented at the GreenePortal website (http://
tako.cpmc.columbia.edu/Tools/).

Author Summary
Bats are important reservoirs for emerging zoonotic viruses
with significant impact on human health including
lyssaviruses, filoviruses, henipaviruses and coronaviruses.
Opportunities for transmission to humans are particularly
prominent in countries like Bangladesh, where people live
in close association with bats. Whereas previous studies ofbats have employed assays that test for known pathogens,
we present the first application of an unbiased molecularapproach to pathogen discovery in this reservoir for
emerging zoonotic disease. Unbiased pyrosequencing ofserum from Pteropus giganteus bats enabled identificationof a novel flavivirus related to Hepatitis C and GB viruses.
Viral nucleic acid was present in 5 of 98 (5%) sera, and in
the saliva of one animal. Sequence identification of two
strains of the virus, tentatively named GBV-D, suggests P.
giganteus as a natural reservoir. Detection of viral nucleic
acid in saliva provides a plausible route for zoonotic
transmission. Phylogenetic analysis indicates that GBV-D isancestral to GBV-A and -C, and separate from the recently
classified genus Hepacivirus. Our findings provide new
insight into the range of known hosts for GB-like viruses
and demonstrate the power of unbiased sequencing tocharacterize the diversity of potentially zoonotic pathogens carried by bats and other reservoirs.

Genome sequencing
Multiple forward and reverse primers for RT-PCR (available
upon request) were designed using the sequences obtained by
UHTS in order to fill gaps between fragments. Amplifications
were performed with Bio-X-act (Bioline, London, UK) according
to manufacturer’s protocols. Products were size fractionated byelectrophoresis and directly sequenced in both directions with ABIPRISM Big Dye Terminator 1.1 Cycle Sequencing kits (PerkinElmer Applied Biosystems, Foster City, CA, USA) at a commercial
facility (Genewiz, South Plainfield, NJ, USA). Additional methods
applied to obtain the genome sequence included touch-down
PCR [30], 2-step walking PCR [31], and 39- and 59- RACE
(Invitrogen).

conducted following Wildlife Trust institutional guidelines under
IACUC approval G2907 issued by Tufts New England Medical
Center, Boston, Massachusetts.

Quantitative real-time PCR
A real time Taqman PCR assay was developed to screen bat
samples for GBV-D. Reactions were performed in a 25 mL volume
by using commercial Taqman Universal Master Mix (Applied
Biosystems, Foster City, CA, USA). Primers and probe were
designed to target a 60 nt region in the NS4A gene region: Fadiforward, 59- gCAgCTgCgTgTgCCA; Fadi-reverse, 59- ACACCCATgATgTTACCACgAC; Fadi-probe, 59- FAM- AggACCCggTCgCTCCAgCA-T-BQX (TIB Molbiol, Adelphia, NJ, USA).
Cycling conditions were: 50uC for 2 min, and 95uC for 10 min,followed by 45 cycles at 95uC for 15 sec and 60uC for 1 min.
Thermal cycling was performed in an ABI 7300 real-time PCRsystem (Applied Biosystems).

Bat sample collection
As part of a longitudinal surveillance study of Nipah virus inbats, 98 free-ranging P. giganteus bats were caught from a colony ofapproximately 1800 individuals in the Faridpur district ofBangladesh in December 2007 (Figure 1). Each bat wasanesthetized using isoflurane gas; morphometric measurements(weight, forearm length, head length, and body condition) weretaken and bats were aged [10]. Each bat was marked for future
identification using an RFID microchip (AVID corp, www.avidid.
com) implanted subcutaneously between the scapulae. Three mLof blood were collected and placed into serum separator tubes(vacutainer; Becton Dickinson, Franklin Lakes, NJ, USA). Serum
was allowed to separate overnight at 4uC then drawn off without
centrifugation and immediately frozen using a liquid nitrogen dry
shipper. To inactivate potentially infectious agents, serum samples
were heat-treated at 56uC for 30 min and then stored at 270uC.
For RNA extraction, 250 mL of serum was added to 750 mL TriReagent LS (Molecular Research Center, Cincinnati, OH, USA).
Saliva was collected from the bat’s throat using a sterile cottonswab. Urine was collected either by catching urine in a 1.0 mL
sterile cryovial while the bat was urinating, or by urethral swab.
Urine and saliva swabs were immediately placed into 1 mL TriReagent LS and frozen in liquid nitrogen.

Serum chemistry
A liver function panel was conducted at the International
Center for Diarrheal Disease Research (Dhaka, Bangaldesh) using
non heat-treated bat sera (Automated Chemistry Analyzer AU
640, Olympus Corporation, Tokyo, Japan). The following
parameters were analyzed: total protein, albumin, globulin,
albumin:globulin ratio, total cholesterol, total bilirubin , alkaline
phosphatase, alanine transferase, aspartate aminotransferase,
gamma glutamyltransferase , and lactate dehydrogenase.

Phylogenetic and sequence analyses
Sequence alignments were generated with ClustalW software[32] and phylogenetic relationships deduced using Geneious
software [33]. Statistical significance was assessed by bootstrap
re-sampling of 1000 pseudoreplicate data sets. Sequence relations
were determined from p-distance matrices calculated with pairwise
deletion for missing data and homogeneous patterns among
lineages based on ClustalW alignments as implemented in MEGA
software [34]. Sliding window similarity analysis was performed
using SimPlot [35]. Potential signalase cleavage sites, glycosylationsites, and phosphorylation sites were analyzed using the respective
prediction servers available at the Center for Biological Sequence
Analysis (http://www.cbs.dtu.dk/services/).

Unbiased high-throughput pyrosequencing (UHTS)
Total RNA from serum was extracted for UHTS analysis toscreen for the presence of microorganisms. Five microliters of
total RNA from each bat were combined into 4 pools: 4 pregnant
bats; 4 non-pregnant female bats, and 2 pools of 4 adult malebats, respectively. Reverse transcription (RT) was performed on
DNase I-treated (DNA-free, Ambion Inc., Austin, TX, USA)RNA pools to generate cDNA using Superscript II RT(Invitrogen, Carlsbad, CA, USA) and random octamers linked
to a defined arbitrary, 17-mer primer sequence tail (MWG,
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Figure 1. Map showing the location of the bat colony in Faridpur district, Bangladesh from which GBV-D was identified.
doi:10.1371/journal.ppat.1000972.g001

male bats that were not included in the initial UHTS pools.
Extracts of saliva from the five positive bats indicated a load of
approximately 200 RNA copies in bat 93; no signal was obtainedwith urine extracts from the five positive bats.

Results
Identification of a GB-like agent from bats
Total RNA from the serum of healthy bats captured at a roostin the Faridpur district of Bangladesh was extracted for UHTS
analysis. Extracts of 16 individual bats were combined into 4 poolsconsisting of 4 pregnant adult bats, 4 non-pregnant adult femalebats, or 264 adult male bats. Each pool yielded between 1,400
and 2,000 assembled contigs or singlton reads (representing
50,000–75,000 reads ranging in size from 31–328 nt). Two readsof 238 and 215 nucleotides (nt) derived from the pregnant bat poolhad distant homology to GBV-A sequences at the deduced aminoacid (aa) level in the E2 and NS4A gene regions respectively(BLASTX); no homology was detected by searches at the nt level(BLASTN; local copy of the executables with standard settings
except that the reward for a nucleotide match was set to 2 insteadof 1). No viral sequences were detected in other pools at the nt or
aa levels. Screening of the individual RNA preparations from thepregnant bat pool using primers derived from the UHTS reads
confirmed the presence of the GBV-like sequence in the serum ofbat 93. A quantitative real time PCR assay indicated a load of
approximately 30 000 RNA copies in bat-93 serum extract, andidentified an additional 4 positive bat sera from the original 98
samples (5/98; 5%), indicating serum loads ranging from 350 to
70,000 RNA copies per assay. These positive samples came fromPLoS Pathogens | www.plospathogens.org

Genomic characterization of GBV-D
Near full-length genome sequence was generated from bat-93
and a second positive serum (bat 68), applying primers crossing
Table 1. Percent sequence similarity between GBV-D (bat-68),-A, -C, and hepaciviruses.

nt*
aa

GBV-D

GBV-D

GBV-C

GBV-A

GBV-B

HCV-1

48

46

39

41

55

41

44

37

38

GBV-C

41

GBV-A

39

47

GBV-B

25

24

24

HVC-1

22

22

22

41
27

*Sequence similarity at nt level in upper right, and at aa level in lower left
portion of the table.
doi:10.1371/journal.ppat.1000972.t001

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Detection of a Novel Flavivirus in Bats

Figure 2. Sliding window similarity analysis between GBV-D and other GBV and hepaciviruses (amino acid sequence; window, 160;
step, 20).
doi:10.1371/journal.ppat.1000972.g002

N-terminal NS2 motifs (pfam 01538) (Figure 2, Figure 3).
However, despite similarity to pfam 01538 no signal sequencecompatible with cleavage at A759/A was found; cleavage may
occur at G826/R, which combined with potential signalasecleavage at A584/F may indicate the existence of a heavily
glycosylated potential 26 kDa product instead of the p7 transmembrane protein identified in HCV [37–39] or the 13 kDa
variant described in GBV-B [40,41]. Conserved C-terminal motifsof the autocatalytic NS2/NS3 endoprotease domain are compatible with NS2/NS3 cleavage at S1067/A and comparable to other
GBV and HCV [42]. Figure 3 indicates potential cleavage sites
for NS3 (peptidase S29, pfam 02907; DEAD box helicase, pfam
07652; helicase C, pfam 00271), NS4A (pfam 01006), NS4B (pfam01001), NS5A (domain-1a zinc finger, pfam 08300; domain-1b,
pfam 08301), and NS5B (pfam 00998).
Conserved aa motifs were recognized in NS proteins. RNAdependent RNA polymerase (RdRp) motifs in RdRp block III thatare conserved with respect to other GBV and hepaciviruses wereidentified in NS5B (Figure 3) [43–46]. Potential phosphorylationsites are present at multiple serine (9), threonine (14) and tyrosine (4)residues in NS5A, compatible with its possible function as aphosphorylation-regulated mediator of viral replication [47]. However, significant conservation of primary sequence is not obvious
for phosphorylation sites, proline-rich, or interferon-sensitivity

gaps between UHTS reads as well as touch-down PCR [30],2-step walking PCR [31], and 39- and 59-RACE (Invitrogen)protocols. The two genome sequences were 96% identical at the nt
level (GenBank Accession nos. GU566734 and GU566735),
indicating two strains of the same virus. Comparison of deduced
polyprotein sequence to other GBV and hepaciviruses indicatedhighest nt and aa sequence identities to GBV-A and -C (Table 1,
Figure 2). The genomic sequence of the GBV-like virus identified
in P. giganteus bats, tentatively named GBV-D, comprises 9,633 nt
with 52 nt of potentially 59-untranslated region (UTR), one
continuous open reading frame (ORF) of 9318 nt (3106 aa) and
265 nt of 39-UTR (Figure 3).
Mature structural proteins in GB viruses, as well as otherflaviviruses, are the product of cleavage by host signal peptidase[36]. In GBV-D the first potential signal sequence cleavage site ispresent after a stretch of 57, largely basic aa (6 kDa, pI = 12),
followed by sequence homologous to E1 (pfam 01539, http://
pfam.sanger.ac.uk/) (Figure 3). The single glycosylation site
N177IT present in that sequence is located in a position
comparable to GBV-C, -A, -B and HCV glycosylation sites.
Identification of the downstream E2 termini is less apparent as thenext 580 aa contain multiple potential signal sequences and 10
potential glycosylation sites that indicate no homology tohepaciviral E2/NS1 (pfam 01560), until the sequence aligns with

Figure 3. Genomic organization of GBV-D, a novel flavivirus identified in the sera of frugivorous bats in Bangladesh. Arrows,
glycosylation sites; solid diamond, active center sites H921, E1011, and C1032 in the autocatalytic NS2/NS3 endoprotease domain; triangle, catalytic triad
H1123, D1147, S1204 of NS3 serine protease; rectangle, NS3 helicase and DEAD-like helicase motifs; open diamond, zinc finger motif; and NS5
polymarase motifs A (T2744VDAICFDSCIT), B (R2802ASGVLTTSSSNCISSFLKVSAAC), C (F2835LIHGDDVMII), D (L2876DTAQSCSA),and E (H2900YFLSTDFR)
motifs.
doi:10.1371/journal.ppat.1000972.g003

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Figure 4. Phylogenetic relationship of GBV-D to other GBV and hepaciviruses. GBV-D amino acid sequences for A: NS5B, B: NS3, and C: thepolyprotein (PPT) were analyzed in comparison to representative sequences of GBV-A, -B, -C and hepatitis C viruses. GenBank accession numbers for

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Detection of a Novel Flavivirus in Bats

the respective sequences are indicated. Entebbe bat virus was used as an outgroup; distance in substitutions per site is indicated by scale bars;
percent bootstrap support for values greater than 85% is indicated at respective nodes.
doi:10.1371/journal.ppat.1000972.g004

determining region motifs [48–50]. The C-terminal portion of NS3
has homology to conserved NTPase/helicase motifs [51]; the Nterminal portion includes conserved active triad residues H1123,
D1147, S1204 of serine protease [52], the viral protease responsible forcleavage of mature non-structural proteins [53]. Likewise, the active
triad H991, E1011, C1032 of the cis-acting protease activity in the Cterminal portion of NS2 is conserved with respect to other GBV andHCV [42]. The only other discernable motif identified was a wellconserved N75 C/D C motif at the N-terminus of E1 (Figure 3) [54].

Serum chemistries
A liver serum chemistry panel was conducted on sera from 15
bats, the five GBV-D infected and 10 non-infected animals.
Standard assays to detect hepatitis and/or impaired liver function
were performed [55]. Levels of total protein, alanine transferase,
aspartate aminotransferase and total cholesterol were withinpublished ranges reported for P. giganteus, except for bat 33(infected) and bat 73 (uninfected), which had modest elevation in
aspartate aminotransferase. Reference values for albumin, globulin, albumin:globulin ratio, total bilirubin, alkaline phosphatase,
gamma glutamyltransferase and lactate dehydrogenase are notavailable for P. giganteus, however, values were comparable to thosereported for other Pteropus species [56]. Mean values did not
significantly differ between infected and uninfected bats (Table 2).

Phylogenetic analysis
Phylogenetic analysis of GBV-D was performed in comparison
to selected representatives of GBV-A, GBV-B, GBV-C and HCV.
Analysis of NS5B aa sequence (Figure 4A) confirmed a closer
relationship of GBV-D to GBV-A and -C than to GBV-B or HCV
as also indicated by pairwise sequence comparisons (Table 1).
The same relationships were also apparent when NS3, or thecomplete polyprotein sequence were analyzed (Figure 4B and C,
respectively). All three trees show GBV-D consistently at the rootof the GBV-A/-C viruses, indicating an independent phylogenetic
clade compatible with a separate species distinct from the recently
created genus Hepacivirus [16].

Discussion
Molecular analyses of sera from Pteropus giganteus bats from
Faridpur, Bangladesh led to the identification of a 9,633 nt sequence
consistent in genomic organization with known GBV and otherspecies within the family Flaviviridae [16]. Whereas previous studies ofbats have employed assays that test for known pathogens, ours is the

Table 2. Liver function values from Pteropus giganteus bats.

T.Prot(g/dL)

Alb(g/dL)Glob

A:GT. Chol(mg/dL)T. Bili(mg/dL)

Alk
Phos

ALT
(U/L)

AST (U/L)

GGT
(U/L)

LDH
(mg/dL)

Ref Range (human)

64.0–82.0

34–50

23–35

1.1–1.8

3.6–6.3.01–1930–120.01–41.01–38

9–40

135–225

Ref Range (P. giganteus)

65–84

30–41

1.2–84.6

30.2–141.0

1

86

50.7

35.3

1.44

1.65

1.8

1572..4

13.9

67.6

87.90

53.4

3

79.7

49.4

30.3

1.63

2.74

2.1

605.7

20.2

67.4

49.20

95

4

70.4

42.9

27.5

1.56

0.5

2.3

532.1

12.3

66.1

75.40

76.7

21

76

48

28

1.71

1.06

1.2

794.9

23.8

83.7

35.70

244.5
96

0–2.3

Bat ID

23

71.5

46.2

25.3

1.83

1.49

1.7

1650

12

115.1

102.00

33

80.5

43.8

36.7

1.19

1.09

2.1

891.6

22.2

146

32.10

752.4

36

89.9

51

38.9

1.31

1.22

2.5

580.4

11.1

109.4

69.00

114.4

37

76.6

47

29.6

1.59

0.85

1.3

205.8

12.6

50.3

72.50

133.9

39

70.4

43.3

27.1

1.6

0.5

1.8

328.7

20.5

77.8

31.00

82.6

68

78.9

44.9

34

1.32

2.54

1.2

813.3

7.9

65.6

46.10

111.8

73

82.4

46.1

36.3

1.27

1.17

1.8

958.8

19.7

185.4

37.00

859.4

82

72.3

43.8

28.5

1.54

0.5

1.4

458.4

22.1

82.1

48.50

350.3

84

82.6

46.2

36.4

1.27

1.43

2.7

923.6

22.9

107.6

46.40

176

93

82.1

47.5

34.6

1.37

1.46

2.6

262

8.6

59.9

47.10

105.2

96

76.5

46.8

29.7

1.58

0.5

1.1

574.6

20.9

79.4

101.80203.7

Mean uninfected

78.73

47.15

31.58

1.518

1.226

1.81

763.9

18.71

97.55

60.85

227.53

Mean infected

77.7

45.22

32.48

1.406

1.288

1.9

540.96

12.72

77.58

54.64

236

STdv uninfected

6.52

2.63

4.71

0.19

0.68

0.52

391.61

4.62

35.31

27.40

239.64

STdv infected

4.56

1.99

3.80

0.17

0.78

0.62

311.19

5.71

38.77

18.63

289.40No indication of hepatitis or impaired liver function was observed; no significant differences between mean values for infected (bold) or non-infected bats wereapparent.
The following parameters were analyzed: Total Protein (T. Prot); Albumin (Alb), Globulin (Glob), Alb:Glob ratio, Total Cholesterol (T. Chol); Total Bilirubin (T. Bili); Akaline
Phosphatase (Alk. Phos); Alanine Transferase (ALT); Aspartate Aminotransferase (AST); Gamma Glutamyltransferase (GGT) and Lactate Dehydrogenase (LDH).
doi:10.1371/journal.ppat.1000972.t002

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first report of an unbiased molecular approach to pathogen discovery
in this important reservoir of emerging infectious diseases. The
modest yield of novel microbial sequences may reflect the choice ofsample (e.g., serum vs feces, tissue or another specimen), competition
between host and microbial template during unbiased amplification,
or both. Efforts to address template competition are under way that
include subtraction of host nucleic acids or the use of semi-random
primers that do not amplify host sequences. Such efforts will likely
enhance the sensitivity and throughput of unbiased sequencing
technologies for pathogen discovery.
The discovery of this chiropteran flavivirus broadens both thetaxonomical and geographical distribution of GB-like viruses. Threetypes of GB viruses have been described: GBV-A, -B and -C[18,19,24,25,54,57]. GBV-B, which has never been found in
humans and was only reported in captive tamarins after serial
passage of the original human GB serum [58], is most closely related
to HCV and was recently classified together with HCV into a new
genus, Hepacivirus, within the family Flaviviridae [16]. GBV-A and -C
remain unclassified members of the family. GBV-A have been
isolated from several New World monkeys. Different genotypes
appear to be associated with specific monkey species of the genera
Saguinus, Callithrix (Callitrichidae family) and Aotus (Aotidae family),
without any clinical signs associated with infection [24,54,57].
GBV-C have been isolated from humans with non-A-E hepatitis;
however, its pathogenicity is unknown and the virus is widespread in
the human population [21,59–61]. Population studies showed that
GB viruses are enzoonotic and species-specific within both Old and
New World nonhuman primates as well as humans, and have likely
co-evolved with their hosts over long periods of time [62].
Previously, the only GBV found in the Old world was GBV-C
from chimpanzees (in Africa) and humans. Although GBV-C were
found in humans, GB viruses have not been previously reported inprimates or other animals on the Indian subcontinent.
GBV-C and -A are remarkable for a truncated or missing capsid(C) protein [18,19]. Due to exhaustion of our samples we wereunable to complete assessment of the 59-terminal sequence;
nonetheless, RACE experiments suggest that GBV-D likely codes
for a short basic peptide, instead of a full-length C protein. The firstmethionine (M1) predicts a peptide of 57 aa (pI = 12); however, themore favorable Kozak context [63] of M3 indicates a 55 aa peptide.
After signalase cleavage from the polyprotein precursor, this peptide
may be functional, possibly influencing maturation of, or directly
binding to, the E1 and/or E2 glycoproteins.
Phylogenetic analyses of NS5B, NS3 and complete polyprotein
sequence place GBV-D at the root of the GBV-A and -C clades andare consistent with a model wherein GBV-D is ancestral to GBV-A
and -C clades. Mixed relationships indicative of recombination
events [64] were not evident (Figure 2, Figure 4). Both pteropidbats and chimpanzees are restricted to the Old World. While therange of chimpanzees (Africa) and P. giganteus (the Indian
subcontinent) do not overlap, it is possible that other primate
species in Bangladesh or India, such as macaques, or other fruit batsin Africa such as Eidelon spp., whose range overlaps that ofchimpanzees, may carry related viruses. While GBV-A is only
known from primates of the New World, an African origin has beensuggested for GBV-C based on a 12-aa indel sequence in NS5A
[65]. Although the NS5A sequence of GBV-D, similar to that of
GBV-A, appears elongated in the indel region, compatible with
their respective earlier phylogenetic branching compared to GBVC, little sequence conservation is observed in that region.
The bats in this study, like primates infected with their associated
GBV [66], all appeared to be healthy. The lack of chemical evidenceof hepatic inflammation or dysfunction suggests that this virus may
not target hepatic cells in bats. This is consistent with the behavior of
GBV-A in its natural primate hosts [54]. In contrast, elevatedalanine transferase levels and mild hepatitis are observed inexperimental infections of macaques with GBV-C isolates fromhumans [67]. Five percent of the bats we studied were infected with
one of at least two different strains of GBV-D, which suggests
widespread viral circulation within this species. The observation thatbats are asymptomatically infected with diverse strains that constitutea distinct phylogenetic clade is compatible with a co-evolutionary
relationship between GBV and their hosts [57,62], and supports the
hypothesis that P. giganteus bats may be a natural reservoir for GBVD. In one case we were able to detect GBV-D nucleic acid in saliva.
This suggests a potential route for viral transmission via fighting or
grooming behavior, or via food shared by bats.
Pteropus giganteus is a frugivorous bat species that carries NiV, azoonotic paramyxovirus [10,11]. This species lives in close
association with humans in Bangladesh and bats have been
observed drinking from (and urinating into) date palm sap
collecting pots [14]. Human consumption of contaminated palm
juice is proposed to be a major route of NiV transmission [68].
Although it is unclear whether infectious virus was present in bat
saliva, the observation that saliva can contain GBV-D nucleic
acids provides a biologically plausible mechanism for transmission
from infected bats to other hosts. While it is currently unknown
whether GBV-D virus occurs in humans, up to 20% of non-A-E
hepatitis cases remain unexplained [19].

Acknowledgments
We thank the Forestry Department of Bangladesh for permission to
conduct this research; Md. Sheikh Gofur and Md. Pitu Biswas for help insampling bats; A. Bennett, A. Tashmukhamedova, and R. Tokarz fortechnical support, and K. Olival for critical comments on the manuscript.

Author Contributions
Conceived and designed the experiments: JHE TB PD WIL. Performed
the experiments: JHE PLQ DV SH. Analyzed the data: JHE PLQ TB CS
OJ DV SKH ME PD. Contributed reagents/materials/analysis tools: JHE
TB CS SC SAK MJH SKH ME SPL WIL. Wrote the paper: JHE PLQ
TB WIL. Coordinated field and lab activities and logistics for work in
Bangladesh; contributed to paper-writing: MJH. Coordinated permissions,field activities and logistics for work in Bangladesh; contributed to paperwriting: SPL.

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