To the Editor: Filoviruses cause highly lethal hemorrhagic fever in humans and nonhuman
primates, except for Reston Ebolavirus (REBOV), which causes severe hemorrhagic fever
in macaques (
1
,
2
). REBOV epizootics among cynomolgus macaques occurred in 1989, 1990, 1992, and 1996
(
2
) and among swine in 2008 (
3
). African fruit bats have been suggested to be natural reservoirs for Zaire Ebolavirus
and Marburg virus (
4
–
6
). However, the natural reservoir of REBOV in the Philippines is unknown. Thus, we
determined the prevalence of REBOV antibody–positive bats in the Philippines.
Permission for this study was obtained from the Department of Environment and Natural
Resources, the Philippines, before collecting bat specimens. Serum specimens from
141 wild-caught bats were collected at several locations during 2008–2009. The bat
species tested are summarized in the Table. Captured bats were humanely killed and
various tissues were obtained. Carcasses were then provided to the Department of Environment
and Natural Resources for issuance of a transport permit.
Table
REBOV-specific IgG in Rousettus amplexicaudatus bats and other bats, the Philippines*
Bat ID
Collection site
ELISA optical density
IFA titer
REBOV NP
REBOV GP
REBOV NP
REBOV GP
1539
FD
2.13
–0.21
1,280
<20
1632
FQ1
0.88
0.2
<20
<20
1642
FQ1
0.36
5.22
<20
20
1643
FQ1
1.26
0.92
<20
<20
1651
FQ1
1.61
1.02
<20
<20
1657
FQ1
–0.45
1.69
<20
<20
1660
FQ1
3.8
2.51
640
<20
*Cutoff optical density of ELISA was 0.82 (sum of optical densities at serum dilutions
of 1:100, 1:400, 1:1,600, and 1:6,400). Values in boldface are positive results. REBOV,
Reston Ebolavirus; Ig, immunoglobulin; IFA, indirect immunofluorescence assay; ID,
identification; NP, nucleoprotein; GP, glycoprotein; FD, forest of Diliman at the
University of the Philippines Diliman campus; FQ1, forest at the Agricultural College
in Province of Quezon, the Philippines. The other 9 R. amplexicaudatus bats collected
at FQ1 had negative results for all assays. The following bat species also had negative
results: 5 Eonycteris spelaea, 35 Cynopterus brachyotis, 38 Ptenochirus jagoli, 6
Haplonycteris fischeri, 2 Macroglossus minimus, 2 Rhinolophus rufus, 1 Rhinolophus
arcuatus, 9 Emballonura alecto, 2 Pipistrellus javanicus, 5 Scotophilus kuhlii, 8
Miniopterus australis, 8 M. schreibersi, 1 M. tristis tritis, 1 Hipposideros diadema,
1 Myotis macrotarsus, and 1 bat of unknown species.
We used immunoglobulin (Ig) G ELISAs with recombinant nucleoprotein (NP) and glycoprotein
(GP) of REBOV (
7
) to determine REBOV antibody prevalence. REBOV NP and GP were expressed and purified
from Tn5 cells infected with recombinant baculoviruses AcResNP and AcResGPDTM, which
express NP and the ectodomain of GP with the histidine tag at its C-terminus. We also
used histidine-tagged recombinant Crimean-Congo hemorrhagic fever virus NP as a negative
control antigen in the IgG ELISA to confirm specificity of reactivity.
In IgG ELISAs for bat specimens, positive results were detected by using rabbit anti-bat
IgG and horseradish peroxidase–conjugated anti-rabbit IgG. Anti-bat (Rousettus aegyptiacus)
rabbit IgG strongly cross-reacts with IgGs of other bat species, including insectivorous
bats (
8
). Bat serum samples were 4-fold serially diluted (1:100–1:6,400) and tested by using
IgG ELISAs. Results of IgG ELISAs were the sum of optical densities at serum dilutions
of 1:100, 1:400, 1:1,600, and 1:6,400. Cutoff values (0.82 for both IgG ELISAs) were
determined by using serum specimens from REBOV antibody–negative bats.
Among 16 serum samples from R. amplexicaudatus bats, 5 (31%) captured at either the
forest of Diliman (14°38′N, 121°2′E) or the forest of Quezon (14°10′N, 121°50′E) had
positive results in the IgG ELISA for REBOV NP, and 5 (31%) captured at the forest
of Quezon had positive results in the IgG ELISA for REBOV GP. The REBOV NP antibody–positive
bats serum samples were confirmed to be NP antibody positive in the IgG ELISA by using
glutathione-S-transferase–tagged partial REBOV NP antigen (
9
). Three samples had positive results in both IgG ELISAs (Table). Serum samples from
other bat species had negative results in IgG ELISAs.
All bat serum samples were also tested by indirect immunofluorescence assays (IFAs)
that used HeLa cells expressing NP and GP (
10
). In the IFAs, 2 samples from R. amplexicaudatus bats captured at the forest of Diliman
and the forest of Quezon had high titers (1,280 and 640, respectively) of NP-specific
antibodies, and 1 sample from an R. amplexicaudatus bat captured at the forest of
Quezon had a positive result in the GP-specific IFA (titer 20). All IFA-positive samples
were also positive in the IgG ELISA (Table).
The forest of Diliman is ≈30 km from the monkey facility and the Bulacan farm where
REBOV infections in monkeys and swine, respectively, were detected. The forest of
Quezon is ≈60 km from the monkey facility. Samples from other bat species had negative
results in IFAs. We also performed heminested reverse transcription PCR specific for
the REBOV NP gene with spleen specimens from all 16 R. amplexicaudatus bats but failed
to detect any REBOV-specific amplicons.
REBOV-specific antibodies were detected only in R. amplexicaudatus bats, a common
species of fruit bat, in the Philippines. In Africa, R. aegyptiacus bats, which are
genetically similar to R. amplexicaudatus bats, have been shown to be naturally infected
with Zaire Ebolavirus and Marburg virus. Thus, R. amplexicaudatus bats are a possible
natural reservoir of REBOV. However, only 16 specimens of R. amplexicaudatus bats
were available in this study, and it will be necessary to investigate more specimens
of this species to detect the REBOV genome or antigens to conclude the bat is a natural
reservoir for REBOV.
We have shown that R. amplexicaudatus bats are putatively infected with REBOV or closely
related viruses in the Philippines. Antibody-positive bats were captured at the sites
near the study areas, where REBOV infections in cynomolgus monkeys and swine have
been identified. Thus, bats are a possible natural reservoir of REBOV. Further analysis
to demonstrate the REBOV genome in bats is necessary to conclude that the bat is a
reservoir of REBOV.