Volume 17, Number 8—August 2011
Reston Ebolavirus Antibodies in Bats, the Philippines
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.
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.
We thank the staff at the Special Pathogens Laboratory, National Institute of Infectious Diseases, and Maiko Endo for taking care of bats at the university farm, and Edison Cosico and Eduardo Eres for collecting the wild bats in the Philippines.
This study was supported in part by a grant-in-aid from the Ministry of Health, Labor and Welfare of Japan and the Japan Society for the Promotion of Science KAKENHI.
- Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Fulhorst CF, Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996. J Infect Dis. 1999;179(Suppl 1):S115–9.
- Morikawa S, Saijo M, Kurane I. Current knowledge on lower virulence of Reston Ebola virus [in French]. Comp Immunol Microbiol Infect Dis. 2007;30:391–8.
- Barrette RW, Metwally SA, Rowland JM, Xu L, Zaki SR, Nichol ST, Discovery of swine as a host for the Reston ebolavirus. Science. 2009;325:204–6.
- Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, Fruit bats as reservoirs of Ebola virus. Nature. 2005;438:575–6.
- Pourrut X, Souris M, Towner JS, Rollin PE, Nichol ST, Gonzalez JP, Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus. BMC Infect Dis. 2009;9:159.
- Towner JS, Amman BR, Sealy TK, Carroll SA, Comer JA, Kemp A, Isolation of genetically diverse Marburg viruses from Egyptian fruit bats. PLoS Pathog. 2009;5:e1000536.
- Saijo M, Niikura M, Ikegami T, Kurane I, Kurata T, Morikawa S. Laboratory diagnostic systems for Ebola and Marburg hemorrhagic fevers developed with recombinant proteins. Clin Vaccine Immunol. 2006;13:444–51.
- Omatsu T, Ishii Y, Kyuwa S, Milanda EG, Terao K, Yoshikawa Y. Molecular evolution inferred from immunological cross-reactivity of immunoglobulin G among Chiroptera and closely related species. Exp Anim. 2003;52:425–8.
- Ikegami T, Saijo M, Niikura M, Miranda ME, Calaor AB, Hernandez M, Immunoglobulin G enzyme-linked immunosorbent assay using truncated nucleoproteins of Reston Ebola virus. Epidemiol Infect. 2003;130:533–9.
- Ikegami T, Saijo M, Niikura M, Miranda ME, Calaor AB, Hernandez M, Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells. Microbiol Immunol. 2002;46:633–8.