Filoviridae, a family of single-stranded non segmented RNA viruses, comprises two genera, marburgvirus (MARV) and ebolavirus (EBOV), the latter comprising Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Cote d'Ivoire ebolavirus (CIEBOV), Reston ebolavirus (REBOV) and Bundibungyo ebolavirus (BEBOV) [1,2]. Filoviruses cause hemorrhagic fever outbreaks in humans, with high case fatality rates (except for REBOV). Seventeen outbreaks and two isolated cases of human EBOV infection have been reported, in Democratic Republic of Congo (DRC) (1976, 1977, 1995, 2007, 2008), Sudan (1976, 1979, 2004), Gabon (1994, 1996, 1997, 2001), Republic of Congo (RC) (2001, 2003, 2005), Ivory Coast (1995) and Uganda (2000, 2007) [2-4]. Three outbreaks and four isolated cases of human MARV infection have been reported, in Germany and the former Yugoslavia (1967), Zimbabwe (1975), Kenya (1980, 1987), DRC (1998-1999), Angola (2005) and Uganda (2007) [5]. Consistent with this epidemiological pattern, ecologic niche modeling of outbreaks and isolated cases has suggested that Ebola hemorrhagic fever occurs in the rain forests of central and western Africa, and Marburg in more open areas of central and western Africa [6,7]. In Gabon and RC, most human ZEBOV outbreaks have occurred through contact with infected animal carcasses, especially great apes and duikers [8]. Bats were recently identified as potential reservoir species of these filoviruses [3,9,10]. During the ZEBOV outbreaks that occurred between 2001 and 2003 in Gabon and RC, numerous animals were captured in the search for reservoir species. Of the 1024 small mammals sampled, including 679 bats, ZEBOV-specific antibodies were detected in the serum of 16 (8%) of 192 fruit bats belonging to three species, namely Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata; ZEBOV RNA was detected in pooled liver and spleen samples from 13 (5%) of 279 such bats [11]. Sequence analysis indicated that bats of these species might serve as a ZEBOV natural host. A large-scale serological survey of bats belonging to these three species was then conducted, with 1390 specimens captured between 2003 and 2006 in three regions of Gabon and in the epidemic border region with RC. The prevalence of ZEBOV-specific IgG was about 5% in bats from all four regions, and fell to about 1% at all four sites a year after the most recent Ebola outbreak, which occurred in 2005 in RC [12]. These results indicate that ZEBOV-specific antibodies are widely distributed in these bats in Gabon and RC, supporting their potential reservoir status. As a Marburg outbreak occurred in 2005 in Angola, 800 km from the Gabon border, we also tested bats for signs of MARV infection. Of 438 bats collected in Gabon and RC, MARV-specific IgG was detected in the serum of 29 of 242 specimens of Rousettus aegyptiacus, and MARV nucleotide sequences were detected in four members of 283 bats of the same species, suggesting that R. aegyptiacus bats may be a MARV reservoir [13].

In the present study, we documented the distribution of ZEBOV- and MARV-specific antibodies in bat communities living in forest areas of Gabon and northern RC. We report detailed serologic results for 2147 bats belonging to at least nine species sampled between 2003 and 2008.

The aim of this study was to elucidate the reported presence of ZEBOV- and MARV-specific antibodies in bats in Gabon. We had previously detected ZEBOV-specific IgG (in 16 of 192 bats) and ZEBOV RNA (in 13 of 279 bats) in E. franqueti, H. monstrosus and M. torquata specimens captured in the Ebola outbreak zone of northern Gabon and Congo between 2001 and 2003, and deduced that these three bat species might represent a ZEBOV reservoir [11]. Phylogenetic analyses of the seven sequences obtained from the 13 ZEBOV RNA positive bats showed very few differences relative to known human sequences (approximately 1% of nucleotide positions in the L gene), confirming that Ebola virus detected in bats corresponded to the ZEBOV species. We subsequently detected MARV-specific IgG (in 29 of 242 bats) and MARV RNA (in 4 of 283 bats) in specimens of R. aegyptiacus captured in southern and southwestern Gabon in 2005 and deduced that this species might represent a MARV reservoir. Phylogenetic analysis of the MARV sequences obtained from the bats, in the N and VP 35 genes, showed approximately 5% nucleotide divergence relative to the other western African Marburg virus lineage, Angola, a difference far smaller than the 15% diversity previously observed among East African Marburg virus isolates. This pointed to the existence of a geographic cluster including Angolan human MARV strains and Gabonese bat strains [13]. No blood samples were available from ZEBOV PCR-positive bats identified during our previous studies. However, all MARV PCR-positive bats were also positive for anti-MARV IgG [13].

Here we report the results of a large serological survey of ZEBOV and MARV in bats living in three regions of Gabon and northwest RC. In addition to the bats studied before, we examined additional specimens for ZEBOV infection (238 for IgG, 1468 for RNA) and for MARV (1438 for both IgG and RNA). We detected both ZEBOV-specific IgG (4%) and MARV-specific IgG (1%) in all four regions, confirming that the two viruses cocirculate in Gabon.

This is the first study to provide evidence of simultaneous ZEBOV and MARV circulation in bat populations in the same country. Several human ZEBOV outbreaks have occurred in Gabon, but no MARV outbreaks have been reported.

Interestingly, of the nine species reported here, specimens of six species (E. franqueti, H. monstrosus, M. torquata, M. pusillus, R. aegyptiacus and Mops condylurus) had ZEBOV-specific IgG and specimens of four species (R. aegyptiacus, H. monstrosus, E. franqueti, and M. pusillus) had MARV-specific IgG (Table 1). ZEBOV seroprevalence rates were high in four species, while a high MARV seroprevalence rate was found in only one species (R. aegyptiacus). Together with the elevated antibody titers (at least 1:400) found in these bats, these finding indicate that these bats have frequent contacts with ZEBOV and MARV.

In an earlier study in DRC, members of three bat species were found to be seropositive for MARV (R. aegyptiacus and two members of the Microchiroptera group: Rhinolophus eloquens and Miniopterus inflatus) [14], possibly because they roosted in close proximity at the capture sites. In Gabon, no specimens belonging to these last two species were captured during our field studies. Surprisingly, ZEBOV-specific IgG was also frequent in R. aegyptiacus (8%). No specimens of this species were sampled between 2001 and 2003 in northern Gabon/Congo [11,15]. ZEBOV seropositivity in R. aegyptiacus bats likely reflects true viral infection, although passive antigenic stimulation cannot be ruled out. In Gabon, Megachiroptera members, including R. aegyptiacus, E. franqueti, M. torquata and H. monstrosus bats, consume the same fruits (especially Ficus spp). Bats of different species tend to visit the same trees, and interspecies virus transmission (or simple antigenic stimulation) could occur via infected saliva deposited on fruits. Hendra virus and Nipah virus transmission has been suggested to occur in this way between bats belonging to Asian Pteropus species [16].

Among IgG-positive R. aegyptiacus specimens, 21 (42%) were positive for MARV, 24 (48%) were positive for ZEBOV, and 6 (10%) were positive for both viruses. Although dual infection and/or antigenic stimulation could explain the concomitant presence of ZEBOV- and MARV-specific IgG in individual bats, serological cross-reactions between Ebola and Marburg antigens cannot be ruled out.

The ZEBOV seroprevalence rates were very similar (4% to 5%) in the four sampling regions (Figure 1). By contrast, the MARV seroprevalence rate was higher (about 2%) in the three regions where R. aegyptiacus was abundantly captured. Although some groups of R. aegyptiacus may occasionally roost outside in the trees, the bats of this species usually roost in natural or man-made cavities (caves, mines, tombs, etc.) and search for food at night. The daily flight distances are unknown. Among the 299 R. aegyptiacus specimens sampled in Gabon, the MARV-specific IgG prevalence rate was significantly higher in the animals captured in caves (14%) than in those captured elsewhere (4%) (Table 3). This difference could be due to a higher circulation of MARV between the bats living in the caves. Similarly, a high MARV-specific IgG prevalence rate (about 20%) has been reported in DRC in a cave-captured population of R. aegyptiacus [14]. Together, these findings point to the existence of two different ecological biotopes for Marburg and Ebola viruses: one linked to bats roosting in caves (Rousettus or Microchiroptera species) and the other linked to bats roosting in trees in the open forest. The ecology of H. monstrosus depends on water supplies, and these bats are known to migrate locally, especially along rivers [17]. Thus, the geographic distribution of ZEBOV in its reservoir species may be very broad, encompassing the forested areas of Africa, especially close to rivers, as previously reported [6,7]. In contrast, the distribution of Marburg virus would be determined by the ecology (horizontal and vertical distribution) of R. aegyptiacus bats and the presence of caves. Our serological results strongly suggest that ZEBOV and MARV overlap in all four regions we sampled in Gabon and RC.

The prevalence of ZEBOV-specific IgG fell from about 5% in 2003 and 8% in 2005 in Mbomo and 7% in Haut-Ogooué (during ZEBOV outbreaks) to 2% at both sites in 2006 (one year after the last outbreak), before rising again to 4% in Haut-Ogooué in 2008 (Table 2). Similar seasonal variations in the IgG prevalence of other viral pathogens (European Bat Lyssavirus-1, Hendra virus, etc.) have also been described in bats [18,19]. These fluctuations could be due to factors such as changes in bat numbers (due to mortality, migration, influx of young animals, etc.), immunological parameters (antibody persistence) and environmental changes (fructification, etc.). Long-term studies of seasonal variations in the ZEBOV-specific IgG prevalence in bat populations might help to understand the dynamics of human Ebola outbreaks.

The higher prevalence of ZEBOV-specific IgG in pregnant females (8%) than in non pregnant females (3%) (Table 5) suggests that EBOV transmission between males and females could occur by contact with infected saliva (bites, licking) or by mating. Pregnant female Pteropus scapulatus bats in Australia were recently shown to be more susceptible than their non pregnant counterparts to Hendra virus infection, and it has been suggested that Hendra virus transmission may occur by contact with the infected placenta [19].

We confirm the cocirculation of Ebola and Marburg viruses in Gabon. To date, Gabon is the only country where bat reservoirs of both viruses have also been identified. Of all the bats sampled in significant numbers in this survey, only specimens of Rousettus aegyptiacus were found to harbor both MARV- and ZEBOV-specific IgG, suggesting that this species may be a natural host of both viruses. The presence of MARV in Gabon, where no human Marburg hemorrhagic fever outbreaks have been reported, represents a potential and previously unrecognized threat to humans. As the MARV seroprevalence was significantly higher in R. aegyptiacus bats captured inside caves than elsewhere, caves and their surroundings should be considered as high-risk areas.

The authors declare that they have no competing interests.

Designed and performed the experiments: XP and EL. Analyzed the data: XP, EL, MS, PR, JT, SN and JPG. Statistical analysis MS. Contributed reagents/materials: PR, SN and JT. Wrote the paper: XP and EL. All authors read and approved the final version of the manuscript.

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