Since the first recognized Ebolavirus disease (EVD) outbreak in humans in 1976 in the Democratic Republic of Congo (DRC), a total of 31 outbreaks have now been recognized in Africa [1
]. The majority remained limited in the number of infected individuals and in the geographic spread, but the outbreaks in West Africa (December 2013 to March 2016) and in Eastern DRC (August 2018 to June 2020) clearly showed that they can also become epidemic and infect thousands of individuals over large geographic areas [4
]. In addition, the frequency of EVD outbreaks also increased over the last decades. With twelve outbreaks, DRC is one of the most affected countries. Since May 2018, the country has been continuously facing the circulation of the virus during four consecutive outbreaks [3
]. The largest EVD outbreak occurred in the eastern provinces of North Kivu and Ituri over almost two years (July 2018–June 2020). It infected 3470 people and 2299 (66.2%) patients died [6
]. The eleventh outbreak occurred in the Equateur province between June and November 2020, close to areas of the 2018 outbreak in the same province [2
]. On the 7th February 2021, a new resurgence has been declared in North Kivu, involving 11 cases to date [3
Except for a few recent outbreaks linked to a resurgence in humans [9
], EVD outbreaks are most likely the result of independent zoonotic transmission event(s) and bats are considered as reservoir species [11
]. Although there is no direct evidence of exposure to infected bats, two EVD outbreaks have been suspected to be linked to bats; i.e., in Luebo (DRC) in 2007 and the major outbreak in West Africa in 2013 [12
]. Other findings increase the likelihood of the role of bats in the filovirus ecology. For example, the detection of another filovirus, Marburg virus (MARV), in bats across Africa (Uganda, RDC, Kenya, South Africa, Gabon, Zambia, Sierra Leone) [15
]. The detection of other filoviruses in bats, such as Lloviu virus in Europe [26
], filoviruses in bats from China [28
] and the Philippines [30
] or Bombali virus (BOMV) in insectivorous bats in Africa provides additional evidence that filoviruses, including Ebola virus, have a Chiropteran origin [32
]. So far, MARV is the only pathogenic filovirus isolated from bats [18
], and Egyptian fruit bats (Rousettus aegyptiacus
), identified as the reservoir, seem to support virus replication with no apparent disease [35
Zaire Ebola virus (EBOV) RNA and antibodies were detected in three frugivorous bat species (Epomops franqueti
, Hypsignathus monstrosus
and Myonycteris torquata
) during EVD outbreaks in 2003 in Gabon and the Republic of Congo [13
]. Surveys of bats in West, Central and East Africa have revealed the presence of antibodies to Ebolaviruses in at least eight frugivorous and one insectivorous species (Mops condylurus
]. Only a few studies have been performed on bats and ebolaviruses in DRC [40
]. Our previous serological survey in the Western province of Bas Congo where no previous EVD outbreaks have been reported, investigated a total of 830 bats, including 428 frugivorous and 402 insectivorous bats, [40
]. We identified antibodies in E. helvum
but no viral RNA was detected.
Bats may represent a source of pathogen spillover into human populations in many African countries through their hunting and butchering for bushmeat or through indirect exposure to fruits contaminated by infected saliva, urine or faeces [44
]. Nevertheless, intermediate amplifying hosts may also play a major role in the emergence of EVD outbreaks, as illustrated in Gabon and Ivory Coast where apes have been confirmed as the source of infection in several outbreaks [11
]. Similar to humans, bats can also transmit their viruses to other mammals through contact with fruits contaminated with secretions or by hunting, for example, non-human primates (monkeys and bonobos) are reported to hunt bats [48
Today, the ecology of Ebolaviruses is still poorly understood and the role of bats in outbreaks needs to be further clarified. In order to increase the chances to detect viral RNA and Ebola antibodies, we investigated the presence of RNA from filoviruses and Ebola antibodies in bats from DRC collected during the EVD outbreaks in the Equateur and North Kivu provinces and in other areas that have already experienced outbreaks.
Although there is evidence for the involvement of bats in the ecology of Ebola viruses, their exact role is still not elucidated. Several studies reported presence of antibodies but only one study in Gabon identified viral RNA in addition to antibodies in a handful of animals from three species of fruit bats [13
]. To clarify the meaning of Ebola virus antibodies, it is essential to document the extent to which viral RNA and shedding can be detected in species with antibodies.
In order to increase our chances to identify viral RNA in bats, we focused our efforts on studying bats as early as possible during outbreaks and tested samples from 741 bats collected during two recent EVD outbreaks and 266 from two regions that have already experienced EVD outbreaks in DRC. Overall, samples were thus collected from 1007 bats, for 903 the species identification was confirmed by sequence analyses, swabs of 676 bats were tested by RT-PCR for the presence of viral RNA, and 925 bats were tested for the presence of antibodies to Ebolavirus. We also focused on frugivorous bats because previous studies on more than 8000 bats sampled in Africa, showed higher rates of Ebolavirus antibodies in frugivorous than in insectivorous bats. Today, antibodies to Zaire Ebolavirus have been detected in eight species of frugivorous bats (E. franqueti, H. monstrosus, M. torquata, E. helvum, E. gambianus, R. aegyptiacus, M. pusillus, L. angolensis)
and only in one genus (Mops
sp) of insectivorous bats [13
]. Unfortunately, we were unable to document the presence of viral RNA in oral and/or rectal swabs from the 604 frugivorous and 72 insectivorous bats sampled during two EVD outbreaks.
Our study includes 197 samples from the three species previously shown to harbor viral RNA, i.e.; Epomops franqueti
(n = 160), Myonycteris torquata
(n = 31) and Hypsignathus monstrosus
(n = 6). Compared to the study conducted during EVD outbreaks in 2003 in Gabon and the Republic of Congo, the total number of samples that we tested here is comparable for E. franqueti
(160 versus 117 in Gabon), but lower for H. monstrosus
(6 versus 21) and M. torquata
(31 versus 141) [13
]. Unlike studies in Gabon, where viral RNA was detected in 4.6% (13/279) of the samples from these three species, all of our 197 samples were negative. It is important to note that oral and rectal swabs were tested from bats that were released after sampling, while in the study from Gabon, organs (i.e., liver, spleen, kidney.) of euthanized bats were tested [13
]. It is most likely that viral loads are lower in swabs than in organs and influence the result of the PCR tests. So far, infectious ebolavirus has never been isolated from bats.
Ebolaviruses are most likely cleared from their hosts and can therefore only be detected for a limited period of time. Despite the fact that we collected samples during outbreaks, sampling started several weeks after the zoonotic transmission events to the index case, either directly from bats to humans or via an intermediate mammal host. It is therefore possible that the bats sampled in the study have already cleared their viruses. For example, experimental inoculation of R. aegyptiacus
bats with Zaire Ebolavirus showed antibody development but the detection of viral RNA or shedding was infrequent [36
]. In contrast, inoculation of the same species with the Marburg virus showed viremia in organs and oral and rectal shedding. The study showed also that MARV can be horizontally transmitted between bats through direct or indirect contact with infectious body fluids [35
]. This also suggests that the virus may be transmitted to other animals, including humans, by the same mechanisms.
It is also important to note that in the study conducted by Leroy and colleagues in Gabon, no single bat was simultaneously positive for antibodies and RT-PCR [13
]. This strengthens the case that active viral replication leading to PCR positivity is transient in infected bats and that virus is quickly cleared, with minimal, if any, overlap with seropositivity. Although, in the study of Forbes et al. on Mops
bats in Kenya, antibodies to EBOV VP40 antigen were detected in bats that tested positive by PCR for the presence of Bombali virus [52
]. Whether the rates of viral replication and infectivity differ between the different bat species in which antibodies to Ebola viruses have been identified needs to be further investigated.
The presence of Ebola antibodies confirms that bats are able to handle infection without serious signs of disease, which has already been experimentally documented, as well as for MARV [36
]. Despite their remarkable immunity, in the case of Ebolavirus, it is still questionable whether bats are the natural host or if they are maybe an intermediate host. It would thus be worth studying the bat biology more in detail and consider for example signs of the Ebolaviruses in their food or interaction with other susceptible mammals, as putative sources of the presence of ebolavirus in bats.
Our study focused on sampling sessions in areas during outbreaks or with previous outbreaks. We did not take into account the potential variation of virus infection and shedding over the seasons and reproduction period. For example, a study on MARV demonstrated pulses of virus circulation in juvenile Egyptian fruit bats correlating to breeding cycles. Juvenile bats are thus the predominant animals within a population that are susceptible to new infections, and significant increases in MARV infection have been demonstrated within this naïve group [19
]. In contrast, the majority of bats in our survey were adults (97.4%) potentially reducing the likelihood to detect virus.
Studying antibodies provides information on a previous acute infection. Using our previously defined positivity criteria, i.e., the simultaneous presence of antibodies against the GP and NP proteins, we only observe one bat (0.2%) out of the 575 frugivorous bats tested for antibodies in outbreak areas that were positive for Sudan Ebolavirus, while the Zaire Ebolavirus was responsible for the current outbreaks. The sample was collected in the North Kivu province. In our previous studies using the same serological assay, we showed that the extent of antibodies to Ebolaviruses varied among species. The highest rates were observed in E. helvum
, H. monstrosus
, M. pusillus
and R. aegyptiacus
. Except for M. pusillus
(n = 124), we only had few samples of these species in outbreak areas, 19 E. helvum
, 6 H. monstrosus
, and 5 R. aegyptiacus
, thus reducing the likelihood to identify positive samples. Nevertheless, we identified positive antibody samples in E. helvum
in our previous study in the western part of DRC and in other areas in Cameroon and Guinea where no EVD outbreak has been documented, illustrating that Ebolaviruses can circulate over a large geographic area across Africa between EVD outbreaks [40
]. Previous studies have also shown antibodies against the Sudan virus in several species of frugivorous bats [39
Although 12 of the 31 documented EVD outbreaks occurred in DRC, there are still a relatively limited number of studies reporting Ebola viruses in bats in the country. With this new study, more than 3000 samples from bats have now been studied in DRC at eight different areas with seven that are in locations with known outbreaks [40
]. In all studies, insectivorous bats were negative for antibodies or viral RNA when tested. Unfortunately, the early studies conducted after original outbreaks around Tandala and Yambuku in 1979 and 1980 and in Kikwit in 1995, only collected a small number of frugivorous bats; 26/426 and 123/539 and all were negative for antibodies and/or virus isolation [41
]. We report here the first study which tested, by molecular and serological assays, a large number of bats collected during ongoing EVD outbreaks in DRC. Today, only a few other studies have been conducted during outbreaks, i.e., one in Gabon and one in southeast Guinea, where the west African outbreak started [13
These studies in DRC and in other areas of Africa illustrate the difficulty of documenting the role of bats as a reservoir of Ebolaviruses and their role in the ecology of these viruses. The challenges relate to the difficulties of sampling bats in their natural environment, identifying viral RNA in a bat since it is very likely that bats can clear the virus and to the different assays used for antibody testing and to the different antibody detection tests and positivity criteria used. By analogy with observations in EVD survivors, we used the simultaneous presence of antibodies against NP and GP as a criterion of positivity in order to increase specificity as described previously [40
]. However, it cannot be excluded that this criterion is too strict, because the dynamics of antibodies directed against different antigens is not known in bats.
Future studies should probably focus on regular surveys of bat colonies suspected of being infected in order to increase the likelihood of detecting viral RNA. The longitudinal following may be even more relevant regarding the previously described seasonality of MARV, another filovirus [19
]. Given the increasing frequency of EVD outbreaks and their potential impact, studies on the ecology and animal reservoir of Ebolaviruses are now urgently needed.