Extensive Survey and Analysis of Factors Associated with Presence of Antibodies to Orthoebolaviruses in Bats from West and Central Africa.

The seroprevalence to orthoebolaviruses was studied in 9594 bats (5972 frugivorous and 3622 insectivorous) from Cameroon, the Democratic Republic of Congo (DRC) and Guinea, with a Luminex-based serological assay including recombinant antigens of four orthoebolavirus species. Seroprevalence is expressed as a range according to different cut-off calculations. Between 6.1% and 18.9% bat samples reacted with at least one orthoebolavirus antigen; the highest reactivity was seen with Glycoprotein (GP) antigens. Seroprevalence varied per species and was higher in frugivorous than insectivorous bats; 9.1-27.5% versus 1.3-4.6%, respectively. Seroprevalence in male (13.5%) and female (14.4%) bats was only slightly different and was higher in adults (14.9%) versus juveniles (9.4%) (p < 0.001). Moreover, seroprevalence was highest in subadults (45.4%) when compared to mature adults (19.2%), (p < 0.001). Our data suggest orthoebolavirus circulation is highest in young bats. More long-term studies are needed to identify birthing pulses for the different bat species in diverse geographic regions and to increase the chances of detecting viral RNA in order to document the genetic diversity of filoviruses in bats and their pathogenic potential for humans. Frugivorous bats seem more likely to be reservoirs of orthoebolaviruses, but the role of insectivorous bats has also to be further examined.

Seroprevalence of IgG Antibodies Against Multiple Arboviruses in Bats from Cameroon, Guinea, and the Democratic Republic of Congo.

Background: Emergence of mosquito-borne arboviruses has caused significant public health burden. The life cycle of arboviruses comprises sylvatic and urban cycles, including a wildlife reservoir, a human host, and an arthropod vector. However, many questions remain on the sylvatic cycles of arboviruses. In this study, we investigate the prevalence of IgG antibodies to arboviruses of public health importance in African bats. Material and Methods: We collected dried blood spots from bats in Cameroon, Guinea, and the Democratic Republic of the Congo (DRC). To detect IgG antibodies to 10 antigens of 6 arboviruses (Dengue, Zika, West Nile, Usutu, Chikungunya, and O'nyong nyong viruses), we adapted a previously validated multiplex detection assay based on the Luminex technology. Results: We tested samples from 2579 bats, representing 1917 frugivorous and 641 insectivorous bats distributed in 7 families and 21 species. Overall, 218/2579 (8.45%) bat samples reacted with at least 1 of the 10 antigens tested. The highest prevalence was observed against Usutu virus with 2.3% (59/2579), followed by 1.9% (49/2579) and 1.35% (35/2579) for the Dengue virus serotypes 4 and 3, respectively. The global seroprevalence varied by country and collection site: 11% (151/1376) in Cameroon, 3.5% (20/565) in DRC, and 7.3% (47/638) in Guinea. The highest rates were observed in Hypsignathus monstrosus (17.9%), Rousettus aegyptiacus (16.4%), and Eidolon helvum (10.7%), and in species from the insectivorous Molossidae family (7.8-8.9%). Finally, we observed changes in seroprevalence over the year in E. helvum and H. monstrosus colonies, which could be related to population structure. Conclusion: On more than 2500 bat samples tested, we showed variable IgG seroprevalences against multiple arboviruses. Overall, the prevalence of IgG antibodies of 8.45% against arboviruses found in bats suggest that they could play a role in arboviruses cycles in the wild, in addition to other animal species.

Investigating the Circulation of Ebola Viruses in Bats during the Ebola Virus Disease Outbreaks in the Equateur and North Kivu Provinces of the Democratic Republic of Congo from 2018.

With 12 of the 31 outbreaks, the Democratic Republic of Congo (DRC) is highly affected by Ebolavirus disease (EVD). To better understand the role of bats in the ecology of Ebola viruses, we conducted surveys in bats during two recent EVD outbreaks and in two areas with previous outbreaks. Dried blood spots were tested for antibodies to ebolaviruses and oral and rectal swabs were screened for the presence of filovirus using a broadly reactive semi-nested RT-PCR. Between 2018 and 2020, 892 (88.6%) frugivorous and 115 (11.4%) insectivorous bats were collected. Overall, 11/925 (1.2%) to 100/925 (10.8%) bats showed antibodies to at least one Ebolavirus antigen depending on the positivity criteria. Antibodies were detected in fruit bats from the four sites and from species previously documented to harbor Ebola antibodies or RNA. We tested for the first time a large number of bats during ongoing EVD outbreaks in DRC, but no viral RNA was detected in the 676 sampled bats. Our study illustrates the difficulty to document the role of bats as a source of Ebolaviruses as they might clear quickly the virus. Given the increasing frequency of EVD outbreaks, more studies on the animal reservoir are urgently needed.

Multiplex detection of antibodies to Chikungunya, O'nyong-nyong, Zika, Dengue, West Nile and Usutu viruses in diverse non-human primate species from Cameroon and the Democratic Republic of Congo.

BACKGROUND: Epidemic arbovirus transmission occurs among humans by mosquito bites and the sylvatic transmission cycles involving non-human primates (NHPs) still exists. However, limited data are available on the extent in NHPs infections and their role. In this study, we have developed and validated a high-throughput serological screening tool to study the circulation of multiple arboviruses that represent a significant threat to human health, in NHPs in Central Africa. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins NS1, envelope domain-3 (DIII) for the dengue (DENV), yellow fever (YFV), usutu (USUV), west nile (WNV) and zika (ZIKV) and envelope 2 for the chikungunya (CHIKV) and o'nyong-nyong (ONNV) were coupled to Luminex beads to detect IgG directed against these viruses. Evaluation of test performance was made using 161 human sera of known arboviral status (66 negative and 95 positive). The sensitivity and specificity of each antigen were determined by statistical methods and ROC curves (except for ONNV and USUV). All NS1 antigens (except NS1-YFV), CHIKV-E2 and WNV-DIII had sensitivities and specificities > 95%. For the other DIII antigens, the sensitivity was low, limiting the interest of their use for seroprevalence studies. Few simultaneous reactions were observed between the CHIKV+ samples and the NS1 antigens to the non-CHIKV arboviruses. On the other hand, the DENV+ samples crossed-reacted with NS1 of all the DENV serotypes (1 to 4), as well as with ZIKV, USUV and to a lesser extent with YFV. A total of 3,518 samples of 29 species of NHPs from Cameroon and the Democratic Republic of Congo (DRC) were tested against NS1 (except YFV), E2 (CHIKV/ONNV) and DIII (WNV) antigens. In monkeys (n = 2,100), the global prevalence varied between 2 and 5% for the ten antigens tested. When we stratified by monkey's biotope, the arboreal species showed the highest reactivity. In monkeys from Cameroon, the highest IgG prevalence were observed against ONNV-E2 and DENV2-NS1 with 3.95% and 3.40% respectively and in DRC, ONNV-E2 (6.63%) and WNV-NS1 (4.42%). Overall prevalence was low in apes (n = 1,418): ranging from 0% for USUV-NS1 to 2.6% for CHIKV-E2. However, a very large disparity was observed among collection site and ape species, e.g. 18% (9/40) and 8.2% (4/49) of gorillas were reactive with CHIKV-E2 or WNV-NS1, respectively in two different sites in Cameroon. CONCLUSIONS/SIGNIFICANCE: We have developed a serological assay based on Luminex technology, with high specificity and sensitivity for simultaneous detection of antibodies to 10 antigens from 6 different arboviruses. This is the first study that evaluated on a large scale the presence of antibodies to arboviruses in NHPs to evaluate their role in sylvatic cycles. The overall low prevalence (<5%) in more than 3,500 NHPs samples from Cameroon and the DRC does not allow us to affirm that NHP are reservoirs, but rather, intermediate hosts of these viruses.

Extensive Serological Survey of Multiple African Nonhuman Primate Species Reveals Low Prevalence of Immunoglobulin G Antibodies to 4 Ebola Virus Species.

Bats are considered a reservoir species for Ebola viruses, but nonhuman primates (NHPs) have represented a source of infection in several outbreaks in humans. Here we report serological screening of blood or fecal samples from monkeys (n = 2322) and apes (n = 2327). Thirty-six NHP species from Cameroon, Democratic Republic of the Congo, and Ivory Coast were tested with a sensitive and specific Luminex-based assay for immunoglobulin G antibodies to 4 Ebola virus species. Using the simultaneous presence of antibodies to nucleoproteins and glycoproteins to define positivity, we showed that specific Ebola virus antibodies are not widespread among NHPs. Only 1 mustached monkey (Cercopithecus cephus) from Cameroon was positive for Sudan ebolavirus. These observations support that NHPs are most likely intermediate hosts for Ebola viruses. With the increasing frequency of Ebola outbreaks, it is crucial to identify the animal reservoir and understand the ecology of Ebola viruses to inform disease control.

Survey of Ebola Viruses in Frugivorous and Insectivorous Bats in Guinea, Cameroon, and the Democratic Republic of the Congo, 2015-2017.

To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015-2017 from countries in Africa that have had previous Ebola outbreaks (Guinea, the Democratic Republic of the Congo) or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from >12 frugivorous and 27 insectivorous species; 2-37 (0.05%-0.92%) bats were seropositive for Zaire and 0-30 (0%-0.75%) bats for Sudan Ebola viruses. We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.