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

Raisa Raulino, Guillaume Thaurignac, Alpha Keita, Amandine Esteban, Souana Goumou, Ramadan Diallo, Placide Mbala Kingebeni, Abdoulaye Toure, Eric Delaporte, Steve Ahuka-Mundeke, et al.

► To cite this version:

Raisa Raulino, Guillaume Thaurignac, Alpha Keita, Amandine Esteban, Souana Goumou, et al. "Seroprevalence of IgG Antibodies Against Multiple Arboviruses in Bats from Cameroon, Guinea, and the Democratic Republic of Congo." Vector Borne and Zoonotic Diseases, 2022, 22 (4), pp.252-262. 10.1089/vbz.2021.0076. hal-05313404

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1 Seroprevalence of IgG antibodies against multiple arboviruses in bats from Cameroon,

2 Guinea, and the Democratic Republic of Congo

3

4

5 Raisa Raulino¹, Guillaume Thaurignac¹, Alpha K. Keita¹,², Amandine Esteban¹, Souana

6 Goumou², Ramadan Diallo³, Simon-Pierre Ndimbo-Kumugo⁴,⁵, Innocent Ndong Bass⁶, Placide

7 Mbala Kingebeni⁴,⁵, Abdoulaye Toure²,⁷,⁸, Eric Delaporte¹, Steve Ahuka-Mundeke⁴,⁵, Jean-

8 Jacques Muyembe Tamfum ⁴,⁵, Eitel Mpoudi-Ngole⁶#, Martine Peeters¹*, Ahidjo Ayouba¹*

9

10 ¹ TransVIHMI ; University of Montpellier, Institut de Recherche pour le Développement,
11 INSERM, Montpellier, France

12 ² Centre de Recherche et de Formation en Infectiologie de Guinée (CERFIG), Conakry,
13 Guinea.

14 ³ Laboratoire de Virologie, Ministère de l'Elevage et des Productions Animales, Republic of
15 Guinea

16 ⁴ National Institute of Biomedical Research (INRB), Kinshasa, Democratic Republic of the
17 Congo

18 ⁵ Service de Microbiologie, Cliniques Universitaires de Kinshasa, Kinshasa, Democratic
19 Republic of Congo.

20 ⁶ Laboratoire de Virologie, Institut de Recherches Médicales et d'Études des Plantes
21 Médicinales (IMPM), Yaoundé, Cameroon-IRD, Yaounde, Cameroon.

22 ⁷ Université de Conakry, Conakry, Guinée

23 ⁸ Institut National de Sante Publique (INSP), Conakry, Guinée

24 #: Deceased

25 * : Co-supervised the work
1 **Running Title:** Antibodies to arboviruses in African bats.

2

3 **Authors email addresses:**

4 Raisa Raulino: raisa.raulino@ird.fr; ORCID:0000-0001-9006-3483

5 Guillaume Thaurignac: guillaume.thaurignac@ird.fr

6 Alpha K. Keita: alpha-kabinet.keita@ird.fr

7 Amandine Esteban: amandine.esteban@ird.fr

8 Souana Goumou: souana.goumou@cerfig.org

9 Ramadan Diallo: dialloramadan1954@gmail.com

10 Simon-Pierre Ndimbo-Kumugo: simonp_ndimbok@yahoo.fr

11 Innocent Ndong Bass: ssabgnodn@yahoo.com

12 Placide Mbala Kingebeni: placidembala@inrb.net

13 Abdoulaye Toure: abdoulaye.toure@ird.fr

14 Eric Delaporte: eric.delaporte@ird.fr

15 Steve Ahuka-Mundeke: amstev04@yahoo.fr

16 Jean-Jacques Muyembe Tamfum: jjmuyembet@gmail.com

17 Eitel Mpoudi-Ngole: empoudi2001@yahoo.co.uk

18 Martine Peeters: martine.peeters@ird.fr

19 Ahidjo Ayouba: ahidjo.ayouba@ird.fr; ORCID: 0000-0002-5081-1632
# 1 ABSTRACT

# 3 Background

4 Emergence of mosquito-borne arboviruses have caused significant public health burden. The
5 life cycle of arboviruses comprises sylvatic and urban cycles, including a wildlife reservoir, a
6 human host and an arthropod vector. However, many questions remain on the sylvatic cycles
7 of arboviruses. Here, we investigate the prevalence of IgG antibodies to arboviruses of public
8 health importance in African bats.

# 9 Material and methods

10 We collected dried blood spots from bats in Cameroon, Guinea and the Democratic Republic
11 of the Congo (DRC). To detect IgG antibodies to ten antigens of six arboviruses (Dengue, Zika,
12 West Nile, Usutu, Chikungunya and O'nyong nyong viruses) we adapted a previously validated
13 multiplex detection assay based on the Luminex technology.

# 14 Results

15 We tested samples from 2,579 bats, representing 1,917 frugivorous and 641 insectivorous bats
16 distributed in seven families and 21 species. Overall, 218/2579 (8.45%) bat samples reacted
17 with at least one of the ten antigens tested. The highest prevalence was observed against Usutu
18 virus with 2.3% (59/2579), followed by 1.9% (49/2579) and 1.35% (35/2579) for the Dengue
19 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 Democratic Republic of the
20 Congo and 7.3% (47/638) in Guinea. The highest rates were observed in *Hypsignathus
21 monstrosus* (17.9%), *Rousettus aegyptiacus* (16.4%) and *Eidolon helvum* (10.7%) and in
22 species from the insectivorous Molossidae family (7.8%-8.9%). Finally, we observed changes
23 in seroprevalence over the year in *E. helvum* and *H. monstrosus* colonies which could be related
24 to population structure.
25
## 1 Conclusion

On more than 2,500 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.

Keywords: arboviruses, bats, Africa, antibodies, Luminex.
# 1 INTRODUCTION

The number and impact of emerging infectious diseases (EIDs) has significantly increased over time. The large majority of EIDs have a zoonotic origin, and more than 70% originate from wildlife (Jones et al., 2008). Among them, mosquito-borne arboviruses constitute a significant threat to public health in worldwide. Arboviruses are predominantly RNA viruses and approximately one third have pathogenic potential for humans, such as Dengue, Zika, West Nile, Usutu, Chikungunya, O'nyong-nyong and many other viruses (Guzmán et al., 2020). The life cycle of arboviruses comprises in general a sylvatic or enzootic cycle and a urban cycle, that include a wildlife reservoir, a human host and an arthropod vector in a complex interplay. Many arboviruses causing epidemics today have their origin in Africa or were first identified in Africa; i.e. Yellow Fever, West Nile, Zika, Chikungunya and Usutu viruses (Shope, 1994, Dick, 1953, Mason and Haddow, 1957). For many of them, like Zika, Dengue, or Chikungunya, non-human primates (NHPs) were considered to play a major role in sylvatic cycles, but this was based on limited data. We recently showed that the observed seroprevalence for arboviruses on a large number of NHPs in Africa was not sufficient for them to be considered as the main reservoir species. Thus, other species most likely also play a role to maintain these viruses in the sylvatic cycles in Africa (Valentine et al., 2019, Raulino et al., 2021).

Bats are known to harbor many viruses and arboviruses have also been detected in bats (Luis et al., 2013, Melaun et al., 2014, Fagre and Kading, 2019, Ayhan et al., 2021, Calderon A, 2016). Bats represent 20% of all mammalian species, with more than 1400 species worldwide, have high population densities and roosting behavior that favor the likelihood of transmission of pathogens (Omatsu et al., 2007). The majority of studies reporting on arboviruses in bats are from southern America (Hernández-Aguilar et al., 2021, Thompson et
al., 2015, Ubico and McLean, 1995). Very few studies have been conducted in Africa, the majority were done before 1970 and reported antibodies to Zika virus or Yellow Fever in several bat species in east Africa (Williams et al., 1964, Simpson et al., 1968).

The limited data available on this important question is probably due to lack of assays for bat's immune typing, the difficulty of arbovirus serology because of antigenic proximity among different arboviral lineages and limited sample volumes that can be drawn from bats. Recently, our group has developed a high throughput serological tool to detect IgG antibodies to different arboviruses of global interest that circumvent some of the above mentioned difficulties (Raulino et al., 2021). In the present report, we adapted this serological tool for the detection of IgG antibodies to six arboviruses (Chikungunya, O'nyong nyong, Dengue, Zika, West Nile and Usutu viruses) and tested bat samples collected in Cameroon, Guinea and DRC. Our data showed that seroprevalence varied by country, by collection site, by bat families and species, and ranged from less to 1% to up to 20%. These findings suggest that bats could play a role in sylvatic life cycles of some arboviruses.
# 1 MATERIAL AND METHODS

## 2 Bat sampling and sites

Samples from free-ranging frugivorous and insectivorous bats were collected between 2016 and 2019 from in Cameroon, DRC and Guinea in the framework of a study on Ebola and other viruses in bats. Bats were captured using mist nets or harp traps in roosting and foraging sites and immediately released after sampling as previously described (De Nys et al., 2018). Dried blood spots (DBS) were prepared on Whatman 903 filter paper (GE Healthcare, Feasterville-Trevose, PA, USA) from whole blood samples collected by venipuncture of the propatagial or brachial vein. DBS samples were preserved as described in De Nys, et al. 2018, stored in the filed at ambient temperature and then at -20°C. For each sample, information on capture site (GPS coordinates (Supplementary Table S5), ecological environment), capture method, morphology (body measurements, weight, color), sex, age class (adult, juvenile), reproduction cycle and visual species identification were recorded in the field. Permissions to perform this research were obtained from administrative authorities of Cameroon, Guinea and the DRC.

## 16 Detection of antibodies to arboviruses

Samples were tested with our previously developed assay based on the Luminex technology with sensitivity and specificity and accuracy > 95% on a reference panel of well documented human samples (Raulino et al., 2021). We used 10 commercially available recombinant proteins from six arboviruses: CHIKV-E2 (Chikungunya virus Envelope protein 2), ONNV-E2 (O'nyong nyong virus envelope protein 2), DENV-NS1 (Dengue virus-Nonstructural protein 1) (1 to 4 serotypes), USUV-NS1 (Usutu virus-Non Structural protein 1), WNV-NS1 (West Nile virus Non Structural protein 1), WNV-DIII (West Nile virus Domain-3 protein) and ZIKV-NS1 (Zika virus Non-Structural protein 1)(Raulino et al. 2021). The assay was performed as previously described and optimized for bats using goat anti-bat biotin-labeled
IgG (Euromedex, France) and Streptavidin-R- phycoerythrin (Fisher Scientific/Life Technologies, Illkirch, France) (Raulino et al., 2021). Whole blood was reconstituted from DBS as previously described and a final plasma dilution 1/200 was used (Ayouba et al., 2019). The results were expressed as median fluorescence intensity (MFI) per 50 beads. Each plate included a blank well, positive samples for Chikungunya, Dengue, Zika and West Nile viruses and also 2 negative samples to validate inter-assay repeatability.

## Calculation of cut-off values for the different antigens

In the absence of positive and negative control samples from bats, we analyzed our data with different statistical methods to determine MFI *cut-off* values for each antigen as previously described (Ayouba et al., 2019, Raulino et al., 2021, Peel et al., 2013, Gilbert et al., 2013, De Nys et al., 2018). We used a change-point analysis with the R package "changepoint" and calculated one single shift in the arithmetic mean with the AMOC (at most one change) method (Lardeux et al., 2016, Killick and Eckley, 2014). We also used fitted univariate distributions (binomial and negative exponential distributions) to our data and defined the cut-off based on a 0.001 risk of error. Data were bootstrapped 10,000 times and averaged for each antigen. Analyses were done with R software version 3.3.6. We then compared the *cut-off* values identified by the 3 different methods and calculated their mean as a consensus *cut-off* that we used in this study (Supplementary Table S1). A sample was considered reactive if MFI was above the *cut-off* value.

## Molecular confirmation of bat species

Species identification recorded in the field was molecularly confirmed on a subset of samples by sequence analysis of an 800 bp fragment of the mitochondrial cytochrome b (CytB) like in our previous studies (De Nys et al., 2018, Lacroix et al., 2020). Sequences were submitted to
1 NCBI for BLAST analysis to identify the most similar bat species. For samples with none or

2 low similarity (<97%) hits with species in Genbank, phylogenetic tree analysis was done using

3 maximum likelihood methods implemented in PhyML (Guindon and Gascuel, 2003) with

4 reference sequences in order to approach genus identification. To discriminate between

5 *Epomophorus gambiensis* and *Micropteropus pusillus*, we used also morphologic details on

6 forearm and weight measurements because species identification was not possible based on

7 CytB sequences (Nesi et al., 2011). Species identification was extrapolated by per capture

8 method, GPS coordinate and morphological descriptions for the other samples.

9

10
# 1 RESULTS

## 2 Distribution and diversity of bat species

We tested a total of 2,579 bats captured in 21 different sites in Guinea (n=8), Cameroon (n=4) and DRC (n=9) (Figure 1). The samples were from bats living in urban areas (52%), villages (26%), forest (10.5%) and caves (10.9%). For 2,313 (89.6%) samples, the species identification in the field was confirmed by sequence analysis, however it was not possible to clearly discriminate between certain *Chaerophon* and *Mops* species from the *Molossidae* family based on CytB sequences and morphological data. We tested at least 12 different species of frugivorous bats, and six different families of insectivorous bats. Details on the different bat species that were collected in each country are shown in Table 1.

Overall, 1193 bats were male, 1381 female and for five data were not available. Among 11 females, 77% were adults, 8.0% were reproductively immature and 14% were juveniles. For 12 males, 70%, 12% and 17% were adults, reproductively immature and juveniles respectively. For the remaining samples information on age and sex were not available.

## 16 Evidence of antibodies to Alphaviruses and Flaviviruses in bats

Antibodies to alphavirus (Chikungunya and O'nyong-nyong) and flaviviruses (Dengue 1 to 4, Usutu, West Nile and Zika) tested in this study were observed in *Eidolon helvum*, *Epomophorus gambianus*, *Epomops sp*, *Hypsignathus monstrosus*, *Lissonycteris angolensis*, *Micropteropus pusillus*, *Myonycteris torquarta*, *Rousettus aegyptiacus*, *Hipposideros sp*, *Miniopterus sp*, *Chaerephon sp*, *Mops sp* and *Rhinolophus sp* bats from the three countries (Figure 1). Overall, 8.45% (218/2,579) of the samples reacted with at least one arbovirus antigen (Table 2). Globally, 2.3% of the bats had antibodies to Usutu virus, 0.85% to 1.35% to serotypes 1 to 4 of Dengue virus and the lowest proportions were observed for Zika virus (0.54%). Global rates and stratified per virus varied among countries and within countries. In
Cameroon, 11% (151/1,376) of bat samples had antibodies to arboviruses, ranging between 0.58% for Zika virus to 4.0% for Usutu virus. In Guinea, we also observed antibodies to all viruses with a total seroprevalence of 7.3% (47/638) and a predominance to O'nyong-nyong (2.35%). In DRC, 3.5% (20/565) of the bat samples tested had antibodies with a predominance to antigen NS1 of West Nile virus, and in contrast to the two other countries, no antibodies to Chikungunya virus were observed.

Antibodies to at least one arbovirus were observed in 14 (70%) of the 21 species tested, i.e. in all species with sufficient samples (>20) (Table 3). The highest rates were observed among the following frugivorous bat species, *Hypsignathus monstrosus* (17.9%), *Rousettus aegyptiacus* (16.4%) and *Eidolon helvum* (10.7%) and in species from the insectivorous *Molossidae* family, *Mops* sp (8.9%) and *Mops/Chaerophon* sp (7.8%). *Eidolon helvum* is a common species, which showed IgG antibodies against all viruses tested, but with different positivity rates for each virus in different sites tested, which is probably related to differences in the numbers of samples tested. *Hypsignathus monstrosus* is also a common species, but large numbers were only tested in Cameroon, and antibodies were observed against all viruses tested with 10% of samples reactive to Usutu virus. In *Rousettus aegyptiacus*, that were highly represented in the sample set from Guinea, antibodies against almost all viruses were detected reaching 8.9% for O'nyong-nyong virus. Also, in bats from the Molossidae family, antibodies against almost all alphaviruses and flaviviruses were observed with 7.4% of samples reactive against to serotype 4 of Dengue in Cameroon. In other species, for example *Micropteropus pusillus*, IgG antibodies were only observed against flavivirus, and in *Myonycteris torquata* only IgG antibodies to alphavirus were detected (Table 3). Overall, antibodies were observed in 10% (192/1917) of frugivorous bats, versus 3.9% (25/641) in insectivorous bats. Frugivorous bats had antibodies to all viruses tested, and in insectivorous bats no antibodies were observed against the Usutu and to antigen NS1 of West Nile virus (Table 3). Only for the serotype 2 of
1 Dengue virus, the difference between the positivity in frugivorous and insectivorous was
2 statistically significant (p=0.028; OR:4.5626; CI 95%: 1.41-39.7).

3 Ten percent of frugivorous and insectivorous bats captured in urban areas had antibodies
4 to arboviruses versus 6.6% in villages. *Eidolon helvum* bats were captured in cities, villages
5 and forests with the following positivity rates, 10.7%, 18% and 9.8% respectively. Similar rates
6 were observed for *Hypsignathus monstrosus* captured in urban areas (18.5%) and villages
7 (16.6%). *Rousettus aegyptiacus* bats were also positive in all settings with rates between 11.5%
8 to 30%. Most insectivorous bats were captured in more remote areas and showed lower rates,
9 except for species of the Molossidae family, with 7.7% of *Chaerephon/Mops*, that were
10 captured in villages having antibodies to arboviruses (**Table 4**). The difference in positivity
11 rates of antibodies to arboviruses between frugivorous and insectivorous might reflect the
12 different biotopes. **Supplementary Table S2** shows in detail the reactivity with the different
13 viruses according to the ecological settings independently from the species. Overall, antibodies
14 to almost all viruses studied are observed in all settings, although at different rates with a trend
15 of lower rates in caves as compared to the three other settings.

16

17 *Antibodies in E. helvum and H. monstrosus according to seasons, age and reproductive cycle*

18 The 815 *Eidolon helvum* and 227 of the 249 *Hypsignathus monstrosus* samples in
19 Cameroon, were part of colonies roosting in Yaoundé and foraging in neighboring areas.
20 Samples were collected on a monthly basis between December 2018 and November 2019. For
21 *E. helvum*, we tested 221 juvenile, 218 reproductively immature and 378 adult bats, and
22 comparable seroprevalence rates were observed in all age categories ranging from 10.1% to
23 11.1%. For *H. monstrosus*, differences according to age category were observed, with highest
24 proportion of positives among adults (31/132, 23.5%), 10/72 (13.9%) of juveniles and lowest
25 proportions in reproductively immature (1/22; 4.6%) (Figure 2). Seropositivity rates were also
significantly higher in adult *H. monstrosus* as compared to adult *E. helvum* bats, 23.5% versus 11.1% respectively (p-value <0.0001) (Supplementary Table S3). We also examined whether antibody status can vary in females during gestation and lactation. Among the female *H. monstrosus* bats, 2/11 (18.1%) of the gestating bats were positive versus 28.5% (18/63) of the other adult female bats, and for *E. helvum* the opposite trend was observed with 18.1% (2/11) gestating bats positive versus 9.3% (15/160) for the other adult female bats. The differences observed were not significant statistically. Similarly, there were also differences between female adults during lactation period, as shown in **Figure 2**, but they were also not statistically significant. Taken together, the data from the *E. helvum* and *H. monstrosus* bat colonies showed fluctuations over time with a minimum rate in June (4.26%) and maximum in February (34.78%) (Supplementary Table S4), however, the number of samples are low for certain months and the population structure at a given period could influence positivity rates as shown above.

14

15 *Simultaneous reaction to multiple alphavirus and flavivirus in bats*

We observed simultaneous reactivity to antigenically-related viruses, but also to genetically diverse and less antigenically related viruses (Table 5). The highest simultaneous reactivity was observed for NS1 antigens of the four Dengue virus serotypes, reaching 68% between the serotypes 1 and 4 of Dengue virus. The results showed also that 30% of the E2 of Chikungunya virus positive samples reacted with E2 of O'nyong-nyong virus, and 5% with NS1 of Usutu virus and to the serotype 3 of Dengue virus. Among the 31 positive samples of O'nyong-nyong virus, 6 (19%) had simultaneous reactions with the E2 of Chikungunya virus. For the other antigens, simultaneous reactions with other antigens were in general below 15%.

16

17

18

19

20

21

22

23

24

25
# 1 DISCUSSION

The objective of this study was to estimate the prevalence of antibodies to arboviruses of public health importance in a large number of bats in Central and West Africa using the same serological assay. We tested 2,579 samples from at least 21 bat species, representing to our knowledge one of the most comprehensive report on arboviruses in bats in Africa. Our data showed spatial and temporal variation of IgG antibodies to diverse arboviruses. We observed an overall seroprevalence to arboviruses of 8.45%. This is lower than what was reported from (the few) previous seroprevalence studies in African bats, but different serological methods were used (i.e. agglutination versus Luminex EIA), the geographical location and the study period can also have an impact (Shepherd and Williams, 1964).

Bats are flying mammals that nightly fly up to 20 km for foraging and up to hundreds or thousands of kilometers during seasonal migrations for some species (Schloesing et al., 2020, Roberts et al., 2012). Comparing prevalence in different collection sites is thus only meaningful if they are sufficiently distant. Here, almost all our study sites are 20-300 km apart (see Figure 1). IgG antibodies to at least one arbovirus antigen were detected in 17 of the 21 sites investigated and, in the four sites with no positive samples, the sample size was low (i.e. < 50). Furthermore, the positivity rate profile for antibodies to arboviruses was statistically different according to the ecological settings (see Supplementary Table S2). These difference could be related to bat species, environment, human factors or a combination of those. For example, in Yaoundé and Obala where most bat samples studied were collected are urbanized areas, that could favored a vector population to interact between bats and humans in the same environment, thus contributing to sustain the transmission cycle of arboviruses.

Similar to other studies, we found variable prevalence by bat species, most likely corresponding to different ecology and exposure to arthropod vectors (Shepherd and Williams, 1964, Kading et al., 2018, Hernández-Aguilar et al., 2021). We observed the highest
1 prevalences in three frugivorous bat species; *Eidolon helvum*, *Hypsignatus monstrosus* and
2 *Rousettus aegytiacus*. *E. helvum* is widespread in equatorial Africa
3 (https://animaldiversity.org/accounts/Eidolon_helvum/; accessed July, 23rd, 2021), and is
4 roosting in both urban and rural areas representing thus a good intermediate between sylvatic
5 and urban cycles of arboviruses. Combined entomological and bat ecology studies are therefore
6 urgently needed for a better understanding of the role of Chiroptera in the lifecycle of
7 arboviruses in Africa.

8 Contrasting with the recurrent outbreaks of Chikungunya virus in DRC (Selhorst et al.,
9 2020), we did not observe IgG antibodies to Chikungunya virus in bat samples from this
10 country, which might be due to the overall limited number of bat samples tested in this large
11 country. Nevertheless, antibodies to Chikungunya virus have been readily detected in free-
12 raging NHPs in DRC, but in different areas as where we tested bats in this study (Raulino et
13 al., 2021). It is also possible that bats are not equally exposed to the same array of mosquito
14 vectors than NHPs that are involved in the sylvatic cycles of arboviruses as suggested by others
15 (Simo et al., 2019, Guzmán et al., 2020, Selhorst et al., 2020, Pastorino et al., 2004).

16 Usutu virus has been isolated from mosquitoes, birds and some mammals in Europe and
17 in some countries in Africa such as Senegal, Central African Republic, South Africa and
18 Uganda but data on Usutu virus in Africa are still very limited (Diagne et al., 2019, Clé et al.,
19 2019, Nikolay et al., 2011). Here, we observed antibodies against to Usutu virus, indicative of
20 the circulation of this virus in at least three African fruit bat species (see Table 3). To the best
21 of our knowledge, this is the first evidence of Usutu virus circulation in bats from Africa,
22 suggesting that they might contribute to the enzootic cycle of Usutu virus in Africa but
23 additional investigations to clarify the roles of bats in the viral cycle in Africa is needed.

24 The 12-month longitudinal follow-up of bat colonies in the same geographical location,
25 allowed to study impact of age, reproduction cycle and eventual seasonality. We observed
1 higher prevalence in adults than in immature or juvenile adults for *H. monstrosus* species, but
2 not for *E. helvum*, suggesting a possible difference in behavior and ecology and subsequent
3 exposure to vectors. During the female reproductive cycles, i.e. gestation or lactation, no
4 statistically significant difference was observed for both species.

5 Nevertheless, our work has some limitations, first of all the lack of confirmation of some
6 of the seroreactivity by affinity-based virus neutralization assay, affinity measurement assays
7 which was hampered by the limited volumes of blood samples, i.e. only a few drops of blood
8 on filter paper. Another limitation is the absence of sequence confirmation of circulation of
9 arboviruses that induced IgG antibodies detected during this work. Arbovirus RNA can only be
10 detected during a limited period during acute infection, therefore prospective studies are needed
11 that should include blood samples in adequate storage conditions for molecular analysis.

12

13 **Conclusion**

14 Overall, the presence of antibodies suggests that bats could play a role in the sylvatic cycle of
15 arboviruses in addition to non-human primates and other mammals or animal species.
16 Population size and the number of susceptible NHP at any time, are most likely not high enough
17 to maintain sylvatic arbovirus cycles and therefore other species like bats with large numbers
18 of offspring, i.e. naïve animals at any time, could thus also play also a role in sylvatic cycles.
19 Moreover, several bat species live close to human settlements and create thus opportunities for
20 contacts via an arthropod vector. More studies are still needed to document diversity of animal
21 species susceptible to arboviruses and their role in sylvatic cycles and emergence of new viruses
22 in humans.

23

24 **Acknowledgements**
1 We thank the staff and SIV team from Projet PRESICA (Innocent Ndong Bass, Aime Mebenga,

2 Joseph Moudindo, Thomas Atemkem and Flaubert Mba Djondzo) for the collection of samples

3 and logistical support in Cameroon. In addition, we thank the field staff from DRC (Mubonga

4 Mukulumanya, Lunguya-Metila Octavie, Guy Midingi and Mbenzo-Abokome Valentin).

5 **Authorship Confirmation Statement**

6 All authors participated to the work presented here and all approved the final version of the
7 manuscript.

8 - Conceived the study: AA, MP.

9 - Collected samples: SPNK, INB, RD, SG, AKK.

10 - Performed experiments: RR, GT, AE.

11 - Analyzed the data: RR, GT, AA MP.

12 - Drafted the manuscript: RR, AA, MP.

13 - Get funding: AA, MP.

14 - Reviewed preliminary and final versions of the manuscript: All the authors

15

16 **Author Disclosure Statement**

17 - All the authors declared no conflicting financial interests.

18

19 **Funding information**

20 RR received the PhD grant from INSERM and MUSE. A.K.K. was supported by fellowships

21 from IRD and the University of Montpellier (MUSE, ANR-16-IDEX-0006). This work was

22 supported by EBO-SURSY project funded by the European Union (FOOD/2016/379-660, by

23 **Research Fund Flanders (FWO, grant number G054820N)**, and by a grant from Montpellier

24 University of Excellence (MUSE) (AbroSud project). The funders had no role in study design,

25 data collection and analysis, decision to publish, or preparation of the manuscript.
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10

11
1 **Author for Correspondence**

2 Dr Ahidjo Ayouba

3 IRD-TransVIHMI

4 911, avenue Agropolis

5 34394 Montpellier cedex 5

6 France

7 Email: Ahidjo.ayouba@ird.fr

8 Phone:+33467416305
<figure><img src="image_2.png" /><figcaption>Figure 1: Bat collection sites and presence of arboviruses in Cameroon, Guinea and DRC.</figcaption></figure>

The maps represent Africa and the 3 countries (Guinea, top; Cameroon, center; and the DRC, bottom) where studied samples have been collected. Grey dots indicate sampling sites where no antibodies to arboviruses were detected; red dots, sites where antibodies to alphaviruses were detected; blue dots, sites where antibodies to flaviviruses were detected; red dots with blue circles, sites were antibodies to flavi- and alpha viruses were detected. Site abbreviations: Cameroon - Village: CP/Campo; MB/Mambele, Urban area: OB/Obala ; YD/Yaoundé. DRC - Caves: BM/Boma; KP/Kimpse; ZG/Zongo, Forest: ZG/Zongo; BD/Boende, Urban area: BN/Beni; BK/Bikoro; BM/Boma; KP/Kimpse; KM/Komanda; MG/Mangina, Village: BD/Boende; KI/Kikiwit. Guinea - Caves: KK/Kankan; KN/Kindia; MC/Macenta; MM/Mamou; SI/Siguiri, Forest: MC/Macenta, Urban area: KN/Kindia; MM/Mamou; Villages: BF/Boffa; KK/Kankan; KN/Kindia; KD/Koundara; LL/Lola; MC/Macenta; MM/Mamou; SI/Siguiri.
<figure><img src="image_3.png" /><figcaption>Figure 2: Percentage of positive samples in colonie of bats, according to age and reproductive cycle. The figure represents the proportions of positive samples in two species (H.monstrosus and E.helvum) of fruit bats followed-up over a year in Yaoundé, Cameroon. Panels A and B (top) represent the proportions of positive samples at three categories of age. Panels C and D (bottom) compare the proportion of positive samples in the same two bat species, stratified by female reproduction/lactation status. (ns) stands for non-significant statistically (p-value above 0.05).</figcaption></figure>

383x244mm (72 x 72 DPI)
<table><caption>**Table 1:** Number of different bat families and species collected in each country.</caption><thead><tr><th>Family/<br>genus/species</th><th>Cameroon<br>n (%)</th><th>DRC<br>n (%)</th><th>Guinea<br>n (%)</th><th>Total<br>n (%)</th></tr></thead><tbody><tr><td><strong>Pteropodidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Casinycteris arginnis</td><td>1 (0.07)</td><td>4 (0.7)</td><td>-</td><td>5 (0.19)</td></tr><tr><td>Eidolon helvum</td><td>815 (59)</td><td>153 (27)</td><td>20 (3.1)</td><td>988 (38)</td></tr><tr><td>Epomophorus gambianus</td><td>-</td><td>41 (7.2)</td><td>44 (6.8)</td><td>85 (3.2)</td></tr><tr><td>Epomops sp*</td><td>121 (8.8)</td><td>56 (9.9)</td><td>21 (3.2)</td><td>198 (7.6)</td></tr><tr><td>Hypsignathus monstrosus</td><td>249 (18)</td><td>5 (0.8)</td><td>2 (0.3)</td><td>256 (9.9)</td></tr><tr><td>Lissonycteris angolensis</td><td>41 (2.9)</td><td>6 (1.0)</td><td>3 (0.4)</td><td>50 (1.9)</td></tr><tr><td>Megaloglossus woermanni</td><td>6 (0.43)</td><td>5 (0.8)</td><td>-</td><td>11 (0.4)</td></tr><tr><td>Micropteropus pusillus</td><td>5 (0.36)</td><td>95 (16)</td><td>19 (2.9)</td><td>119 (4.6)</td></tr><tr><td>Myonycteris torquata</td><td>26 (1.8)</td><td>31 (5.4)</td><td>-</td><td>57 (2.2)</td></tr><tr><td>Nanonycteris veldkampii</td><td>-</td><td>-</td><td>1 (0.1)</td><td>1 (0.03)</td></tr><tr><td>Rousettus aegyptiacus</td><td>1 (0.07)</td><td>-</td><td>145 (22)</td><td>146 (5.6)</td></tr><tr><td>Scotonycteris zenkeri</td><td>1 (0.07)</td><td>-</td><td>-</td><td>1 (0.03)</td></tr><tr><td><strong>Subtotal frugivorous bats</strong></td><td><strong>1266 (92)</strong></td><td><strong>396 (70)</strong></td><td><strong>255 (33)</strong></td><td><strong>1917 (74)</strong></td></tr><tr><td><strong>Hipposideridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Hipposideros sp</td><td>1 (0.07)</td><td>102 (18)</td><td>78 (12)</td><td>181 (7.0)</td></tr><tr><td><strong>Miniopteridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Miniopterus sp</td><td>-</td><td>49 (8.6)</td><td>5 (0.7)</td><td>54 (2.0)</td></tr><tr><td><strong>Molossidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Chaerephon sp</td><td>-</td><td>2 (0.3)</td><td>154 (24)</td><td>156 (6.0)</td></tr><tr><td>Chaerephon or Mops</td><td>94 (6.8)</td><td>-</td><td>35 (5.4)</td><td>129 (5.0)</td></tr><tr><td>Mops sp</td><td>-</td><td>-</td><td>56 (8.7)</td><td>56 (2.1)</td></tr><tr><td><strong>Nycteridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Nycteris sp</td><td>-</td><td>1 (0.1)</td><td>2 (0.3)</td><td>3 (0.11)</td></tr><tr><td><strong>Rhinolophidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Rhinolophus sp</td><td>-</td><td>7 (1.2)</td><td>35 (5.4)</td><td>42 (1.6)</td></tr><tr><td><strong>Vespertilionidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Neoromicia sp</td><td>-</td><td>4 (0.7)</td><td>-</td><td>4 (0.15)</td></tr><tr><td>Scotophilus sp</td><td>-</td><td>3 (0.5)</td><td>13 (2.0)</td><td>16 (0.6)</td></tr><tr><td><strong>Subtotal insectivorous bats</strong></td><td><strong>95 (6.9)</strong></td><td><strong>168 (29)</strong></td><td><strong>378 (59)</strong></td><td><strong>641 (24)</strong></td></tr><tr><td><strong>Indeterminate species</strong></td><td>15 (1.0)</td><td>1 (0.1)</td><td>5 (0.7)</td><td>21 (0.8)</td></tr><tr><td><strong>Total</strong></td><td><strong>1376 (53.3)</strong></td><td><strong>565 (21.9)</strong></td><td><strong>638 (24.7)</strong></td><td><strong>2579 (100)</strong></td></tr></tbody></table>

<footnote>*In Cameroon and DRC, *Epomops franqueti*, in Guinea *Epomops buetikoferi*</footnote>
<table><caption>**Table 2:** Prevalence of antibodies to Alphaviruses (CHIKV and ONNV) and Flaviruses (ZIKV, DENV1-4, USUV and WNV) in bats in the different collection sites shown in Figure 1.</caption><thead><tr><th></th><th></th><th>CHIKV_E2</th><th>ONNV_E2</th><th>ZIKV_NS1</th><th>DENV1_NS1</th><th>DENV2_NS1</th><th>DENV3_NS1</th><th>DENV4_NS1</th><th>USUV_NS1</th><th>WNV_NS1</th><th>WNV_D3</th><th>Total *positive (%)</th></tr></thead><tbody><tr><td colspan="13" style="text-align: center;">N Positive (% / CI)</td></tr><tr><td>Cameroon</td><td>n</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CP</td><td>81</td><td>1 (1.23)</td><td>1 (1.23)</td><td>-</td><td>1 (1.23)</td><td>2 (2.46)</td><td>-</td><td>1 (1.23)</td><td>-</td><td>-</td><td>-</td><td>4 (4.9)</td></tr><tr><td>MB</td><td>143</td><td>1 (0.69)</td><td>2 (1.39)</td><td>-</td><td>2 (1.39)</td><td>-</td><td>1 (0.69)</td><td>7 (4.89)</td><td>-</td><td>-</td><td>1 (0.69)</td><td>11 (7.6)</td></tr><tr><td>OB</td><td>295</td><td>2 (0.67)</td><td>1 (0.33)</td><td>2 (0.67)</td><td>1 (0.33)</td><td>1 (0.33)</td><td>1 (0.33)</td><td>2 (0.67)</td><td>4 (1.35)</td><td>2 (0.67)</td><td>4 (1.35)</td><td>15 (5.0)</td></tr><tr><td>YD</td><td>857</td><td>11 (1.3)</td><td>9 (1.0)</td><td>6 (0.7)</td><td>16 (1.9)</td><td>19 (2.2)</td><td>18 (2.1)</td><td>26 (3.0)</td><td>52 (6.0)</td><td>9 (1.0)</td><td>5 (0.6)</td><td>121 (14.1)</td></tr><tr><td>SUB-TOTAL</td><td>1376</td><td>15 (1.0 / 0.66 - 1.80)</td><td>13 (0.94 / 0.55 - 1.61)</td><td>8 (0.58 / 0.29 - 1.15)</td><td>20 (1.4 / 0.94 - 2.24)</td><td>22 (1.6 / 1.05 - 2.41)</td><td>20 (1.4 / 0.94 - 2.24)</td><td>36 (2.6 / 1.89 - 3.61)</td><td>56 (4.0 / 3.14 - 5.25)</td><td>11 (0.8 / 0.44 - 1.43)</td><td>10 (0.72 / 0.39 - 1.34)</td><td>151 (11)</td></tr><tr><td colspan="13" style="text-align: center;">N Positive (% / CI)</td></tr><tr><td>DRC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BN</td><td>77</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (1.29)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (1.2)</td></tr><tr><td>BK</td><td>43</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (2.32)</td><td>-</td><td>-</td><td>1 (2.32)</td><td>2 (4.6)</td></tr><tr><td>BD</td><td>136</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (0.73)</td><td>3 (2.20)</td><td>1 (0.73)</td><td>-</td><td>2 (1.51)</td><td>6 (4.4)</td></tr><tr><td>BM</td><td>41</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0 (0.0)</td></tr><tr><td>KI</td><td>42</td><td>-</td><td>1 (2.38)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (2.3)</td></tr><tr><td>KP</td><td>43</td><td>-</td><td>1 (2.32)</td><td>1 (2.32)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2 (4.7)</td></tr><tr><td>KM</td><td>20</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0 (0.0)</td></tr><tr><td>MG</td><td>21</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0 (0.0)</td></tr><tr><td>ZG</td><td>142</td><td>-</td><td>1 (0.70)</td><td>1 (0.70)</td><td>1 (0.70)</td><td>1 (0.70)</td><td>2 (1.40)</td><td>1 (0.70)</td><td>1 (0.70)</td><td>6 (4.22)</td><td>-</td><td>8 (5.6)</td></tr><tr><td>SUB-TOTAL</td><td>565</td><td>0 (0.0 / 0.00 - 0.68)</td><td>3 (0.53 / 0.18 - 1.55)</td><td>2 (0.35 / 0.09 - 1.29)</td><td>1 (0.17 / 0.03 - 1.00)</td><td>1 (0.17 / 0.03 - 1.00)</td><td>4 (0.70 / 0.27 - 1.81)</td><td>5 (0.88 / 0.37 - 2.06)</td><td>2 (0.35 / 0.09 - 1.29)</td><td>6 (1.06 / 0.48 - 2.30)</td><td>3 (0.53 / 0.18 - 1.55)</td><td>20 (3.5)</td></tr><tr><td colspan="13" style="text-align: center;">N Positive (% / CI)</td></tr><tr><td>Guinea</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BF</td><td>93</td><td>-</td><td>1 (1.07)</td><td>1 (1.07)</td><td>-</td><td>1 (1.07)</td><td>5 (5.37)</td><td>1 (1.07)</td><td>-</td><td>-</td><td>1 (1.07)</td><td>9 (9.6)</td></tr><tr><td>KK</td><td>91</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (1.09)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2 (2.19)</td></tr><tr><td>KN</td><td>114</td><td>2 (1.75)</td><td>3 (2.63)</td><td>-</td><td>1 (0.87)</td><td>1 (0.87)</td><td>1 (0.87)</td><td>1 (0.87)</td><td>-</td><td>-</td><td>-</td><td>6 (5.2)</td></tr><tr><td>KD</td><td>7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0 (0.0)</td></tr><tr><td>LL</td><td>36</td><td>-</td><td>1 (2.77)</td><td>1 (2.77)</td><td>-</td><td>-</td><td>1 (2.77)</td><td>-</td><td>1 (2.77)</td><td>2 (5.55)</td><td>1 (2.77)</td><td>5 (13.8)</td></tr><tr><td>MC</td><td>98</td><td>-</td><td>7 (7.14)</td><td>-</td><td>-</td><td>1 (1.02)</td><td>3 (3.06)</td><td>4 (4.08)</td><td>-</td><td>-</td><td>3 (3.06)</td><td>12 (12)</td></tr><tr><td>MM</td><td>99</td><td>3 (3.00)</td><td>2 (2.00)</td><td>1 (1.00)</td><td>-</td><td>1 (1.00)</td><td>-</td><td>1 (1.00)</td><td>-</td><td>-</td><td>-</td><td>8 (8.0)</td></tr><tr><td>SG</td><td>100</td><td>-</td><td>1 (1.00)</td><td>1 (1.00)</td><td>-</td><td>1 (1.00)</td><td>1 (1.00)</td><td>1 (1.00)</td><td>-</td><td>-</td><td>1 (1.00)</td><td>4 (4.0)</td></tr><tr><td>SUB-TOTAL</td><td>638</td><td>5 (0.78 / 0.33 - 1.83)</td><td>15 (2.35 / 1.42 - 3.85)</td><td>4 (0.62 / 0.24 - 1.61)</td><td>1 (0.15 / 0.02 - 0.89)</td><td>6 (0.94 / 0.43 - 2.04)</td><td>11 (1.75 / 0.96 - 3.07)</td><td>8 (1.25 / 0.63 - 2.46)</td><td>1 (0.15 / 0.02 - 0.89)</td><td>2 (0.31 / 0.08 - 1.14)</td><td>8 (1.25 / 0.63 - 2.46)</td><td>47 (7.3)</td></tr><tr><td>Total</td><td>2579</td><td>20 (0.77 / 0.50 - 1.20)</td><td>31 (1.2 / 0.84 - 1.71)</td><td>14 (0.54 / 0.32 - 0.91)</td><td>22 (0.85 / 0.56 - 1.29)</td><td>29 (1.12 / 0.78 - 1.62)</td><td>35 (1.35 / 0.97 - 1.89)</td><td>49 (1.9 / 1.44 - 2.51)</td><td>59 (2.3 / 1.77 - 2.94)</td><td>19 (0.73 / 0.47 - 1.15)</td><td>21 (0.81 / 0.53 - 1.25)</td><td>218 (8.45)</td></tr></tbody></table>

CHIKV, Chikungunya virus; ONNV, O'nyong nyong virus; ZIKV, Zika virus; DENV, Dengue virus; USUV, Usutu virus; WNV, West Nile virus; CI = 95% confidence interval; *total means number of samples reactive with at least one antigen
<table><caption>**Table 3:** Seroprevalence of IgG antibodies to arboviruses in bat species.</caption><thead><tr><th>Species / genus</th><th>N tested</th><th>CHIK_E2</th><th>ONNV_E2</th><th>ZIKV_NS1</th><th>DENV1_NS1</th><th>DENV2_NS1</th><th>DENV3_NS1</th><th>DENV4_NS1</th><th>USUV_NS1</th><th>WNV_NS1</th><th>WNV_D3</th><th>N Pos£</th><th>% ; CI95%</th></tr><tr><th></th><th></th><th colspan="11">N positive (%)</th></tr></thead><tbody><tr><td>Casinycteris sp</td><td>5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; 0-43</td></tr><tr><td>Eidolon helvum</td><td>988</td><td>9 (0.91)</td><td>9 (0.91)</td><td>5 (0.50)</td><td>16 (1.61)</td><td>21 (2.12)</td><td>16 (1.61)</td><td>26 (2.63)</td><td>32 (3.23)</td><td>15 (1.51)</td><td>9 (0.91)</td><td>106</td><td>10.7 ; 8.9-12</td></tr><tr><td>Epomophorus gambianus</td><td>85</td><td>-</td><td>1 (1.17)</td><td>-</td><td>-</td><td>-</td><td>1 (1.17)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2</td><td>2.35 ; 0.6-8.1</td></tr><tr><td>Epomops sp*</td><td>198</td><td>1 (0.52)</td><td>-</td><td>3 (1.51)</td><td>-</td><td>-</td><td>-</td><td>1 (0.52)</td><td>-</td><td>-</td><td>1 (0.52)</td><td>6</td><td>3.0 ; 1.3-6.4</td></tr><tr><td>Hypsignathus monstrosus</td><td>256</td><td>3 (1.17)</td><td>2 (0.78)</td><td>3 (1.17)</td><td>3 (1.17)</td><td>3 (1.17)</td><td>7 (2.76)</td><td>6 (2.34)</td><td>26 (10.1)</td><td>4 (1.56)</td><td>2 (0.78)</td><td>46</td><td>17.9 ; 13-23</td></tr><tr><td>Lissonycteris angolensis</td><td>50</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (2.0)</td><td>-</td><td>-</td><td>-</td><td>1</td><td>2.0 ; 0.3-10</td></tr><tr><td>Megaloglossus woermanni</td><td>11</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; 0-25</td></tr><tr><td>Micropteropus pusillus</td><td>119</td><td>-</td><td>-</td><td>1 (0.84)</td><td>-</td><td>-</td><td>1 (0.84)</td><td>1 (0.84)</td><td>1 (0.84)</td><td>-</td><td>1 (0.84)</td><td>5</td><td>4.2 ; 1.8-9.4</td></tr><tr><td>Myonycteris torquarta</td><td>57</td><td>1 (1.75)</td><td>2 (3.5)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>5.2 ; 1.8-14</td></tr><tr><td>Nanonycteris veldkampii</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; §</td></tr><tr><td>Rousettus aegyptiacus</td><td>146</td><td>4 (1.37)</td><td>13 (8.96)</td><td>1 (0.68)</td><td>1 (0.68)</td><td>3 (2.06)</td><td>4 (2.73)</td><td>5 (3.44)</td><td>-</td><td>-</td><td>4 (1.37)</td><td>24</td><td>16.4 ; 11-23</td></tr><tr><td>Scotonycteris zenkeri</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; §</td></tr><tr><td><strong>Frugivorous bats</strong></td><td><strong>1917</strong></td><td><strong>17 (0.88)</strong></td><td><strong>27 (1.4)</strong></td><td><strong>13 (0.67)</strong></td><td><strong>20 (1.04)</strong></td><td><strong>27 (1.4)§</strong></td><td><strong>28 (1.46)</strong></td><td><strong>40 (2.0)</strong></td><td><strong>59 (3.0)</strong></td><td><strong>19 (0.99)</strong></td><td><strong>17 (0.88)</strong></td><td><strong>192</strong></td><td><strong>10 ; 8.7-11</strong></td></tr><tr><td>Hipposideros sp</td><td>181</td><td>-</td><td>1 (0.55)</td><td>-</td><td>-</td><td>1 (0.55)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (0.55)</td><td>3</td><td>1.65 ; 0.5-4.7</td></tr><tr><td>Miniopterus sp</td><td>54</td><td>-</td><td>-</td><td>1 (1.85)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>1.85 ; 0.3-9.7</td></tr><tr><td>Chaerephon sp</td><td>156</td><td>1 (0.64)</td><td>1 (0.64)</td><td>-</td><td>-</td><td>-</td><td>1 (0.64)</td><td>2 (1.28)</td><td>-</td><td>-</td><td>-</td><td>5</td><td>3.2 ; 1.3-7.2</td></tr><tr><td>Chaerephon or Mops</td><td>129</td><td>1 (0.77)</td><td>2 (1.55)</td><td>-</td><td>2 (1.55)</td><td>-</td><td>1 (0.77)</td><td>7 (5.42)</td><td>-</td><td>-</td><td>-</td><td>10</td><td>7.75 ; 4.2-13</td></tr><tr><td>Mops sp</td><td>56</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (1.92)</td><td>4 (7.69)</td><td>-</td><td>-</td><td>-</td><td>1 (1.92)</td><td>5</td><td>8.9 ; 3.8-19</td></tr><tr><td>Nycteris sp</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; 0-56</td></tr><tr><td>Rhinolophus sp</td><td>42</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (2.38)</td><td>1</td><td>2.38 ; 0.4-12</td></tr><tr><td>Neoromicia sp</td><td>4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; 0-49</td></tr><tr><td>Scotophilus sp</td><td>16</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0</td><td>0.0 ; 0-19</td></tr><tr><td><strong>Insectivorous bats</strong></td><td><strong>641</strong></td><td><strong>2 (0.31)</strong></td><td><strong>4 (0.62)</strong></td><td><strong>1 (0.15)</strong></td><td><strong>2 (0.31)</strong></td><td><strong>2 (0.31)§</strong></td><td><strong>6 (0.93)</strong></td><td><strong>9 (1.4)</strong></td><td><strong>0 (0.0)</strong></td><td><strong>0 (0.0)</strong></td><td><strong>3 (0.46)</strong></td><td><strong>25</strong></td><td><strong>3.9 ; 2.6-5.7</strong></td></tr><tr><td>Ideterminate species</td><td>21</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 (4.76)</td><td>1</td><td>4.8 ; 0.8-22</td></tr></tbody></table>

<footnote>*In Cameroon and DRC, *Epomops franqueti*, in Guinea *Epomops buetikoferi*; $ = p-value=0.02825$ ; CI= confidence interval; §= confidence interval was not calculated; CHIKV, Chikungunya virus; ONNV, O'nyong nyong virus; ZIKV, Zika virus; DENV, Dengue virus; USUV, Usutu virus; WNV, West Nile virus, £ number positive with at least one antigen.</footnote>
<table><caption>**Table 4:** Proportion of samples with antibodies to arbovirus for each species according ecological site where they were captured.</caption><thead><tr><th></th><th>Caves</th><th>Forest</th><th>Urban area</th><th>Village</th></tr><tr><td></td><td colspan="4" style="text-align: center;">N pos / N tested (%)</td></tr></thead><tbody><tr><td><strong>Pteropodidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Casinycteris sp</td><td>-</td><td>0/4 (0.0)</td><td>-</td><td>-</td></tr><tr><td>Eidolon helvum</td><td>0/2 (0.0)</td><td>14/143 (9.8)</td><td>88/821 (10.7)</td><td>4/22 (18)</td></tr><tr><td>Epomophorus gambianus</td><td>0/1 (0.0)</td><td>-</td><td>1/42 (2.4)</td><td>1/42 (2.4)</td></tr><tr><td>Epomops sp*</td><td>-</td><td>0/11 (0.0)</td><td>4/89 (4.5)</td><td>2/98 (2.0)</td></tr><tr><td>Hypsignathus monstrosus</td><td>-</td><td>0/5 (0.0)</td><td>42/227 (18.5)</td><td>4/24 (16.6)</td></tr><tr><td>Lissonycteris angolensis</td><td>-</td><td>1/3 (33)</td><td>0/43 (0.0)</td><td>0/4 (0.0)</td></tr><tr><td>Megaloglossus woermanni</td><td>-</td><td>-</td><td>0/1 (0.0)</td><td>0/10 (0.0)</td></tr><tr><td>Micropteropus pusillus</td><td>0/3 (0.0)</td><td>0/39 (0.0)</td><td>3/61 (4.9)</td><td>2/16 (12.5)</td></tr><tr><td>Myonycteris torquarta</td><td>-</td><td>0/12 (0.0)</td><td>1/27 (3.7)</td><td>1/18 (5.5)</td></tr><tr><td>Nanonycteris veldkampii</td><td>-</td><td>-</td><td>-</td><td>0/1 (0.0)</td></tr><tr><td>Rousettus aegyptiacus</td><td>7/23 (30)</td><td>5/41 (12)</td><td>3/26 (11.5)</td><td>9/56 (16)</td></tr><tr><td>Scotonycteris zenkeri</td><td>-</td><td>-</td><td>-</td><td>0/1 (0.0)</td></tr><tr><td><strong>Subtotal Frugivorous bats</strong></td><td><strong>7/29 (24)</strong></td><td><strong>20/258 (7.7)</strong></td><td><strong>142/1337 (10.6)</strong></td><td><strong>23/293 (7.9)</strong></td></tr><tr><td><strong>Hipposideridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Hipposideros sp</td><td>3/154 (1.9)</td><td>0/6 (0.0)</td><td>0/19 (0.0)</td><td>0/2 (0.0)</td></tr><tr><td><strong>Miniopteridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Miniopterus sp</td><td>1/51 (1.9)</td><td>0/3 (0.0)</td><td>-</td><td>-</td></tr><tr><td><strong>Molossidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Chaerephon sp</td><td>0/2 (0.0)</td><td>-</td><td>0/1 (0.0)</td><td>5/153 (3.3)</td></tr><tr><td>Chaerephon or Mops</td><td>-</td><td>-</td><td>-</td><td>10/129 (7.7)</td></tr><tr><td>Mops sp</td><td>-</td><td>-</td><td>-</td><td>5/56 (8.9)</td></tr><tr><td><strong>Nycteridae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Nycteris sp</td><td>0/2 (0.0)</td><td>0/1 (0.0)</td><td>-</td><td>-</td></tr><tr><td><strong>Rhinolophidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Rhinolophus sp</td><td>1/38 (2.6)</td><td>0/4 (0.0)</td><td>-</td><td>-</td></tr><tr><td><strong>Vespertilionidae</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Neoromicia sp</td><td>-</td><td>-</td><td>-</td><td>0/4 (0.0)</td></tr><tr><td>Scotophilus sp</td><td>-</td><td>-</td><td>0/2 (0.0)</td><td>0/14 (0.0)</td></tr><tr><td><strong>Subtotal Insectivorous bats</strong></td><td><strong>5/245 (2.0)</strong></td><td><strong>0/14 (0.0)</strong></td><td><strong>0/22 (0.0)</strong></td><td><strong>20/376 (5.3)</strong></td></tr><tr><td>Indeterminate species</td><td>0/5 (0.0)</td><td>-</td><td>0/1 (0.0)</td><td>1/15 (6.6)</td></tr><tr><td><strong>Total/ (% [95%CI])</strong></td><td><strong>12/281 (4.2 [2.4-7.3])</strong></td><td><strong>20/272 (7.35 [4.8-11])</strong></td><td><strong>142/1360 (10.4 [8.9-12])</strong></td><td><strong>44/666 (6.6 [4.9-8.7])</strong></td></tr></tbody></table>

<footnote>*In Cameroon and DRC, *Epomops franqueti*, in Guinea *Epomops buetikoferi*</footnote>
<table><caption>**Table 5:** Proportion of simultaneous reactivity to antigenically-related virus and less antigenically related ones.</caption><thead><tr><th></th><th>N tested</th><th>CHIK_E 2</th><th>ONNV_E 2</th><th>ZIKV_NS 1</th><th>DV1_NS 1</th><th>DV2_NS 1</th><th>DV3_NS 1</th><th>DV4_NS 1</th><th>USUV_NS 1</th><th>WNV_NS 1</th><th>WNV_D 3</th></tr><tr><th></th><th></th><th colspan="10">N pos (%)</th></tr></thead><tbody><tr><td>CHIK_E2</td><td>n=20</td><td></td><td>6 (30)</td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>1 (5)</td><td>0 (0)</td><td>1 (5)</td><td>0 (0)</td><td>0 (0)</td></tr><tr><td>ONNV_E2</td><td>n=31</td><td>6 (19)</td><td></td><td>0 (0)</td><td>0 (0)</td><td>0 (0)</td><td>2 (6.4)</td><td>3 (9.6)</td><td>0 (0)</td><td>0 (0)</td><td>4 (12)</td></tr><tr><td>ZIKV_NS1</td><td>n=14</td><td>0 (0)</td><td>0 (0)</td><td></td><td>1 (7)</td><td>1 (7)</td><td>0 (0)</td><td>1 (7)</td><td>1 (7)</td><td>1 (7)</td><td>2 (14)</td></tr><tr><td>DV1_NS1</td><td>n=22</td><td>0 (0)</td><td>0 (0)</td><td>1 (4.5)</td><td></td><td>12 (54)</td><td>3 (13)</td><td>15 (68)</td><td>1 (4.5)</td><td>0 (0)</td><td>0 (0)</td></tr><tr><td>DV2_NS1</td><td>n=29</td><td>0 (0)</td><td>0 (0)</td><td>1 (3.4)</td><td>12 (41)</td><td></td><td>4 (13)</td><td>12 (41)</td><td>1 (3.4)</td><td>0 (0)</td><td>0 (0)</td></tr><tr><td>DV3_NS1</td><td>n=35</td><td>1 (2.8)</td><td>2 (5.7)</td><td>0 (0)</td><td>3 (8.5)</td><td>4 (11)</td><td></td><td>5 (14)</td><td>11 (31)</td><td>7 (20)</td><td>2 (5.7)</td></tr><tr><td>DV4_NS1</td><td>n=49</td><td>0 (0)</td><td>3 (6.1)</td><td>1 (2)</td><td>15 (30)</td><td>12 (24)</td><td>5 (10)</td><td></td><td>3 (6.1)</td><td>7 (20)</td><td>1 (6.1)</td></tr><tr><td>USUV_NS 1</td><td>n=59</td><td>1 (1.6)</td><td>0 (0)</td><td>1 (1.6)</td><td>1 (1.6)</td><td>1 (1.6)</td><td>11 (18)</td><td>3 (5.0)</td><td></td><td>7 (11)</td><td>1 (1.6)</td></tr><tr><td>WNV_NS1</td><td>n=19</td><td>0 (0)</td><td>0 (0)</td><td>1 (5.2)</td><td>0 (0)</td><td>0 (0)</td><td>7 (36)</td><td>1 (5.2)</td><td>7 (36)</td><td></td><td>1 (5.2)</td></tr><tr><td>WNV_D3</td><td>n=21</td><td>0 (0)</td><td>3 (14)</td><td>2 (9.5)</td><td>0 (0)</td><td>0 (0)</td><td>2 (9.5)</td><td>3 (14)</td><td>1 (4.76)</td><td>1 (4.76)</td><td></td></tr></tbody></table>
**Figure 1:** Bat collection sites and presence of arboviruses in Cameroon, Guinea and DRC.

The maps represent Africa and the 3 countries (Guinea, top; Cameroon, center; and the DRC, bottom) where studied samples have been collected. Grey dots indicate sampling sites where no antibodies to arboviruses were detected; red dots, sites where antibodies to alphaviruses were detected; blue dots, sites where antibodies to flaviviruses were detected; red dots with blue circles, sites where antibodies to flaviviruses were detected. Site abbreviations:

**Cameroon** – Village: CP/Campo; MB/Mambele, Urban area: OB/Obala; YD/Yaoundé. **DRC** – Caves: BM/Boma; KP/Kimpse; ZG/Zongo, Forest: ZG/Zongo; BD/Boende, Urban area: BN/Beni; BK/Bikoro; BM/Boma; KP/Kimpse; KM/Komanda; MG/Mangina, Village: BD/Boende; KI/Kikiwit. **Guinea** – Caves: KK/Kankan; KN/Kindia; MC/Macenta; MM/Mamou; SI/Siguiri, Forest: MC/Macenta, Urban area: KN/Kindia; MM/Mamou; Villages: BF/Boffa; KK/Kankan; KN/Kindia; KD/Koundara; LL/Lola; MC/Macenta; MM/Mamou; SI/Siguiri.

**Figure 2:** Percentage of positive samples in colonie of bats, according to age and reproductive cycle.

The figure represents the proportions of positive sample in two species (*H.monstrosus* and *E.helvum*) of fruit bats followed-up over a year in Yaoundé, Cameroon. Panels A and B (top) represent the proportions of positive samples at three categories of age. Panels C and D (bottom) compare the proportion of positive samples in the same two bat species, stratified by female reproduction/lactation status. (ns) stands for non-significant statistically (p-value above 0.05).
<table><caption>**Supplementary Table S1:** Cut-off values in MFI (Medium Fluorescence Intensity) used to discriminate between antibody positive and negative bat samples.</caption><thead><tr><th></th><th>Change Point</th><th>Binomial</th><th>Exponential</th><th>Mean</th></tr></thead><tbody><tr><td>CHIK_E2</td><td>609</td><td>138</td><td>89</td><td>279</td></tr><tr><td>ONNV_E2</td><td>747</td><td>253</td><td>162</td><td>387</td></tr><tr><td>ZIKV_NS1</td><td>223</td><td>46</td><td>30</td><td>100</td></tr><tr><td>DENV1_NS1</td><td>391</td><td>168</td><td>106</td><td>222</td></tr><tr><td>DENV2_NS1</td><td>473</td><td>248</td><td>153</td><td>291</td></tr><tr><td>DENV3_NS1</td><td>29</td><td>17</td><td>14</td><td>20</td></tr><tr><td>DENV4_NS1</td><td>268</td><td>223</td><td>146</td><td>212</td></tr><tr><td>USUV_NS1</td><td>548</td><td>237</td><td>102</td><td>296</td></tr><tr><td>WNV_NS1</td><td>926</td><td>168</td><td>93</td><td>396</td></tr><tr><td>WNV_D3</td><td>356</td><td>67</td><td>58</td><td>160</td></tr></tbody></table>
<table><caption>**Supplementary Table S2 :** Percentage of samples with antibodies to the different arboviruses according to ecological setting where they were captured</caption><thead><tr><th></th><th>N pos/N tested</th><th>%</th><th>p-value</th><th>CHIK_E2</th><th>ONNV_E2</th><th>ZIKV_NS1</th><th>DENV1_NS1</th><th>DENV2_NS1</th><th>DENV3_NS1</th><th>DENV4_NS1</th><th>USUV_NS1</th><th>WNV_NS1</th><th>WNV_D3</th></tr></thead><tbody><tr><td>Caves</td><td>12/281</td><td>4,7</td><td></td><td>2 (0.71)</td><td>6 (2.1)</td><td>1 (0.35)</td><td>0 (0.0)</td><td>2 (0.71)</td><td>1 (0.35)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>2 (0.71)</td></tr><tr><td>Forest</td><td>20/272</td><td>7,3</td><td></td><td>0 (0.0)</td><td>5 (1.8)</td><td>1 (0.36)</td><td>1 (0.36)</td><td>1 (0.36)</td><td>3 (1.1)</td><td>5 (1.8)</td><td>2 (0.73)</td><td>6 (2.2)</td><td>3 (1.1)</td></tr><tr><td>Urban area</td><td>142/1360</td><td>10,4</td><td>&gt;0,001</td><td>15 (1.1)</td><td>10 (0.73)</td><td>8 (0.58)</td><td>17 (1.25)</td><td>20 (1.5)</td><td>21 (1.5)</td><td>29 (2.1)</td><td>56 (4.1)</td><td>11 (0.8)</td><td>10 (0.73)</td></tr><tr><td>Village</td><td>44/666</td><td>6,6</td><td></td><td>3 (0.45)</td><td>10 (1.5)</td><td>4 (0.73)</td><td>4 (0.73)</td><td>7 (1.0)</td><td>10 (1.5)</td><td>15 (2.2)</td><td>1 (0.15)</td><td>2 (0.3)</td><td>6 (0.9)</td></tr></tbody></table>

(%) the percentage of positive samples
<table><caption>**Supplementary Table S3:** Number and percentage of samples with antibodies to arboviruses in colonies of *Eidolon helvum* and *Hypsignatus monstrosus* bats according to age category and reproductive status for adult females.</caption><thead><tr><th></th><th colspan="2">Hypsignatus monstrosus</th><th colspan="2">Eidolon helvum</th><th>Inter-species</th></tr><tr><th></th><th>N tested</th><th>N pos (% ; 95%CI)</th><th>N tested</th><th>N pos (% ;95%CI)</th><th>p-value</th></tr></thead><tbody><tr><td>Juvenile</td><td>72</td><td>10 (13.9 ; 7.7-23.7)</td><td>221</td><td>24 (10.9 ; 7.4-15.0)</td><td>0.52593</td></tr><tr><td>Immature adult *</td><td>22</td><td>1 (4.6 ; 0.8-21)</td><td>218</td><td>22 (10.1 ; 6.7-14.8)</td><td>0.70359</td></tr><tr><td>Adult *</td><td>132</td><td>31 (23.5 ; 17-31)</td><td>376</td><td>42 (11.1 ; 8.3-14.7)</td><td>0.000413</td></tr><tr><td>F-gestation</td><td>11</td><td>2 (18.1 ; 5.1-47)</td><td>11</td><td>2 (18.1 ; 5.1-47)</td><td>1</td></tr><tr><td>F-no gestation</td><td>63</td><td>18 (28.5 ; 17-38)</td><td>160</td><td>15 (9.3 ; 5.7-14)</td><td>0.00061</td></tr><tr><td>F-lactation</td><td>21</td><td>8 (38.0 ; 20-59)</td><td>65</td><td>7 (10.7 ; 5.3-20)</td><td>0.00776</td></tr><tr><td>F-no lactation</td><td>58</td><td>12 (20.6 ; 12-32)</td><td>125</td><td>10 (8.0 ; 4.4-14)</td><td>0.02549</td></tr></tbody></table>

95% CI= confidential interval; * indicated a *p-value* less than 0.05 among Immature adult and Adult on *Hypsignatus monstrosus*
<table><caption>**Supplementary Table S4:** Seroprevalence of antibodies to arbovirus in colonies of *Eidolon helvum* and *Hypsignatus monstruosus* bats from Yaoundé and Obala over the months.</caption><thead><tr><th colspan="3">E. helvum</th><th colspan="2">H. monstrosus</th><th colspan="3">Total</th><th rowspan="2">Rains mm</th></tr><tr><th>Months</th><th>N Tested</th><th>N pos (%)</th><th>N Tested</th><th>N pos (%)</th><th>N Tested</th><th>N pos (%)</th><th>CI 95%</th></tr></thead><tbody><tr><td>Dec-18</td><td>20</td><td>3 (15.0)</td><td>34</td><td>6 (17.7)</td><td>54</td><td>9 (16.7)</td><td>9.0 ; 28</td><td>13</td></tr><tr><td>Jan-19</td><td>95</td><td>10 (10.5)</td><td>40</td><td>10 (25.0)</td><td>135</td><td>20 (14.8)</td><td>9.7 ; 21</td><td>18</td></tr><tr><td>Feb-19</td><td>8</td><td>0 (0.0)</td><td>15</td><td>8 (53.3)</td><td>23</td><td>8 (34.8)</td><td>18 ; 55</td><td>26</td></tr><tr><td>Mar-19</td><td>52</td><td>6 (11.5)</td><td>25</td><td>3 (12.0)</td><td>77</td><td>9 (11.7)</td><td>6.2 ; 20</td><td>56</td></tr><tr><td>Apr-19</td><td>47</td><td>6 (12.8)</td><td>16</td><td>1 (6.3)</td><td>63</td><td>7 (11.1)</td><td>5.4 ; 21</td><td>104</td></tr><tr><td>May-19</td><td>149</td><td>22 (14.8)</td><td>21</td><td>5 (23.8)</td><td>170</td><td>27 (15.9)</td><td>11 ; 20</td><td>95</td></tr><tr><td>Jun-19</td><td>81</td><td>3 (3.7)</td><td>13</td><td>1 (7.7)</td><td>94</td><td>4 (4.3)</td><td>1.6 ; 10</td><td>83</td></tr><tr><td>Jul-19</td><td>130</td><td>21 (16.2)</td><td>5</td><td>0 (0.0)</td><td>135</td><td>21 (15.6)</td><td>10 ; 22</td><td>41</td></tr><tr><td>Sep-19</td><td>50</td><td>5 (10.0)</td><td>19</td><td>4 (21.1)</td><td>69</td><td>9 (13.0)</td><td>7.0 ; 22</td><td>144</td></tr><tr><td>Oct-19</td><td>69</td><td>7 (10.1)</td><td>17</td><td>3 (17.7)</td><td>86</td><td>10 (11.6)</td><td>6.4 ; 20</td><td>106</td></tr><tr><td>Nov-19</td><td>114</td><td>5 (4.4)</td><td>22</td><td>1 (4.6)</td><td>136</td><td>6 (4.4)</td><td>2.0 ; 9.3</td><td>56</td></tr></tbody><tfoot><tr><td>Total</td><td>815</td><td>88 (10.8)</td><td>227</td><td>42 (18.5)</td><td>1042</td><td>130 (12.5)</td><td>10-14</td><td></td></tr></tfoot></table>
<table><caption>**Supplementary Table S5 : GPS coordinates of the study sites in Cameroon, Guinea and the DRC**</caption><thead><tr><th>Sites</th><th>Latitude (N or S)</th><th>GPS</th><th>Longitude (E or W)</th><th>GPS</th></tr></thead><tbody><tr><td colspan="5"><strong>CMR</strong></td></tr><tr><td>Campo</td><td>N</td><td>2,37278</td><td>E</td><td>10.10434</td></tr><tr><td>Mambele</td><td>N</td><td>2,44172</td><td>E</td><td>15.43393</td></tr><tr><td>Obala</td><td>N</td><td>4,0306</td><td>E</td><td>11.53149</td></tr><tr><td>Yaoundé</td><td>N</td><td>3,86401</td><td>E</td><td>11.48763</td></tr><tr><td colspan="5"><strong>DRC</strong></td></tr><tr><td>Beni</td><td>N</td><td>0.4898</td><td>E</td><td>29.45981</td></tr><tr><td>Bikoro</td><td>S</td><td>0.73664</td><td>E</td><td>18.1378</td></tr><tr><td>Boende</td><td>S</td><td>00.38007</td><td>E</td><td>20.85815</td></tr><tr><td>Boma</td><td>S</td><td>5,57029</td><td>E</td><td>13.045</td></tr><tr><td>Kikwit</td><td>S</td><td>5.03754</td><td>E</td><td>18.81946</td></tr><tr><td>Kimpese</td><td>S</td><td>5,4821</td><td>E</td><td>14,45868</td></tr><tr><td>Komanda</td><td>N</td><td>1.34869</td><td>E</td><td>29.75669</td></tr><tr><td>Mangina</td><td>N</td><td>0.62339</td><td>E</td><td>29.61268</td></tr><tr><td>Zongo</td><td>S</td><td>4,72927</td><td>E</td><td>14.88844</td></tr><tr><td colspan="5"><strong>Guinea</strong></td></tr><tr><td>Boffa</td><td>N</td><td>10.32175</td><td>W</td><td>14.26312</td></tr><tr><td>Kankan</td><td>N</td><td>10.37856</td><td>W</td><td>9.37333</td></tr><tr><td>Kindia</td><td>N</td><td>9.52728</td><td>W</td><td>13.68561</td></tr><tr><td>Koundara</td><td>N</td><td>12.32034</td><td>W</td><td>12.78523</td></tr><tr><td>Lola</td><td>N</td><td>7.91639</td><td>W</td><td>8.49855</td></tr><tr><td>Macenta</td><td>N</td><td>8.53663</td><td>W</td><td>9.50049</td></tr><tr><td>Mamou</td><td>N</td><td>10.2849</td><td>W</td><td>12.23256</td></tr><tr><td>Siguiri</td><td>N</td><td>11.5565</td><td>W</td><td>8.80697</td></tr></tbody></table>