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.

Coronaviruses Are Abundant and Genetically Diverse in West and Central African Bats, including Viruses Closely Related to Human Coronaviruses.

Bats are at the origin of human coronaviruses, either directly or via an intermediate host. We tested swabs from 4597 bats (897 from the Democratic Republic of Congo (DRC), 2191 from Cameroon and 1509 from Guinea) with a broadly reactive PCR in the RdRp region. Coronaviruses were detected in 903 (19.6%) bats and in all species, with more than 25 individuals tested. The highest prevalence was observed in Eidolon helvum (239/733; 39.9%) and Rhinolophus sp. (306/899; 34.1%), followed by Hipposideros sp. (61/291; 20.9%). Frugivorous bats were predominantly infected with beta coronaviruses from the Nobecovirus subgenus (93.8%), in which at least 6 species/genus-specific subclades were observed. In contrast, insectivorous bats were infected with beta-coronaviruses from different subgenera (Nobecovirus (8.5%), Hibecovirus (32.8%), Merbecovirus (0.5%) and Sarbecovirus (57.6%)) and with a high diversity of alpha-coronaviruses. Overall, our study shows a high prevalence and genetic diversity of coronaviruses in bats and illustrates that Rhinolophus bats in Africa are infected at high levels with the Sarbecovirus subgenus, to which SARS-CoV-2 belongs. It is important to characterize in more detail the different coronavirus lineages from bats for their potential to infect human cells, their evolution and to study frequency and modes of contact between humans and bats in Africa.

Dynamics of Antibodies to Ebolaviruses in an Eidolon helvum Bat Colony in Cameroon.

The ecology of ebolaviruses is still poorly understood and the role of bats in outbreaks needs to be further clarified. Straw-colored fruit bats (Eidolon helvum) are the most common fruit bats in Africa and antibodies to ebolaviruses have been documented in this species. Between December 2018 and November 2019, samples were collected at approximately monthly intervals in roosting and feeding sites from 820 bats from an Eidolon helvum colony. Dried blood spots (DBS) were tested for antibodies to Zaire, Sudan, and Bundibugyo ebolaviruses. The proportion of samples reactive with GP antigens increased significantly with age from 0-9/220 (0-4.1%) in juveniles to 26-158/225 (11.6-70.2%) in immature adults and 10-225/372 (2.7-60.5%) in adult bats. Antibody responses were lower in lactating females. Viral RNA was not detected in 456 swab samples collected from 152 juvenile and 214 immature adult bats. Overall, our study shows that antibody levels increase in young bats suggesting that seroconversion to Ebola or related viruses occurs in older juvenile and immature adult bats. Multiple year monitoring would be needed to confirm this trend. Knowledge of the periods of the year with the highest risk of Ebolavirus circulation can guide the implementation of strategies to mitigate spill-over events.

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.

Wide Diversity of Coronaviruses in Frugivorous and Insectivorous Bat Species: A Pilot Study in Guinea, West Africa.

Zoonoses can constitute a threat for public health that can have a global importance, as seen with the current COVID-19 pandemic of severe acute respiratory syndrome coronavirus (SARS-CoV2). Bats have been recognized as an important reservoir of zoonotic coronaviruses (CoVs). In West Africa, where there is a high diversity of bat species, little is known on the circulation of CoVs in these hosts, especially at the interface with human populations. In this study, in Guinea, we tested a total of 319 bats belonging to 14 genera and six families of insectivorous and frugivorous bats across the country, for the presence of coronaviruses. We found CoVs in 35 (11%) of the tested bats-in three insectivorous bat species and five fruit bat species that were mostly captured close to human habitat. Positivity rates varied from 5.7% to 100%, depending on bat species. A wide diversity of alpha and beta coronaviruses was found across the country, including three sequences belonging to SarbeCoVs and MerbeCoVs subgenera known to harbor highly pathogenic human coronaviruses. Our findings suggest that CoVs are widely spread in West Africa and their circulation should be assessed to evaluate the risk of exposure of potential zoonotic CoVs to humans.

Multiplex detection and dynamics of IgG antibodies to SARS-CoV2 and the highly pathogenic human coronaviruses SARS-CoV and MERS-CoV.

BACKGROUND: Knowledge of the COVID-19 epidemic extent and the level of herd immunity is urgently needed to help manage this pandemic. METHODS: We used a panel of 167 samples (77 pre-epidemic and 90 COVID-19 seroconverters) and SARS-CoV1, SARS-CoV2 and MERS-CoV Spike and/or Nucleopcapsid (NC) proteins to develop a high throughput multiplex screening assay to detect IgG antibodies in human plasma. Assay performances were determined by ROC curves analysis. A subset of the COVID-19+ samples (n = 36) were also tested by a commercial NC-based ELISA test and the results compared with those of the novel assay. RESULTS: On samples collected ≥14 days after symptoms onset, the accuracy of the assay is 100 % (95 % CI: 100-100) for the Spike antigen and 99.9 % (95 % CI:99.7-100) for NC. By logistic regression, we estimated that 50 % of the patients have seroconverted at 5.7 ± 1.6; 5.7 ± 1.8 and 7.9 ± 1.0 days after symptoms onset against Spike, NC or both antigens, respectively and all have seroconverted two weeks after symptoms onset. IgG titration in a subset of samples showed that early phase samples present lower IgG titers than those from later phase. IgG to SARS-CoV2 NC cross-reacted at 100 % with SARS-CoV1 NC. Twenty-nine of the 36 (80.5 %) samples tested were positive by the commercial ELISA while 31/36 (86.1 %) were positive by the novel assay. CONCLUSIONS: Our assay is highly sensitive and specific for the detection of IgG antibodies to SARS-CoV2 proteins, suitable for high throughput epidemiological surveys. The novel assay is more sensitive than a commercial ELISA.

Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment.

BACKGROUND: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. METHODS: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. FINDINGS: The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Équateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. INTERPRETATION: The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

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.

Distribution of bat-borne viruses and environment patterns.

Environmental modifications are leading to biodiversity changes, loss and habitat disturbance. This in turn increases contacts between wildlife and hence the risk of transmission and emergence of zoonotic diseases. We analyzed the environment and land use using remote spatial data around the sampling locations of bats positive for coronavirus (21 sites) and astrovirus (11 sites) collected in 43 sites. A clear association between viruses and hosts was observed. Viruses associated to synanthropic bat genera, such as Myotis or Scotophilus were associated to highly transformed habitats with human presence while viruses associated to fruit bat genera were correlated with natural environments with dense forest, grassland areas and regions of high elevation. In particular, group C betacoronavirus were associated with mosaic habitats found in anthropized environments.

Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (ßCoV), respectively. The ßCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat ßCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of ßCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii.

Diversity of bat astroviruses in Lao PDR and Cambodia.

Astroviruses are known to infect humans and a wide range of animal species, and can cause gastroenteritis in their hosts. Recent studies have reported astroviruses in bats in Europe and in several locations in China. We sampled 1876 bats from 17 genera at 45 sites from 14 and 13 provinces in Cambodia and Lao PDR respectively, and tested them for astroviruses. Our study revealed a high diversity of astroviruses among various Yangochiroptera and Yinpterochiroptera bats. Evidence for varying degrees of host restriction for astroviruses in bats was found. Furthermore, additional Pteropodid hosts were detected. The astroviruses formed distinct phylogenetic clusters within the genus Mamastrovirus, most closely related to other known bat astroviruses. The astrovirus sequences were found to be highly saturated indicating that phylogenetic relationships should be interpreted carefully. An astrovirus clustering in a group with other viruses from diverse hosts, including from ungulates and porcupines, was found in a Rousettus bat. These findings suggest that diverse astroviruses can be found in many species of mammals, including bats.