Study site and population
Data for this manuscript, which include viruses detected in bats and rodents and behavioral information on human-wildlife contact were collected from February 2017 to December 2018 in two adjacent villages in the Bono East Region in Ghana (7o 43’N, 1o42’W; Fig. 1). The villages, which have a combined population of 3,754 people (39), were chosen as a site for this study as this region has undergone recent and on-going anthropogenically-induced landscape change characterized by deforestation with a patchwork of protected mixed deciduous forest fragments interspersed among villages, orchards, and agricultural fields (33). The protected forest is a managed sanctuary providing habitat for two revered non-human primate species (black and white colobus (Colobus vellerosus) and Lowe’s mona monkey (Cercopithecus campbelli).
Communities at the site are primarily small-scale smallholder farmers who rely on crop and livestock production and to a lesser degree hunting for their livelihoods. The site is within the forest-savannah transition zone along the vegetative belt of Ghana and is characterized by a moderate climate and fertile soils in which a range of subsistence and cash crops are produced. Cashew nut (Anacardium occidentale), mango (Mangifera sp.), fig (Ficus sp.), and neem (Azadirachta indica) trees provide roosting and foraging habitat for a diversity of fruit bats that are resident or migrating through the area. The Tano sacred grove, a protected area 20 km north of the study site, hosts diverse bat species and the largest colony of straw-colored fruit bats (Eidolon helvum) in Ghana, with an estimated 2 million bats (11, 40). Bats from this grove travel to the surrounding orchards to feed. In addition, several species of rodents make their homes in and around human dwellings and in the agricultural fields at the study site, bringing these wild animals into close contact with community members and their livestock. Farmers in these communities primarily raise local breeds of sheep, pigs, domestic fowl (chickens and ducks), and cattle. We collected data on characteristics of the site to supplement observational data, including habitat types, human population density, species and population estimates of domestic and wild animals present, water sources, anthropogenic changes, and types of human-animal contact.
Wildlife sample collection
Bats and rodents were humanely sampled (and released back to the wild) from February 2017 to December 2018. Sampling occurred during both the rainy and dry seasons targeting a minimum of 100 animals per taxa per season each year. Seasons were classified using precipitation data from the Ghana National Climate Change Committee with the dry season ranging from November to March (mean annual rainfall of 215 mm) and the rainy season from April to October (mean annual rainfall of 1,250 mm) (41).
Bat and rodent sampling
Bats were captured using 6-18 m mist nets. The mist nets were set before dusk and opened for trapping three to four hours before dawn when bats returned to their roosts from foraging. The mist nets were actively monitored throughout the trapping session, and bats were extracted from the net as soon as possible after capture (42) by personnel wearing full personal protective equipment (PPE) for biosafety. Each bat was placed into a porous cotton bag and kept in a cool location until sampling. Rodents were live-captured using Sherman live traps (22.9 x 8.9 x 7.6 cm3) and locally produced wire mesh traps, baited with fish. Rodent traps were set at night in the agricultural fields and with the resident’s permission, around and within houses and outbuildings. Traps were collected in the early morning and placed in a cool, shady location during processing. Rodents were anesthetized with isoflurane using the open drop method (43) for sampling. Rodents and bats were weighed, and morphometric measurements were obtained using calipers, including body length (tip of nose to base of tail), tail length, ear length, and hind foot length for rodents and forearm length for bats. Data on health status, age class, sex, and reproductive status (pregnant, lactating) were also collected.
Oral, urogenital, and fecal swabs were collected in duplicate using sterile, polyester-tipped swabs and placed in viral transport medium (VTM) and TriReagent (Trizol) and stored in liquid nitrogen until transferring to an ultra-low freezer (-80°C). Blood samples were collected from the lateral tail or saphenous vein in rodents and from the brachial or cephalic veins in bats. An aliquot of blood was preserved in VTM and stored in liquid nitrogen until transferring to an ultra-low temperature freezer (-80°C). Serum was also archived from animals for which the blood volume was sufficient (blood samples were not taken in excess 1% of the total body weight). All bats and rodents were temporarily marked with non-toxic nail polish or markers applied to the claws or fur to avoid repeated sampling within the same season’s capture event. The animals were all apparently healthy and released following sampling.
Species identiﬁcation was conﬁrmed by DNA bar coding of the cytochrome b (Cytb) and cytochrome oxidase subunit 1 (CO1) mitochondrial genes (44) for PCR-positive individuals. The PCR amplicons were sequenced and BLASTed against reference sequences in GenBank. Sequences with > 97% sequence identity were classified to the host species. Sequences that did not meet this threshold were classified to the genus level. DNA barcoding was also performed on a subset of the PCR-negative samples.
Community engagement and questionnaire administration
We administered questionnaires among the local community members to learn about people’s awareness and perceptions of zoonotic disease risks and the environmental factors and types of activities in which they engage in that might influence the risk of pathogen transmission from wildlife to humans (human demographics, livelihood activities, types of animal contact, and food safety and sanitation practices).
Prior to initiating the study, the project team met with local officials and community leaders to discuss the goals of the project. With permissions from the local authorities, our team conducted household visits and made announcements in the villages to inform the community members of the study. All messages were communicated in the local dialect using lay language to convey the study purpose, eligibility, potential risks and beneﬁts of participation, and the time during which the study would take place. The team selected all households in the village for participation. Only one person per household was recruited and efforts were made to include participants across a range of ages and gender.
The aims of the study were communicated in the local language, and written informed consent was obtained from all study participants. Questionnaires were administered to collect demographic and livelihood information, travel history, and data on interactions with domestic and wild animals. The questionnaires were written in English and translated into the local language (Twi) during administration.
Virus detection and discovery
Testing of the bat, rodent, and shrew oral and rectal swab samples was performed at the UC Davis One Health Institute Laboratory in Davis, California and Veterinary Services Directorate, Ministry of Food and Agriculture in Ghana. A 250 μl aliquot of each sample was utilized for RNA extraction. RNA was extracted using Direct-Zol RNA columns (Zymo Research Corp), and 8 μl RNA was reverse transcribed into cDNA transcription using Superscript III (Invitrogen Corp, Carlsbad, CA).
The housekeeping gene, b-actin, was targeted as an internal control for the presence of ampliﬁable nucleic acid in the RNA extracts (45). The RNA extracts were then screened via consensus PCR targeting conserved RNA regions for corona- (46, 47), paramyxo- (48), flavi- (49), influenza (50), and filo- (51) viruses. Bands of the expected size for each assay were excised and purified using the Qiaquick kit (Qiagen Inc.). Puriﬁed PCR products were cloned (pCR4-TOPO vector; Invitrogen Corp.) and sequenced (ABI 3730 Capillary Electrophoresis Genetic Analyzer; Applied Biosystems, Inc., Foster City, CA). Sequences were analyzed and edited using Geneious Prime (Version 2019.1.3), uploaded into Genbank, and compared with known sequences. Sequences were classified as belonging to viral taxa according to established cut-offs and methods (52). Virus sequences sharing ≥ 90% identity to another sequence in the GenBank database were classified as a known virus sequence, while viral sequences sharing less than 90% identity to a known sequence were considered novel viruses and named sequentially with other previously unreported virus sequences detected as part of the PREDICT project. Virus isolation was not attempted for any of the positive samples.
Statistical analyses of the survey and virus detection data were performed using R v3.6.0 (53). Responses to the survey were coded, and descriptive statistics were calculated. The frequencies of responses related to hunting and slaughtering of animals were evaluated for differences by gender and age of the respondents using Chi-square tests. Given the high frequency of Kenya bat coronavirus/BtKY56/ detections in Epomophorus gambianus bats, we conducted analyses to explore associations between host demographics, season, and coronavirus positive samples. The Fisher’s exact test was used to compare the proportion of positive Kenya bat coronavirus/BtKY56/BtKY55 results in E. gambianus bats across sex, age class, season, and specimen type. Logistic regression models were then constructed to explore the relationships between these factors and a positive Kenya bat coronavirus/BtKY56/BtKY55 RNA result in the E. gambianus bats.