Metagenomic analysis of coronaviruses in bat fecal matter collected from a human-bat interface around Arua City, West Nile Region, Uganda

Abstract

Background Many recent disease outbreaks in humans had a zoonotic virus etiology. The ongoing coronavirus disease 2019 (COVID-19) pandemic has had devastating health and socioeconomic impacts. Bats are reservoirs for a diverse range of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Human activities, especially at the wildlife inter phase, are at the core of forces driving the emergence of new viral agents. Therefore, strategies aiming to mitigate potential spillover and disease emergence, while supporting the conservation of bats and their important ecological roles are needed

Methods A total of 40 individual bat fecal matter samples were collected and pooled together for each of the 11 sites sampled around Arua city. Samples collected directly into Zymo DNA/RNA Shield Reagent, transported and preserved at −80°C until further analysis. DNA/RNA were extracted using RNeasy Power microbiome Kit and DNeasy® PowerSoil® Kit (QIAGEN, Hilden, Germany) following the manufacturer’s protocol. Purified DNA samples were submitted for sequencing using Illumina MiSeq platform. Bioinformatic analyses were conducted in Biology Laboratory of Bucknell University USA by Professor Ken Field using STAR v. 2.2 pipelines. STAR’s quality module (FastQC v2.08 tool) filtered sequencing reads based on a 25 PHRED score threshold, no ambiguous bases and a 50bp minimum size. Then taxonomy classification was conducted on both trimmed reads (SPAdes v3.13.0). The STAR’s taxonomy module allows for a broad exploration using minimap2 v2.16, Kraken2 v2.0.7, Metaphlan v2.7.7, BWA v0.7.12 and GOTTCHA2 v2.1.5

Results Coronavirus (CoV) RNA was detected in 6 (54.45%) of the 11 different sample pools/sites. Sequence analysis of a 405-nucleotide region of the highly conserved RNA-Dependent RNA polymerase gene (RdRp) showed that all coronaviruses detected in the 6 pools are genetically related to the human common cold coronaviruses 229E9 (NC_002645.1), sharing a 94% nucleotide homology.

We also performed bat species identification using sequence analysis of the 313-nucleotide region of cytochrome C oxidase (COI) subunit 1 fragment. We found that that each of the 11 fecal pools contained 4 different bat species, namely; Eidolon helvum, Epomophorus_gambianus, Epomophorus labiatus and Epomophorus pusillus (formerly Micropteropus pusillus).

Conclusion In six of the bat fecal matter, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E (94% nucleotide identities). All bat fecal matters from which we detected coronaviruses were collected near human settlements, demonstrating the importance of continued surveillance of coronaviruses in bats to better understand the prevalence, diversity, and potential risks for spillover.

DNA barcoding is an important tool in identifying bat species and discovery of new bat species. Barcoding of Arua city bat fecal matter DNA revealed four distinct species based on the results of this study. Future barcode studies of other bat species are important to obtain to complete the Ugandan database for bats