Coronavirus surveillance in wildlife from two Congo basin countries detects RNA of multiple species circulating in bats and rodents

Abstract

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats

“…Analysis of our probe capture data confirmed the presence of several novel coronaviruses in these specimens, as had been previously determined by Kumakamba et al (2021). Our results also suggested the CoVs in these specimens contained spike genes that were highly divergent from any others that have been previously described.…”

“…Bat swab specimens and partial RdRP sequences: Rectal and oral swabs were collected between August 2015 and June 2018 in different locations in DRC from bats that were either captured and released or that were for sale in local markets [Kumakamba 2021]. Swabs were collected into individual 2.0 ml screw-top cryotubes containing 1.5 ml of either Universal Viral Transport Medium (BD) or Trizol® (Invitrogen), stored in liquid nitrogen for transport as soon as practical and later transferred into -80°C freezers.…”

“…Swabs were collected into individual 2.0 ml screw-top cryotubes containing 1.5 ml of either Universal Viral Transport Medium (BD) or Trizol® (Invitrogen), stored in liquid nitrogen for transport as soon as practical and later transferred into -80°C freezers. CoV screening involved two consensus PCR assays targeting the RNA-dependant RNA polymerase (RdRp) performed in Kinshasa, DRC, and commercial Sanger sequencing of amplicons [Quan 2010, Watanabe 2010, Kumakamba 2021]. Bat species were identified by ecologists in the field and verified using a PCR targeting the Cytochrome B gene [Townzen 2008].…”

Enterovirus Sequence Data Obtained from Primate Samples in Central Africa Suggest a High Prevalence of Enteroviruses

“…The samples included primarily oral and rectal swabs, liver and spleen tissue, as well as feces, and were obtained from animals that were trapped and released, animals in captivity, and animals hunted for consumption. RNA was isolated and reverse transcribed ( 4 ), before the samples were screened for enterovirus RNA using a family level consensus PCR targeting the 5′ noncoding region ( 5 ). Both strands of the PCR amplicons were sequenced (Sanger), aligned (ClustalW, Geneious 11.1.3), and subjected to phylogenetic analysis using MrBayes 3.2, employing default parameters and 4 chains of 1,000,000 generations, with final split frequencies below 0.01 ( 6 ).…”

“…Despite having tested many bats in the study, we did not detect enterovirus RNA in any of them. Bats, which are hosts of many zoonotic viruses, including rabies and coronaviruses, can be experimentally infected with enteroviruses, but reports of genuine bat enteroviruses are sparse, unlike reports of other bat picornaviruses ( 4 , 8 , 17 – 22 ). We conclude that Central African bats may either not host many enteroviruses or that the enteroviruses that infect bats are genetically divergent enough from the known species to evade PCR detection with the primers used in this study.…”