Genomic characterization of seven distinct bat coronaviruses in Kenya

Abstract

To better understand the genetic diversity and genomic features of 41 coronaviruses (CoVs) identified from Kenya bats in 2006, seven CoVs as representatives of seven different phylogenetic groups identified from partial polymerase gene sequences, were subjected to extensive genomic sequencing. As a result, 15-16kb nucleotide sequences encoding complete RNA dependent RNA polymerase, spike, envelope, membrane, and nucleocapsid proteins plus other open reading frames (ORFs) were generated. Sequences analysis confirmed that the CoVs from Kenya bats are divergent members of Alphacoronavirus and Betacoronavirus genera. Furthermore, the CoVs BtKY22, BtKY41, and BtKY43 in Alphacoronavirus genus and BtKY24 in Betacoronavirus genus are likely representatives of 4 novel CoV species. BtKY27 and BtKY33 are members of the established bat CoV species in Alphacoronavirus genus and BtKY06 is a member of the established bat CoV species in Betacoronavirus genus. The genome organization of these seven CoVs is similar to other known CoVs from the same groups except for differences in the number of putative ORFs following the N gene. The present results confirm a significant diversity of CoVs circulating in Kenya bats. These Kenya bat CoVs are phylogenetically distant from any previously described human and animal CoVs. However, because of the examples of host switching among CoVs after relatively minor sequence changes in S1 domain of spike protein, a further surveillance in animal reservoirs and understanding the interface between host susceptibility is critical for predicting and preventing the potential threat of bat CoVs to public health.

Fig. 1

Schematic representation of the genome organization of Kenya bat CoVs and representative alpha- and beta-coronaviruses. Shaded boxes represent open reading frames (ORFs) encoding structural proteins and unshaded boxes represent those encoding nonstructural proteins.

Fig. 2

Multiple amino acid sequence alignments showing the putative S1–S2 junctional region of CoV spike protein. The identical amino acids are highlighted in black and the similar amino acids are highlighted in gray. The regions containing S1 GxCx motif, conserved S2 nonamer IPTNFSISI, the furin cleavage site (in MHV, HCoV OC43, and BCoV; underlined), and cathepsin L cleavage site (in SARS-CoV) are indicated.

Fig. 3

Phylogenetic analysis of ORF1b, S, M and N of bat CoVs from Kenya. The unrooted trees are constructed by Maximum likelihood method with 1000 bootstrap replications after ambiguous regions from alignments of ORF1b, S, M, and N are removed. The seven Kenya CoVs are highlighted with solid circles. The genus taxonomy information is shown to the right side of the phylogeny. The maximum likelihood bootstrap is indicated next to the nodes. The scale bar indicates the estimated number of nucleotide substitutions per site.