Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History

Abstract

Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5' and 3' ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination "hot spot" in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances.IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.

Keywords: Africa; HCoV-229E; HCoV-NL63; bats; coronavirus; recombination; zoonoses.

FIG 1

Map of Kenya showing the geographic locations of 30 bat collection sites.

FIG 2

Phylogeny of RdRps of all CoVs discovered in this study. The host (bat genus), number of sequences, and operational classification (lineage) are shown on the right of the tree. Branches that represent the minority host genera within the lineage defined by a single dominant host genus are indicated in red and labeled with a solid red circle. The tree is midpoint rooted for clarity only, and support values are shown only for internal branches.

FIG 3

Phylogenies of RdRp of alphacoronaviruses and betacoronaviruses. The trees are inferred using representative CoV sequences from this study as well as those obtained from GenBank. The sequences are labeled with accession number/strain name, host (species), and geographic origin (three-letter country code). Different colors are used to distinguish the following groups: orange, Kenyan bat CoVs discovered during this study; blue, CoVs identified from nonbat mammals; green, the Perimyotis subflavus virus previously reported to be related to HCoV-NL63; black, the remaining bat viruses. The lineage information for Kenyan CoVs is shown to the right of the phylogeny and matches that in Fig. 2.

FIG 4

Mantel correlograms showing the Kenyan bat CoV RdRp sequences stratified by geographic distances (A) and host genetic distances (B). A Mantel correlation index (r) was calculated for each of the distance classes. Under the null hypothesis of no relationship between the distance matrices, r values would be close to zero. Positive r values suggest smaller genetic distances between case pairs, whereas negative r values suggest larger genetic distances between case pairs. Filled circles, r significantly different from zero; empty circles, r not significantly different from zero. The graph in panel B also shows kernel density plots for intragenus host distance density (solid light gray line) and intergenus host distance density (dotted light gray line). The corresponding y axis for the plot is shown on the right of panel B. The light gray rectangle between the two plots represents the transition area between the intragenus and intergenus host genetic distances.

FIG 5

Genome organization of two bat 229E-like viruses and three bat NL63-like viruses sampled from Kenyan bats. A unified length scale is used for all the genomes. Within each genome, the ORFs (arrow boxes) and ribosomal frameshift sites (vertical lines) are indicated at their corresponding positions.

FIG 6

Phylogenetic analyses of major open reading frames of NL63-like and 229E-like CoVs in the context of alphacoronaviruses revealing evidence of recombination. The names of viruses sequenced in this study are shown in orange. Three potential recombinant genomes, of HCoV-NL63, BtKYNL63-15, and HKU8, are indicated with red circles, blue triangles, and brown squares, respectively.

FIG 7

Relationships between HCoV-NL63 and related viruses at the receptor binding domain. (A) Alignment of NL63-like and 229E-like viruses and related viruses at the receptor binding domain. The positions of three receptor binding motifs (RBMs) are marked with double-arrowed black lines. Residues in the NL63-CoV RBMs that directly contact the ACE2 receptor are marked with red downward arrows. (B) Phylogenetic relationships of NL63-like and 229E-like viruses at the receptor binding domain of HCoV-NL63. The tree is based on an amino acid alignment and midpoint rooted.

FIG 8

Recombination analyses of HCoV-NL63 using Simplot. Genome-scale similarity comparisons of HCoV-NL63 (query) against BtKYNL63-9a (major parental group; blue), BtKYNL63-9b (green), BtKY229E-8 (minor parental group; red), HCoV-229E (orange), BtCoV/FO1A-F2/Hip_aba/GHA/2010 (pink), and alpaca respiratory CoV (brown) were done. A full genome structure, with reference to HCoV-NL63, is shown above the similarity plot, with the positions and boundaries of the major open reading frames indicated. At the beginning of the S gene, the flat line followed by a sudden drop in similarity is due to a gap (deletion within HCoV-229E S gene) in the alignment.