Evidence for an Ancestral Association of Human Coronavirus 229E with Bats

Abstract

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E.

Importance: The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.

FIG 1

Phylogenetic relationships of the genus Alphacoronavirus, HCoV-229E strains, and the novel bat viruses. (A) Bayesian phylogeny of an 816-nucleotide RdRp gene sequence fragment corresponding to positions 13891 to 14705 in HCoV-229E prototype strain inf-1 (GenBank accession no. NC_002645) using a GTR+G+I substitution model. SARS-CoV was used as an outgroup. Viruses with additional sequence information generated in this study are marked with circles (full genome) or triangles (spike gene). Bat viruses detected in our previous studies from Ghana (33) and Gabon are given in cyan (41). (B) Neighbor-joining phylogeny of the same RdRp gene fragment with a nucleotide percentage distance substitution model and the complete deletion option. The tree was rooted against HCoV-NL63. Viruses are colored according to their origin. (C) Bayesian phylogeny of the full spike gene of bat 229E-related CoVs, the alpaca 229E-related CoV, and HCoV-229E strains identified with GenBank accession numbers and year of isolation, using a WAG amino acid substitution model and HCoV-NL63 as an outgroup. The novel bat 229E–related CoVs are shown in boldface and red. Branches leading to the outgroup were truncated for graphical reasons, as indicated by slashed lines. Values at nodes show support of grouping from posterior probabilities or 1,000 bootstrap replicates (only values above 0.7 are shown).

FIG 2

Genome organization of 229E-related coronaviruses and relationships between viruses from bats and humans. (A) 229E-related CoV genomes are represented by black lines; ORFs are indicated by gray arrows. Locations of transcription regulatory core sequences (TRS) are marked by black dots. HCoV-NL63 is shown for comparison. (B) Similarity plots generated using SSE V1.1 (38) with a sliding window of 400 and a step size of 40 nucleotides (nt). The HCoV-229E prototype strain inf-1 was used with the indicated animal viruses.

FIG 3

Bayesian phylogenies of major open reading frames and recombination analysis of HCoV-229E and related animal viruses. (A) Phylogenies were calculated with a WAG amino acid substitution model. The novel bat viruses are shown in red. The alpaca CoV is shown in cyan. Filled circles, posterior probability support exceeding 0.95; the scale bar corresponds to genetic distance. Details on the origin of HCoV-229E strain VFC408, which was generated for this study, can be retrieved from reference . Branches leading the outgroup HCoV-NL63 were truncated for graphical reasons. (B) Bootscan analysis using the Jukes-Cantor algorithm with a sliding window of 1,500 and a step size of 300 nt. The HCoV-220E inf-1 strain was used with animal 229E-related viruses as indicated. (C) Phylogenies of the S1 and S2 subunits were calculated according to panel A. One representative HCoV-229E strain was selected per decade according to reference : GenBank accession no. DQ243974, DQ243964, DQ243984, and DQ243967.

FIG 4

Amino acid sequence alignment of the 5′ ends of the spike genes of HCoV-229E and related animal viruses. Amino acid alignment of the first part of the spike genes of 229E-related CoVs, including four bat 229E-related CoVs, the alpaca 229E-related CoV and the HCoV-229E inf-1 strain, is shown. Conserved amino acid residues are marked in black, and sequence gaps are represented by hyphens.

FIG 5

Nucleotide sequence alignment of the genomic 3′ ends of HCoV-229E and related animal viruses Nucleotide alignment of the genome region downstream of the nucleocapsid gene, including four bat 229E-related CoVs, the alpaca 229E-related CoV, and representative HCoV-229E, are shown, with full genomes identified with GenBank accession number or strain name. Dots represent identical nucleotides, and hyphens represent sequence gaps. Gray bars above alignments indicate open reading frames and the beginning of the poly(A) tail. The putative start and stop codon of ORF8 is in lime green, and the corresponding putative TRS element is in blue. The conserved genomic sequence elements and the highly conserved stem elements forming part of the pseudoknot (PK) are marked with gray and purple background.

FIG 6

Amino acid sequence alignment of the putative ORF8 from a bat 229E-related coronavirus and closest hits from two other hipposiderid bat coronaviruses Conserved amino acid residues between sequence pairs are highlighted in color according to amino acid properties, and sequence gaps are represented by hyphens. The central domain showing higher sequence similarity between compared viruses is boxed for clarity. The 229E-related alphacoronavirus KW2E-F56 from Hipposideros cf. ruber detected in this study is given in red, the alphacoronavirus HKU10 originated from a Chinese H. pomona animal, and the betacoronavirus Zaria originated from a Nigerian H. gigas animal.