Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event

Abstract

Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 x 10(-4) nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.

FIG. 1.

Linear representation of the ORFs of the group 2 coronaviruses and SARS-CoV. Nucleotide insertions (open arrowheads) and deletions (solid arrowheads) in the HCoV-OC43 genome compared to BCoV are shown.

FIG. 2.

Overview of the putative domain organization and potential proteolytic cleavage sites of the HCoV-OC43 replicase polyprotein pp1ab. Cleavage sites that are predicted to be processed by 3C-like protease are indicated by black arrowheads, while potential papain-like protease cleavage sites are indicated by white arrowheads. The following predicted domains are shown: papain-like proteases 1 and 2 (PLP1 and PLP2), X domain (X), putative transmembrane domains 1, 2, and 3 (TM1, TM2, and TM3), 3C-like protease (3CL), growth factor-like domain (GFL), RdRp, metal ion-binding domain (MB), HEL, ATPase, putative 3′-to-5′ exonuclease (ExoN), putative poly(U)-specific endo-RNase (XendoU), and a putative S-adenosylmethionine-dependent ribose 2′-O-methyltransferase (MT).

FIG. 3.

Maizel-Lenk dot matrix plots: the complete genome sequence of HCoV-OC43 is compared to the complete genome sequences of BCoV, MHV, SARS-CoV, HCoV-229E, IBV, and TGEV, respectively. Sequence identities are indicated by a dot.

FIG. 4.

Phylogenetic analysis of the coronavirus ORF1b replicase amino acid sequences. The HCoV-OC43 ORF1b protein (GenBank accession number AY391777) was compared to other coronaviruses and to an equine torovirus as an outgroup. Group 1, HCoV-229E (accession number AF304460), HCoV-NL63 (AY567487), PEDV strain CV777 (AF353511), and TGEV strain Purdue (AJ271965). Group 2, BCoV strain Mebus (U00735), MHV type 2 (MHV-2; AF201929), MHV strain Penn 97-1 (AF208066), and MHV-A59 (X51939). Group 3, IBV strain Beaudette (M95169), IBV strain LX4 (AY338732), IBV strain BJ (AY319651). SARS-CoV strain Frankfurt-1 (AY291315) is not classified in any of these groups but is most closely related to group 2 coronaviruses. Outgroup, equine Berne torovirus (EToV; X52374). Regions that were poorly conserved in the manually edited multiple protein sequence alignment were deleted from the alignment. All columns containing gaps were removed. The resulting alignment included 2,083 characters (1,122 being parsimony informative) and contained the meld of the following HCoV-OC43 fragments: 13686-13721, 13737-13793, 13797-13820, 13857-13889, 13869-13994, 14013-14090, 14127-14174, 14247-14390, 14397-14594, 14598-14756, 14766-14855, 14859-15230, 15243-15443, 15480-15674, 15684-15719, 15729-15764, 15786-15854, 15864-15989, 16023-16358, 16374-16715, 16719-16898, 16902-17093, 17115-17258, 17268-17336, 17340-17363, 17379-17501, 17535-17561, 17568-17825, 17925-18008, 18018-18032, 18069-18101, 18207-18440, 18450-18527, 18531-18563, 18576-18602, 18612-18929, 18942-19010, 19,026-19139, 19143-19259, 19284-19466, 19479-19625, 19686-19793, 20289-20318, 20370-20603, 20625-20708, 20718-20762, 20769-20885, 20907-21008, 21045-21125, 21135-21233, 21252-21296, 21309-21431, and 21438-21476. The frequencies of occurrence of particular bifurcations (percentage of 10,000 bootstrap replicate calculations) are indicated at the nodes.

FIG. 5.

Maximum-likelihood phylogenetic tree of spike gene nucleotide sequences of HCoV-OC43 and several BCoV strains for which the date of isolation was known.

FIG. 6.

Results of the evolutionary rate analysis. Line a, linear regression of root-to-tip divergence (y axis) versus sampling time (x axis). The point at which the regression line crosses the time axis indicates the TMRCA (1891). Line b, maximum-likelihood estimate (1873) with 95% confidence intervals (1815 to 1899) for the TMRCA. Curve c, marginal posterior probability (right y axis) for the TMRCA obtained by using the Bayesian coalescent approach. The vertical bars in the distribution represent the 95% highest posterior density interval. Dates of isolation of HCoV-OC43 and BCoV strains are indicated by grey dots.