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. 2006 Dec 15;364(5):964-73.
doi: 10.1016/j.jmb.2006.09.074. Epub 2006 Oct 3.

Mosaic structure of human coronavirus NL63, one thousand years of evolution

Krzysztof Pyrc  1 , Ronald Dijkman, Lea Deng, Maarten F Jebbink, Howard A Ross, Ben Berkhout, Lia van der Hoek
Affiliations

Mosaic structure of human coronavirus NL63, one thousand years of evolution

Krzysztof Pyrc et al. J Mol Biol. .

Abstract

Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.

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Figures

Figure 1
Figure 1
Molecular variability of HCoV-NL63 along the genome. (a) Frequency of polymorphic sites at the nucleotide level among four isolates of HCoV-NL63. (b)Frequency of polymorphic sites on the synonymous and non-synonymous positions among four isolates of HCoV-NL63. The analysis was done with a 500 nt non-overlapping window.
Figure 2
Figure 2
Discordance in phylogenetic clustering of different isolates of HCoV-NL63 at regions 3K, 6K and 21K. Phylogenetic trees were constructed as described in Materials and Methods using an UPGMA algorithm. The scale bar unit represents a 0.002 substitution per site. The trees were rooted with the sequences of it's closest relative: for 3K and 6K the HCoV-229E and for the 21K region the PEDV. Four completely sequenced isolates are marked with colored boxes, illustrating the discordance in clustering in different regions of the genome.
Figure 3
Figure 3
Discordance in clustering of different isolates of HCoV-NL63 at regions 3K, 6K and 21K. Three alignments of only variable sites subtracted from the original sequence with DnaSP 4.0 software, shows the discordance in clustering at different regions of the genome. Groups A and B were created arbitrarily to show the discordance. Group A was defined as the group that contains the Amsterdam 1 isolate.
Figure 4
Figure 4
Identification of recombination sites in the S gene. The alignment includes only subtracted variable sites. These variable sites were subtracted with DnaSP 4.0 software. The change of color represents the alternation of genetic clustering between isolates. The numbers at the top represent the beginning of the S gene (nt 20,472 in the Amsterdam 1 isolate), coordinates of recombination spots inside the S gene (nt 21,061–21,072 and 21,575–21,576 in the Amsterdam 1 isolate) and the 3′ terminus of the S gene (nt 24,542 in the Amsterdam 1 isolate).
Figure 5
Figure 5
Signs of interspecies recombination in the M gene. Similarity plot (a) and bootscan analysis with the Kimura (two-parameter) distance model, neighbor-joining tree model and 500 bootstrap replicates (b) of the HCoV-NL63 M protein. (c) Analysis of the substitution pattern on the synonymous and non-synonymous level between the M gene of HCoV-NL63 and HCoV-229E. The window used was 40 nucleotides with a step of one nucleotide.
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