Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus

Abstract

A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

Keywords: computer-aided design; coronavirus infections; disease transmission; evolution; high-throughput nucleotide sequencing; infectious; molecular; respiratory tract infections; viruses; zoonoses.

Figure 1

A) Primers designed for reverse transcription and overlapping PCR amplification of the novel coronavirus (CoV). Dots indicate the predicted binding site of each primer along the EMC/2012 genome (x-axis). Gray bars indicate predicted amplicon lengths. Amplicon numbers are indicated beside each set of products. B) PCR products (3 µL of a 25-µL reaction) were resolved by electrophoresis on a 0.6% agarose gel and visualized by ethidium bromide staining. Lane M is the molecular weight marker (sizes indicated at left), Lanes 1–15 show the products of the amplicons depicted in Panel A. Lane C is the reagent PCR control.

Figure 2

A) Sequence differences among EMC/2012, England/Qatar/2012 and England1. The sequences of the 3 genomes were aligned, and differences between the sequence of England/Qatar/2012 and England1 (upper row) or EMC/2012 and England1 (lower row) were tabulated. The colored vertical ticks indicate nucleotide differences (change to A: red, change to T: dark red, change to G: indigo, change to C: medium blue, gap: gray). B) Non-consensus variants detected in the virus sample. The Illumina readset (Illumina, San Diego, CA, USA) for England/Qatar/2012 was mapped to the England/Qatar/2012 genome. Nucleotide positions showing nucleotides that differed from the consensus were tabulated. Colored dots indicate nucleotide positions with >1%–5% (gray), >5%–10% (orange), and >10% (red) nonconsensus variants. Positions with >5% variation and observed nucleotides are as follows: 14311: T, 92.07; G, 7.81; C, 0.12. 18460: C, 94.03; T, 5.97. 18692: G, 85.35; T, 14.65. 22385: G, 83.59; A, 16.41; C, 0.01. 26554: A, 90.85; G, 9.00; T, 0.15. C) Open reading frame (ORF) analysis of England/Qatar/2012. The positions of stop codons in each of the 3 forward ORFs are indicated by vertical black lines; the presence of ORFs of >75 aa are indicated by a closed box. ORF nomenclature is from Van Boheemen et al. (3).

Figure 3

Phylogenetic analyses of coronaviruses. A–F) Maximum-likelihood phylogenies of combined and each individual open reading frame (ORF), including ORF 1ab, S, E, M, and N. Previously defined viral lineages (group 1, 2a, 2b, 2c, 2d, 3, and 4) are highlighted by color blocks and described in (A). G) Phylogenetic analyses on the partial RNA-dependent RNA polymerase sequence region (396 bp) of coronaviruses (CoVs). Partial gene sequences from other CoVs that are closely related to the novel human betaCoVs are included and marked with asterisks. Bootstrap analysis of 1,000 replicates was performed for each phylogeny. The novel human betaCoVs studied here are shown in red. Scale bar indicates nucleotide substitutions per site.

Figure 4

tMRCA analysis across a range of fixed evolutionary rates. The tMRCA of EMC/2012 and England/Qatar/2012 estimated from fixing a range of genomic evolutionary rates (1 × 10⁻⁴, 2 × 10⁻⁴, 5 × 10⁻⁴, 1 × 10⁻³, 2 × 10⁻³, and 5 × 10⁻³ substitutions/site/year) are shown in data points with vertical error bars (95% highest posterior density). Evolutionary rate estimates of human CoV genome and genes in the literature are indicated at the bottom of the plot (mean or point estimate as a dot, 95% CIs of estimate as a square bracket). tMRCA, time to the most recent common ancestor; CoV, coronavirus; SARS, severe acute respiratory syndrome; ORF, open reading frame.