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. 2013 Dec 14;382(9909):1993-2002.
doi: 10.1016/S0140-6736(13)61887-5. Epub 2013 Sep 20.

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study

Matthew Cotten  1 , Simon J Watson  1 , Paul Kellam  2 , Abdullah A Al-Rabeeah  3 , Hatem Q Makhdoom  4 , Abdullah Assiri  3 , Jaffar A Al-Tawfiq  5 , Rafat F Alhakeem  3 , Hossam Madani  4 , Fahad A AlRabiah  6 , Sami Al Hajjar  6 , Wafa N Al-nassir  7 , Ali Albarrak  8 , Hesham Flemban  9 , Hanan H Balkhy  10 , Sarah Alsubaie  11 , Anne L Palser  1 , Astrid Gall  1 , Rachael Bashford-Rogers  1 , Andrew Rambaut  12 , Alimuddin I Zumla  13 , Ziad A Memish  14
Affiliations

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study

Matthew Cotten et al. Lancet. .

Abstract

Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

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Figures

Figure 1
Figure 1
Deep sequencing process Products of RT-PCR amplification of MERS-CoV patient RNA (A). All expected products are 2–3 kb in length, with amplicons 9/11 and 10/11 producing 6 and 4 kb products. DNA marker sizes are shown in kb. (B) 100 000 random reads (a tenth of the entire sample dataset for Al-Hasa_19) were mapped to the Al-Hasa_1_2013 genome. The histogram shows the positions of each read across the genome with 100–1000 reads at each position across the entire genome. The actual coverage is ten times greater, yielding 1000–10 000 coverage. Open reading frame (ORF) map (C) of a successfully assembled Al-Hasa_19 genome. An open box shows open reading frames of 100 codons or greater in length. Stop codons are shown by vertical black lines. The first ATG in each ORF is shown by a vertical red line. MERS-CoV=Middle East respiratory syndrome coronavirus.
Figure 2
Figure 2
Bayesian-inferred phylogeny of all 21 new sequences Combined with the published genomes (EMC/2012 [GenBank number JX869059], Jordan-N3 [KC776174], Munich/AbuDhabi [KF192507], England-Qatar_2012 [KC667074], Al-Hasa_1_2013 [KF186567], Al-Hasa_2_2013 [KF186566], Al-Hasa_3_2013 [KF186565], Al-Hasa_4_2013 [KF186564], and England2-HPA [no number available]). The single letter patient codes from Assiri and colleagues are given where appropriate. Clade A, clade B, and the Al-Hasa cluster are marked with vertical bars. Aminoacid changes along the internal branches were established though likelihood-based ancestral state reconstruction. These are shown above the branches and colour-coded by ORF. The scale bar below the phylogeny shows the genetic distance, in substitutions per site, from the arbitrary midpoint root. Bayesian posterior probabilities for each clade are given above the relevant node. MERS-CoV=Middle East respiratory syndrome coronavirus. ORF=open reading frame.
Figure 3
Figure 3
Time-resolved phylogenetic tree Based on the concatenated coding regions of the MERS-CoV genome. Branch colours show the most probable geographical location for that branch, established with a discrete traits model implemented in BEAST version 1.7.5. Change in branch colour shows a change in geographical location during its evolutionary history. Node labels show the posterior probability for the inferred geographical location at that node. Asterisks show nodes with >0·95 posterior probability support for that clade. The posterior probabilities on the geographical location of the root are Al-Hofuf 0·03, Riyadh 0·48, Buraidah 0·04, Bisha 0·18, Abu Dhabi 0·05, Doha 0·13, Hafr-Al-Batin 0·04, and Makkah 0·04. MERS-CoV=Middle East respiratory syndrome coronavirus
Figure 4
Figure 4
Transmission analysis Proposed transmission network for all available Al-Hasa MERS-CoV genomes. The light blue nodes show genomes with at least one statistically possible transmission linkage to a known MERS-CoV genome (transmission cases). The coloured nodes in the bottom panel (sporadic cases) are MERS-CoV genomes that cannot be linked by direct transmission to any known MERS-CoV case. Genomes are organised by date of sample collection, and stays in hospital A or D are shown by grey horizontal bars. Red squares show potential transmission, either by shared dialysis session or by direct exposure to a known MERS case. Black arrows show proposed transmission pairs supported by the sequence data, dashed red arrows show proposed transmission pairs not supported by the sequence data. The expected number of differences between each pair of sequences was calculated as the product of the interval between sampling, the evolutionary rate of the virus, and the maximum common length between the two virus genomes. Assuming that the number of differences between two sequences over a length of time is Poisson distributed, with λ equal to the expected number of mutations, the probability of getting the observed number of mutations between the two sequences by chance can be calculated from the cumulative density function of the Poisson distribution. A transmission pair was rejected if the cumulative probability value was less than 0·05. A Bonferroni correction was applied to account for multiple comparisons, resulting in an adjusted significance level of 3·85 × 10−3. ASR=ancestral sequence reconstruction as defined in table 2. MERS-CoV=Middle East respiratory syndrome coronavirus.
Figure 5
Figure 5
Single nucleotide differences between the Bisha_1_2012 genome and all other genomes Difference shown by vertical coloured bars. Grey=gap in the query sequence. Orange=change to A. Red=change to T. Blue=change to G. Purple=change to C. The ORF map of the MERS-CoV genome with the major ORFs marked is provided above the figure for reference. MERS-CoV=Middle East respiratory syndrome coronavirus. ORF=open reading frame.
Figure 6
Figure 6
Geographical distribution of genotypes MERS-CoV genotypes (coloured circles with genome names) are shown near the site of probable infection (white-filled circles). The 19 Al-Hasa sequences are shown by a single blue-filled circle. The genetically related genotypes from distinct locations (Bisha_1_2012, Riyadh_1_2012 and Riyadh_3_2013, England_Qatar_2012, Munich_AbuDhabi_2013) are linked with blue lines. MERS-CoV=Middle East respiratory syndrome coronavirus.
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