An observational, laboratory-based study of outbreaks of middle East respiratory syndrome coronavirus in Jeddah and Riyadh, kingdom of Saudi Arabia, 2014

Abstract

Background: In spring 2014, a sudden rise in the number of notified Middle East respiratory syndrome coronavirus (MERS-CoV) infections occurred across Saudi Arabia with a focus in Jeddah. Hypotheses to explain the outbreak pattern include increased surveillance, increased zoonotic transmission, nosocomial transmission, and changes in viral transmissibility, as well as diagnostic laboratory artifacts.

Methods: Diagnostic results from Jeddah Regional Laboratory were analyzed. Viruses from the Jeddah outbreak and viruses occurring during the same time in Riyadh, Al-Kharj, and Madinah were fully or partially sequenced. A set of 4 single-nucleotide polymorphisms distinctive to the Jeddah outbreak were determined from additional viruses. Viruses from Riyadh and Jeddah were isolated and studied in cell culture.

Results: Up to 481 samples were received per day for reverse transcription polymerase chain reaction (RT-PCR) testing. A laboratory proficiency assessment suggested positive and negative results to be reliable. Forty-nine percent of 168 positive-testing samples during the Jeddah outbreak stemmed from King Fahd Hospital. All viruses from Jeddah were monophyletic and similar, whereas viruses from Riyadh were paraphyletic and diverse. A hospital-associated transmission cluster, to which cases in Indiana (United States) and the Netherlands belonged, was discovered in Riyadh. One Jeddah-type virus was found in Riyadh, with matching travel history to Jeddah. Virus isolates representing outbreaks in Jeddah and Riyadh were not different from MERS-CoV EMC/2012 in replication, escape of interferon response, or serum neutralization.

Conclusions: Virus shedding and virus functions did not change significantly during the outbreak in Jeddah. These results suggest the outbreaks to have been caused by biologically unchanged viruses in connection with nosocomial transmission.

Keywords: MERS-coronavirus; nosocomial transmission; outbreak; transmission infection control; virus isolation.

Figure 1.

Summary of features of the outbreak as derived from Jeddah Regional Laboratory file data. A, Overall diagnostic requests. B, Positive cases (y axis = cases per day) in King Fahd Hospital (KFH) vs all other hospitals, recording 3-day intervals starting on March 26 and ending on April 28 2014. C and D, Distribution of cycle threshold (Ct) values in 1056 samples pertaining to investigations in cases in Jeddah without hospital number (n = 18 positive samples) vs 3799 samples with hospital number (n = 150 positive samples). Average Ct values in cases and contacts were 30 and 33.1, respectively (2-tailed t test, P < .009).

Figure 2.

Phylogenetic tree inferred using MrBayes [11] for the concatenated coding regions of 105 Middle East respiratory syndrome coronavirus genomes or partial genomes sampled from humans and camels. We employed a codon position-specific general time reversible (GTR) substitution model with γ-distributed rates among sites. Displayed is the majority consensus of 10 000 trees sampled from the posterior distribution with mean branch lengths. Posterior support is shown for nodes where <0.90. Sequences sampled from camels are denoted with a yellow circle, those from humans with a green circle. Sequences new to this study are labeled in bold. The cluster comprising viruses isolated from the Jeddah/Makkah hospitals in April 2014 are highlighted with a red box and those from the Prince Sultan Military Medical City, Riyadh, in March–April 2014 are highlighted in blue. For comparison, the Al-Hasa 2013 hospital outbreak [12] is highlighted in yellow and the 2013 Hafr-Al-Batin community outbreak [13] in green.

Figure 3.

Growth kinetics of Middle East respiratory syndrome coronavirus (MERS-CoV) isolates EMC/2012, Jeddah_10306, and Riyadh_683 in cell culture. VeroB4 and A459 cells were infected at a multiplicity of infection (MOI) of 1 (A and B, respectively) or MOI of 0.01 (C and D, respectively). Samples from the supernatant were taken at indicated time points, and virus growth was measured by real-time reverse transcription polymerase chain reaction. VeroB4 cells infected at an MOI of 1 (A) showed total cytopathogenic effect (CPE) at 48 hours postinfection (p.i.), terminating the experiment. A459 cells did not show any CPE even when infected at an MOI of 1 at 72 hours p.i. (B). E, Effect of pretreatment of cell cultures with type I interferon (IFN) at low or high dosage. F, Virus-neutralizing effect of human serum with known anti-MERS-CoV neutralizing antibody titer at different dilutions. Abbreviations: GE, genome equivalents; tCPE, total cytopathogenic effect.

Figure 4.

Virus shedding in patients. Cycle threshold (Ct) values during the outbreak in Jeddah. A and B, Frequency distribution of Ct values in Jeddah vs other cities. C, Ct values during the outbreak in Jeddah by week, starting on 26 March 2014.