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Case Reports
. 2016 Nov;2(6):a001214.
doi: 10.1101/mcs.a001214.

Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus

Donghyun Park  1 , Hee Jae Huh  2 , Yeon Jeong Kim  1 , Dae-Soon Son  1 , Hyo-Jeong Jeon  3 , Eu-Hyun Im  4 , Jong-Won Kim  2 , Nam Yong Lee  2 , Eun-Suk Kang  2 , Cheol In Kang  5 , Doo Ryeon Chung  5 , Jin-Hyun Ahn  6 , Kyong Ran Peck  7 , Sun Shim Choi  4 , Yae-Jean Kim  8 , Chang-Seok Ki  2 , Woong-Yang Park  9
Affiliations
Case Reports

Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus

Donghyun Park et al. Cold Spring Harb Mol Case Stud. 2016 Nov.

Abstract

Genome sequence analysis of Middle East respiratory syndrome coronavirus (MERS-CoV) variants from patient specimens has revealed the evolutionary dynamics and mechanisms of pathogenesis of the virus. However, most studies have analyzed the consensus sequences of MERS-CoVs, precluding an investigation of intrapatient heterogeneity. Here, we analyzed non-consensus sequences to characterize intrapatient heterogeneity in cases associated with the 2015 outbreak of MERS in South Korea. Deep-sequencing analysis of MERS-CoV genomes performed on specimens from eight patients revealed significant intrapatient variation; therefore, sequence heterogeneity was further analyzed using _targeted deep sequencing. A total of 35 specimens from 24 patients (including a super-spreader) were sequenced to detect and analyze variants displaying intrapatient heterogeneity. Based on the analysis of non-consensus sequences, we demonstrated the intrapatient heterogeneity of MERS-CoVs, with the highest level in the super-spreader specimen. The heterogeneity could be transmitted in a close association with variation in the consensus sequences, suggesting the occurrence of multiple MERS-CoV infections. Analysis of intrapatient heterogeneity revealed a relationship between D510G and I529T mutations in the receptor-binding domain (RBD) of the viral spike glycoprotein. These two mutations have been reported to reduce the affinity of the RBD for human CD26. Notably, although the frequency of both D510G and I529T varied greatly among specimens, the combined frequency of the single mutants was consistently high (87.7% ± 1.9% on average). Concurrently, the frequency of occurrence of the wild type at the two positions was only 6.5% ± 1.7% on average, supporting the hypothesis that selection pressure exerted by the host immune response played a critical role in shaping genetic variants and their interaction in human MERS-CoVs during the outbreak.

Keywords: recurrent upper and lower respiratory tract infections.

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Figures

Figure 1.
Figure 1.
Reproducibility assessment for the detection of non–consensus variants. Correlation of allele frequencies between sample replicates from (A) Patient 80 and (B) Patient 162. (C) Representative validation by the Sanger sequencing method.
Figure 2.
Figure 2.
Summary of variable sites among 35 samples from 24 patients. At each position, a mixed base is displayed and colored if its frequency was >10%. For mixed bases, the font size reflects the frequency of that base. Mod, moderate; Sev, severe.
Figure 3.
Figure 3.
The fraction of mutant and wild-type alleles at positions 510 and 529 of the spike glycoprotein. To measure the frequencies of the mutant and the wild-type alleles, sequencing reads covering positions 510 and 529 were selected. For each specimen, the read counts supporting each type were divided by the total read counts. The frequencies of the double mutants (D510G, I529T), the single mutants (D510G, I529 and D510, I529T), and the wild-type (D510, I529) alleles are plotted for 35 specimens sorted by sample number.
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