title: Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus.
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.
authors.affiliation: Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea;; Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Company Limited, Seoul 06351, South Korea.
authors.forename: Donghyun
authors.initials: D
authors.lastname: Park
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Hee Jae
authors.identifier: 0000-0001-8999-7561
authors.initials: HJ
authors.lastname: Huh
authors.affiliation: Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea;; Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Company Limited, Seoul 06351, South Korea.
authors.forename: Yeon Jeong
authors.identifier: 0000-0003-2292-1372
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea;; Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Company Limited, Seoul 06351, South Korea.
authors.forename: Dae-Soon
authors.initials: DS
authors.lastname: Son
authors.affiliation: Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea.
authors.forename: Hyo-Jeong
authors.initials: HJ
authors.lastname: Jeon
authors.affiliation: Department of Medical Biotechnology, College of Biomedical Science, and Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 24341, South Korea.
authors.forename: Eu-Hyun
authors.initials: EH
authors.lastname: Im
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Nam Yong
authors.initials: NY
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Eun-Suk
authors.initials: ES
authors.lastname: Kang
authors.affiliation: Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Cheol In
authors.initials: CI
authors.lastname: Kang
authors.affiliation: Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Doo Ryeon
authors.initials: DR
authors.lastname: Chung
authors.affiliation: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.
authors.forename: Jin-Hyun
authors.initials: JH
authors.lastname: Ahn
authors.affiliation: Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Kyong Ran
authors.initials: KR
authors.lastname: Peck
authors.affiliation: Department of Medical Biotechnology, College of Biomedical Science, and Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 24341, South Korea.
authors.forename: Sun Shim
authors.initials: SS
authors.lastname: Choi
authors.affiliation: Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Yae-Jean
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, South Korea.
authors.forename: Chang-Seok
authors.initials: CS
authors.lastname: Ki
authors.affiliation: Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea;; Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.
authors.forename: Woong-Yang
authors.initials: WY
authors.lastname: Park
mesh_terms: D017934:Coronavirus / Q000235:genetics; D018352:Coronavirus Infections / Q000453:epidemiology; D004196:Disease Outbreaks; D005260:Female; D018740:Genetic Heterogeneity; D016679:Genome, Viral / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011991:Receptors, Virus / Q000737:chemistry; D056910:Republic of Korea / Q000453:epidemiology; D017422:Sequence Analysis, DNA; D064370:Spike Glycoprotein, Coronavirus / Q000737:chemistry / Q000235:genetics*; D000073336:Whole Genome Sequencing / Q000379:methods
chemical_list: D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus
keywords: recurrent upper and lower respiratory tract infections