key: cord-0876181-0q92ntsm authors: Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Man Kim, Heui; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon title: Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015 date: 2016-01-03 journal: Emerg Infect Dis DOI: 10.3201/eid2201.151055 sha: cdbf113c93f807ee1853027325c17d3d00dd871c doc_id: 876181 cord_uid: 0q92ntsm An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations. We identified genetic variability of MERS-CoV S genes among infected persons in South Korea. Samples from 7 patients identified as positive for MERS-CoV were sequenced. These patients were identified by using sequences upstream of the envelope protein gene and open reading frame (ORF) 1a in real-time reverse transcription PCRs (5) ( Table 1) . Index case-patient 1 (PAT001) had traveled to Bahrain, the United Arab Emirates, and Saudi Arabia during April 24-May 4, 2015, and became symptomatic on May 11 after his return to South Korea (6) . After he visited a local clinic, his symptoms worsened, and he was hospitalized on May 15. During his hospitalization (May 15-17), PAT001 shared a room with PAT003 and the same ward with PAT009, PAT012, PAT013, and PAT015. PAT042 was admitted to the same hospital on May 19 ( Figure 1 ). PAT010 was the son of PAT003 and had visited his father in the hospital before traveling to China, where he became symptomatic and tested positive for MERS-CoV. PAT002 was the wife of PAT001 and cared for him during his illness. The S gene was amplified directly from nucleic acids extracted from respiratory specimens (6 patients) or a viral isolate (1 patient) (7) by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription was performed by using the Superscript III First-Strand Synthesis System (Life Technologies, Bleiswijk, the Netherlands) and virus-specific reverse primers. cDNA was amplified by using an overlapping PCR to generate products of 600-3,000 bp that covered the entire S gene. Resulting PCR amplicons were sequenced by using Sanger sequencing with an ABI 3730 Analyzer (Applied Biosystems, Foster City, CA, USA) or next-generation sequencing. For next-generation sequencing, PCR products were pooled and fragmented to an average of 300 bp, and a sequencing library was constructed by using the Illumina TruSeq Nano DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing was performed by using the Illumina MiSeq Platform (Illumina). To explore relationships of newly isolated virus strains from South Korea with other MERS-CoV strains, 131 reference MERS-CoV S gene sequences from GenBank and MERS-CoV Sequences June 2015 (http://tinyurl. com/MERS-CoV-4Jun15) (8) and 8 strains from South Korea, including a sequence from PAT010 (ChinaGD01; Chinese Centers for Disease Control and Prevention, Beijing, China), were aligned by using MUSCLE software (9) . This alignment was used for subsequent phylogenetic analysis. A phylogenetic tree was constructed by using the maximum-likelihood method with a bootstrap value of 1,000 and RAxML software (10) . All 8 ORFs from virus isolates obtained during the outbreak in South Korea were most closely related to ORFs of the recently isolated 2015 Riyadh clade, but isolates from South Korea constituted a novel branch, which was supported by a bootstrap value of 87% ( Figure 2 ). Phylogenetic data indicated that virus isolates from other patients originated from virus isolates from the index case-patient. These data also showed that strains detected in 2015 formed 2 groups: KSA-2466-like viruses and KKUH_0734-like viruses. Viruses from South Korea isolated in 2015 clustered with 1 sublineage of KKUH_0734-like viruses from Saudi Arabia. Nucleotide sequence comparisons with 131 reference MERS-CoV S genes showed that the clade from South Korea had highest identity (99.68%-99.9%) with recently circulating strains from Riyadh isolated in 2015. Strains from South Korea had 8 novel nucleotide substitutions (C183G, A409C, T1586C, G1588C, T1848C, G1886A, T3177C, and C3267T) that are unique to the South Korea lineage and share nucleotide substitution T258C with some viruses from Saudi Arabia detected earlier in 2015 ( Table 2 ). T3177C and C3267T mutations were observed only in all viruses from South Korea. Of the 8 nucleotide substitutions, 4 (A409C, T1586C, G1588C, and G1886A) were nonsynonymous and resulted in 4 amino acid changes (S137R, I529T, V530L, and R629H) ( Table 2) . Among these mutations, 2 nonsynonymous variants (S137R and V530L) were identified in isolates from PAT002 after the third passage in Vero cells and were assumed to be cell culture-adaptive mutations (11) . The I529T and V530L mutations were located in the RBD, but not at the RBD-DPP4 receptor interface (3). The R629H mutation was situated outside the RBD. However, on the basis of only these results, we could not determine whether these amino acid substitutions affected receptorbinding affinity between human DPP4 receptor and MERS-CoV S protein. To understand the rate at which virus genetic diversification occurred during the outbreak in South Korea, we used the Bayesian-Markov Chain Monte Carlo method in The S gene was estimated to evolve at mean rate of 6.72 × 10 -3 substitutions/site/year (95% highest posterior density [HPD] 5.59−6.93 × 10 -3 substitutions/site/year). This mutation rate for the S gene was higher than that for complete MERV-CoV genomes in other studies: 1.12 × 10 -3 substitutions/site/year (95% HPD 8.76 × 10 −4 -1.37 × 10 -3 substitutions/site/year) (12) and 9.29 × 10 −4 substitutions/site/ year (95% HPD 7.19 × 10 −4 -1.15 × 10 -3 substitutions/site/ year) (13) . However, more data are required to demonstrate the pattern of MERS-CoV evolution during the outbreak in South Korea because results are limited by a relatively low number of sequences, short selected time points, examination of only the S gene region, and different sequencing methods. Accurate genome sequencing can identify spatiotemporal patterns that help understand dynamics of rapid spread of MERS-CoV infection. We report S glycoprotein gene sequences of MERS-CoV from 8 patients and a strain cultured in Vero cells. Genetic information obtained is useful for understanding the evolutionary history of MERS-CoV. On the basis of our phylogenetic analyses, virus sequences of strains isolated in South Korea in 2015 form a unique clade. Genetic variations elucidated in this study show an unreported sequence in the RBD, which suggests that MERS-CoV circulating in South Korea during the outbreak in 2015 has higher genetic variability and mutation rates. However, we cannot conclude that deleterious effects promoting spread of infection will occur because of these mutations. Additional genetic information will resolve precise characteristics of the MERS-CoV obtained during the outbreak in South Korea. 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