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

Park, Donghyun; Huh, Hee Jae; Kim, Yeon Jeong; Son, Dae-Soon; Jeon, Hyo-Jeong; Im, Eu-Hyun; Kim, Jong-Won; Lee, Nam Yong; Kang, Eun-Suk; Kang, Cheol In; Chung, Doo Ryeon; Ahn, Jin-Hyun; Peck, Kyong Ran; Choi, Sun Shim; Kim, Yae-Jean; Ki, Chang-Seok; Park, Woong-Yang

In this study, we demonstrated the intrapatient heterogeneity of MERS-CoVs, ruling out the possibility of technical noise. Based on the statistical test described above, we first selected 16 non-consensus variant candidates with frequencies significantly higher than those from technical noise. Among those variant candidates, we tested nine candidates by Sanger sequencing and validated seven variants, resulting in a 78% validation rate. After removal of the two nonvalidated candidates, we ultimately listed a total of 14 non-consensus variants, expecting an 89% validation rate. There was a tight correlation between the frequencies of the two variants at positions 22984 and 23041. These results suggest that technical artifacts were highly unlikely to have generated such non-consensus variants. Our analysis was limited to relatively high-frequency variants because the sequencing error rate presents certain limitations for the study of RNA as opposed to DNA viruses. Technical advances such as the use of unique molecular identifiers to remove PCR errors may produce more accurate descriptions of MERS-CoV populations in patients (Kinde et al. 2011) . Despite the limitations, we demonstrated the presence of heterogeneous MERS-CoV populations in clinical samples from MERS-CoV-infected patients.