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

Based on the tight correlation of base frequencies at positions 22984 and 23041, our data suggested that the double mutant carrying D510G and I529T is rare. Therefore, we selected sequencing reads covering both positions in each specimen to measure the frequencies of the double mutants (i.e., D510G, I529T), the single mutants (i.e., D510G, I529 and D510, I529T), and the wild type (i.e., D510, I529). On average, the frequency of the double mutant was only 1.4% ± 0.3%, whereas the single mutants and the wild type were present at 87.7% ± 1.9% and 6.5% ± 1.7%, respectively (Fig. 3) . Recently, both D510G and I529T mutations in RBD were shown to reduce its affinity for human CD26 compared with the wildtype RBD (Kim et al. 2016c ). Although the previous study did not test the binding affinity of the double mutant, in our data the two mutations are mutually exclusive, suggesting that the double mutant severely impairs viral fitness. Notably, although the frequency of each single mutant varied greatly among specimens, the combined frequency of both single mutants was consistently high in most samples. At the same time, the frequency of the wild type was no more than 10% in most samples (31 of 35 specimens). Thus, our data suggest that there was strong selection pressure favoring the variants of the spike glycoprotein with reduced affinity for the host receptor.