| The recent emerging of Sever Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) attracted global attention. The animal-originated feature of the two emerging CoVs raised concerns of the molecular evolution characteristics and propagation characteristics of CoVs. However, as the short prevalent period, it is limited to study the evolution characteristics of these two viruses in natural state. Human CoV-OC43(HCoV-OC43) is one of the most commonly epidemic HCoV, associated with human infections and one of the earliest discovered human CoV, is of animal origin and belongs to β genus as well as SARS-CoV and MERS-CoV. Therefore, the study of its evolutionary characteristics is likely to give clues for revealing the evolutionary characteristics of SARS-CoV, MERS-CoV and other new CoVs with the probability of human infections.Based on HCoV positive respiratory specimens collected from various geographical regions of China during2005to2012, HCoV-OC43-positive samples were confirmed and the sequences of genes(S, RdRp and N gene) or complete genomes were obtained based on PCR methods. Further phylogenetic analysis showed that HCoV-OC43genotype B, C and D were co-epidemic in China and an emerging genotype E was discovered since2010. More specifically, genotype B reemerged in2010after its absence since2006, genotype C was not detected after2006, genotype D was the predominant epidemic strain since2007to2009. The new genotype E emerged in2010generated from the recombination of strains from genotype B, C and D with recombination sites in nsp2/nsp3, nsp6/nsp7, nsp12/nsp13, ns5a/E and M/N junction. These findings imply the possibility that the genotype replacement is one of the major reasons for HCoV-OC43to maintain its epidemic ability among the population. The age distributions in different genotypes were different significantly (P=0.0094). Genotype D showed a broad age range (0.1to90year old), mainly in young adults. Genotype B (0.6-21years old) were mainly in infants and young adults. The three patients positive on genotype C were elder adults with age of58,72and88years old. The patients positive on genotype E (0.8-2.7years old) were all infants.To further reveal the evolutionary variability of HCoV-OC43, molecular clock, selection pressure and the temporal evolution routes of positive selection sites were analyzed to systemically investigate the evolutionary dynamics of each genotype. The results show that in all genotypes of HCoV-OC43, the spike gene of the predominantly epidemic genotype D have a higher rate of base substitutions and the most abundant (twenty-five) positive selection sites, of which seven mutation hotspots on the timing of the separation nodes of different branches. Homologous modeling of the three dimensional structure of its N-terminal domain (NTD) demonstrate that six mutation hotspots located in the N-terminal or around the crucial carbohydrate-binding sites. Regular amino acids mutations in NTD was also existed in genotype B in the re-epidemic strains after2012, indicating that the mutation in NTD may prompt genotype D adapted to the host immune pressure to maintain its epidemic predominance.In summary, this study reveals the epidemic HCoV-OC43genotypes in our country, phylogenetic analysis suggests that genetic recombination dynamics may be the main reason for genotype shift to maintain its dominant epidemic, and mutations of key amino acid sites are the reason why certain genotype could escape immune pressure and circulate for a very long time among the population. The amino acid mutation of NTD region and recombination hotspots may provide an important basis for elucidating the evolutionary characteristics of CoV.
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