[Molecular epidemiological study of human coronavirus OC43 in Shanghai from 2009-2016].

Objective: To understand the epidemiological characteristics of Human coronavirus (HCoV), the patterns of emergence and circulation, and the genotype distribution of human coronavirus OC43 (HCoV-OC43) from November, 2009 to April, 2016 in Shanghai. Methods: A total of 6 059 respiratory specimens, including pharyngeal swab, sputum, nasopharyngeal aspirates and alveolar lavage fluid, as well as relative clinical data were collected from patients with acute respiratory infections from seven sentinel hospitals during November, 2009 to April, 2016 in Shanghai. Respiratory specimens were tested by RT-PCR with HCoV-conserved primers and subsequently genotyped by DNA sequencing. Using specific primers to amplify and sequence full-length Spike (S), RNA-dependent RNA polymerase (RDRP) and nucleocapsid (N) gene from HCoV-OC43 positive samples. Further genotype and phylogenetic analysis of HCoV-OC43 were performed by conducting phylogenetic trees. Results: Among 6 059 patients, the total frequency of HCoV was 63 (1.04%), in which HCoV-OC43 was the most frequently detected species with 34 positive samples, followed by human coronavirus 229E (HCoV-229E) and human coronavirus HKU1 (HCoV-HKU1) with 18 and 10 positive sample respectively. However, other HCoV like human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV), were not been detected, which illustrated that HCoV-OC43 was the dominant subtype. The full-length of S, RDRP and N gene were obtained from 29 HCoV-OC43 positive samples. According to the sequence-analysis, 27 of which was genotype D, 2 of which was genotype B and others genotype, including genotype E, F and G, were not detected. The result indicated that the genotype D may be the dominant genotype. Further analysis of S protein that help HCoV-OC43 to entry host cell and stimulate the host immune system to produce neutralizing antibody found that two important functional domains in S protein, N-terminal domain (NTD) and receptor-binding domain (RBD) contained more amino acid substitution and positive selection sites, accompanied with amino acid insertion/deletion. 13 positive selection sites were all located in the NTD or RBD, 10 of which were located in the NTD and 3 in the RBD. Conclusion: Human coronavirus OC43 was the major circulation human coronaviurs in Shanghai from 2009 to 2016, in which genotype D was the dominant genotype. NTD and RBD regions of the S protein were hypervariable region during HCoV-OC43 evolution, and had amino acid substitutions as well as amino acid insertion/deletion.