Two novel human coronavirus OC43 genotypes circulating in hospitalized children with pneumonia in China

ABSTRACT HCoV-OC43 is one of the mildly pathogenic coronaviruses with high infection rates in common population. Here, 43 HCoV-OC43 related cases with pneumonia were reported, corresponding genomes of HCoV-OC43 were obtained. Phylogenetic analyses based on complete genome, orf1ab and spike genes revealed that two novel genotypes of HCoV-OC43 have emerged in China. Obvious recombinant events also can be detected in the analysis of the evolutionary dynamics of novel HCoV-OC43 genotypes. Estimated divergence time analysis indicated that the two novel genotypes had apparently independent evolutionary routes. Efforts should be conducted for further investigation of genomic diversity and evolution analysis of mildly pathogenic coronaviruses.


Introduction
There are four mildly pathogenic coronaviruses that can infect humans, HCoV-OC43, 229E, NL63 and HKU1, that mainly cause mild and self-limiting respiratory tract infections [1][2][3][4]. These mildly pathogenic coronaviruses were circulated endemically, contributing to episodes of human common cold worldwide and severe infection in immunosuppressed patients and infants [5,6]. HCoV-OC43 is the most prevalent HCoV in respiratory tract infections, which is believed to associate with fatal encephalitis [7]. Gene sequence of HCoV-OC43 showed a high similarity to bovine coronavirus and canine coronavirus [8]. 9-O-acetyl-sialic acid is believed to be the receptor to HCoV-OC43, which is the common receptor to Bovine CoV and HCoV-HKU1 [9].
Due to the capacity of coronaviruses to adapt to different hosts and make high-frequency mutations spontaneously in spike, it is easier for coronavirus to cross species barriers and enhance infectivity through successive generations [10]. Thus, it is urgently necessary to conduct virus genomic diversity and evolution investigation, especially for mildly pathogenic coronaviruses [11]. Here, a cluster of HCoV-OC43 related hospitalized children were enrolled in this study. The further genomic phylogenetic and evolution analysis of obtained sequences suggested two novel HCoV-OC43 genotypes (genotypes J and K) emerged in China. The discovery of novel HCoV-OC43 genotypes in human sheds light on the evolutionary trend and potential health threats of mildly pathogenic coronaviruses.  to determine the most recent common ancestor (tMRCA) of the novel genotypes based on spike gene. Estimation of pairwise genetic distances was performed and analyzed by using the Neighbor-Joining method with 1000 bootstrap replicates in MEGA 5.1 software. Bootscan analysis in SimPlot software was used for potential recombination events detection.
After deleting overrepresented sequences, 38 HCoV-OC43 genome sequences of representative genotypes from the NCBI database with 43 HCoV-OC43 strains enrolled in this study were processed for the investigation of genetic relationship. As shown in Figure 1(A), all available HCoV-OC43 strains on NCBI can be divided into nine genotypes (A to I genotypes). However, two novel genotypes can be revealed after enrolling the genome sequences we acquired. Two novel genotypes, named genotype J and genotype K, occupy relatively independent positions in the phylogenetic tree. The time to a most recent common ancestor for the two genotypes was estimated to be between 4 and 6 years before the identification in 2014-2016. Besides, it is obvious that genotypes J and K were circulating simultaneously in 2019, and genotype K has become the dominant genotype gradually (Supplementary Table 1).
As shown in Figure 1(B), the genetic distance of the genotype J compared with genotypes A, B, C, D, E, F, G, and I were >0.5% (0.005 substitution/site), Phylogenetic trees based on spike and orf1ab gene showed different topologies associated with genotype J (Supplementary Figure 1). Recombination analysis also revealed that genotype J comes from the recombination between genotypes H and I (Figure 1(C), Supplementary Figure 1), while genotype K may be derived from genotype I. Above all, two novel genotypes of HCoV-OC43 associated with pneumonia emerged in China and showed different evolutionary routes.

Discussion
In this study, 43 sequences of HCoV-OC43 were enrolled for phylogenetic and evolution analysis, which revealed 2 novel genotypes circulating in China. HCoV-OC43 was circulated endemically, contributing to episodes of human common cold worldwide and severe infection in immunosuppressed patients and infants. However, the studies about HCoV-OC43 genomic diversity and evolution investigation are still rare due to the absence of enough genome sequence. There is no vaccine or specific treatment to protect against mildly pathogenic coronaviruses. Most people with mildly pathogenic human coronavirus illness will recover spontaneously, but people can have multiple infections in their lifetime. The discovery of two novel genotypes of HCoV-OC43 associated with pneumonia alarmed the significance of the monitoring common coronavirus variants.

Ethics statement
Sample collection and all experiments in the present study were performed with Ethical Approval given by Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.