Prevalence and genetic diversity analysis of human coronaviruses among cross-border children

Liu, Peilin; Shi, Lei; Zhang, Wei; He, Jianan; Liu, Chunxiao; Zhao, Chunzhong; Kong, Siu Kai; Loo, Jacky Fong Chuen; Gu, Dayong; Hu, Longfei

There was virus co-infection between human coronaviruses with other common respiratory diseases. Adenovirus(Adv) and Rhinovirus(RV) were the most common two viruses that concomitantly detected with HCoVs in children younger than 6 years old. A total of 40 RdRp genes, including 20 for OC43, 15 for HKU1, 4 for NL63 and 1 for 229E, and 36 S genes, including 16 for OC43, 16 for HKU1 and 4 for NL63, were sequenced to perform phylogenetic analysis. Since there is a high conservative in RdRp gene, phylogenetic tree was not shown here. Multiple alignments results of Fig. 2 Phylogenetic analysis based on partial S genes of OC43, HKU1 and NL63. (I) Phylogenetic tree of OC43 S genes (2.2 kb) constructed with maximum likelihood; (II) Phylogenetic tree of HKU1 S genes (2.4 kb) constructed with maximum likelihood; (III) Phylogenetic tree of NL63 S genes (4.0 kb) constructed with neighbour-joining. Our samples were indicated with a red spot and others were used as referenced strains from complete genomes in GenBank. The strains indicated with "*" were clustered into genotype E, recombinant of B, C and D. The OC43 and NL63 phylogenetic trees were constructed using BCoV and Bat CoV respectively as outgroup RdRp genes indicated that OC43 and HKU1 possessed 99-100% nt identities. Largest divergences were observed in HKU1 coronaviruses, which possessed 96 -100% nt identities, but sequences detected in this study were 99-100% homologous to the published strains (Table 3) . For the phylogenetic trees constructed based on 31 S genes with a genomic length over 2 kb of four HCoVs, there was a high level of genetic diversity among those HCoVs (Fig. 2) . The OC43 coronaviruses were clustered into clade B (5,41.7%), clade D (6,50%) and clade E(1,8.3%) while none of the strains of genotype A and C was detected (Fig. 2I) . Besides, there was one OC43 sequence (SW1502-30/2015/Shenzhen, China) being clustered with a new recombination genotype E (CH) (Genbank accession no: KP198611.1). Similarly, HKU1 strains in this study were clustered into clade A (7,46.7%) and clade B (8,53.3%) and related to the sequences detected in Beijing and Hong Kong SAR respectively, while no clade C was detected (Fig. 2 II) . NL63 strains in this study were clustered into clade A (1,25.0%) and clade B (3,75.0%), related to strains isolated from USA and Denmark, while no clade C were detected neither (Fig. 2 III) . Moreover, we found nucleotide mutations in some of the samples (Fig. 3) . Three out of 8 OC43 coronaviruses of genotype D had a total of 11 bases substitution in nucleotide position 25,059-25,112 of S genes (Genbank accession number of referenced strain: KF923904.1) (Fig. 3a) . Six out of 8 HKU1 coronaviruses of genotype B were found with an extra insertion in nucleotide position 24,465 of genome leading to an additional amino acid "Threonine" insertion in amino acid position 510 of Spike (Genbank accession of referenced strain: DQ415911.1) (Fig. 3b ).