Molecular epidemiology and evolutionary histories of human coronavirus OC43 and HKU1 among patients with upper respiratory tract infections in Kuala Lumpur, Malaysia

Abstract

Background: Despite the worldwide circulation of human coronavirus OC43 (HCoV-OC43) and HKU1 (HCoV-HKU1), data on their molecular epidemiology and evolutionary dynamics in the tropical Southeast Asia region is lacking.

Methods: The study aimed to investigate the genetic diversity, temporal distribution, population history and clinical symptoms of betacoronavirus infections in Kuala Lumpur, Malaysia between 2012 and 2013. A total of 2,060 adults presented with acute respiratory symptoms were screened for the presence of betacoronaviruses using multiplex PCR. The spike glycoprotein, nucleocapsid and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference.

Results: A total of 48/2060 (2.4 %) specimens were tested positive for HCoV-OC43 (1.3 %) and HCoV-HKU1 (1.1 %). Both HCoV-OC43 and HCoV-HKU1 were co-circulating throughout the year, with the lowest detection rates reported in the October-January period. Phylogenetic analysis of the spike gene showed that the majority of HCoV-OC43 isolates were grouped into two previously undefined genotypes, provisionally assigned as novel lineage 1 and novel lineage 2. Sign of natural recombination was observed in these potentially novel lineages. Location mapping showed that the novel lineage 1 is currently circulating in Malaysia, Thailand, Japan and China, while novel lineage 2 can be found in Malaysia and China. Molecular dating showed the origin of HCoV-OC43 around late 1950s, before it diverged into genotypes A (1960s), B (1990s), and other genotypes (2000s). Phylogenetic analysis revealed that 27.3 % of the HCoV-HKU1 strains belong to genotype A while 72.7 % belongs to genotype B. The tree root of HCoV-HKU1 was similar to that of HCoV-OC43, with the tMRCA of genotypes A and B estimated around the 1990s and 2000s, respectively. Correlation of HCoV-OC43 and HCoV-HKU1 with the severity of respiratory symptoms was not observed.

Conclusions: The present study reported the molecular complexity and evolutionary dynamics of human betacoronaviruses among adults with acute respiratory symptoms in a tropical country. Two novel HCoV-OC43 genetic lineages were identified, warranting further investigation on their genotypic and phenotypic characteristics.

Fig. 1

Annual distribution of HCoV-OC43 and HCoV-HKU1 among adults with acute in Malaysia. The monthly detection of HCoV-OC43 and HCoV-HKU1 (right axis, in bars) and the total number of nasopharyngeal swabs screened (left axis, in solid line) between March 2012 and February 2013 were shown

Fig. 2

Maximum clade credibility (MCC) tree of HCoV-OC43 genotypes. Estimation of the time of the most recent common ancestors (tMRCA) with 95 % highest posterior density (95 % HPD) of HCoV-OC43 genotypes based on the spike gene (S1 domain) (848 bp). Data were analyzed under relaxed molecular clock with GTR + I substitution model and a constant size coalescent model implemented in BEAST. The Malaysian HCoV-OC43 isolates obtained in this study were color-coded and the HCoV-OC43 genotypes (a) to (e) as well as novel lineages 1 and 2 were indicated. The MCC posterior probability values were indicated on the nodes of each genotype

Fig. 3

Maximum clade credibility (MCC) tree of HCoV-HKU1 genotypes. Estimation of the time of the most recent common ancestors (tMRCA) with 95 % highest posterior density (95 % HPD) of HCoV-HKU1 genotypes based on the spike gene (S1 domain) (897 bp). Data were analyzed under relaxed molecular clock with GTR + I substitution model and a constant size coalescent model implemented in BEAST. The Malaysian HCoV-HKU1isolates obtained in this study were color-coded and the HCoV-HKU1 genotypes (a) to (c) were indicated. The MCC posterior probability values were indicated on the nodes of each genotype

Fig. 4

Recombination analyses of HCoV-OC43 novel lineages 1 and 2. Reference strains of HCoV-OC43 genotype A (ATCC VR-759), B (87309 Belgium 2003), and C (HK04-01) were used as the putative parental strains. The bootstrap values were plotted for a window of 160 bp moving in increments of 20 bp along the alignment. Samples 12MYKL0208, Niigata.JPN/11-764, CU-H967_2009, 892A/08 were used as representative sequences for novel lineage 1 in addition to 12MYKL0002, 12MYKL0760 and 12689/12 isolates as representatives for novel lineage 2