Diversity and Evolutionary Histories of Human Coronaviruses NL63 and 229E Associated with Acute Upper Respiratory Tract Symptoms in Kuala Lumpur, Malaysia

Abstract

The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region.

Figure 1.

Annual distribution of HCoV-NL63 and HCoV-229E among adults with acute upper respiratory tract infections in Kuala Lumpur, Malaysia. The total number of nasopharyngeal swabs screened and the monthly distribution of HCoV-NL63 and HCoV-229E between March 2012 and February 2013 were presented.

Figure 2.

Maximum clade credibility tree of HCoV-NL63. Spike gene (S1 domain) sequences (1,383 nucleotides) were analyzed under the relaxed molecular clock with a GTR+I substitution model and a constant size coalescent model implemented in BEAST. Posterior probability values and the estimation of the time of the most recent common ancestors with 95% highest posterior density were indicated on major nodes. The HCoV-NL63 sequences obtained in this study were color coded and HCoV-NL63 genotypes A–C were indicated; green = genotype A, blue = genotype B, and red = genotype C. The recombinant genotype is indicated by purple color. The sampling site for each sequence was indicated by codes for the representation of countries. Country codes are as follows; MY = Malaysia; US = United States; JP = Japan; NL = Netherlands; CN = China.

Figure 3.

Maximum clade credibility tree of HCoV-229E. Spike gene (S1 domain) sequences (855 nucleotides) were analyzed under the relaxed molecular clock with a GTR+I substitution model and a constant size coalescent model implemented in BEAST. Posterior probability values and the estimation of the time to the most recent common ancestors with 95% highest posterior density were indicated on the major nodes. The HCoV-229E sequences obtained in this study were color coded and the HCoV-229E groups 1–4 were indicated green = genotype 1, red = genotype 2, blue = genotype 3, and purple = genotype 4. The sampling site for each sequence was indicated by codes for the representation of countries. Country codes are as follows; MY = Malaysia; US = United States; JP = Japan; NL = Netherlands; CN = China; AU = Australia; IT = Italy.