Transmission and evolutionary dynamics of human coronavirus OC43 strains in coastal Kenya investigated by partial spike sequence analysis, 2015-16.
ABSTRACT: Human coronavirus OC43 (HCoV-OC43) is a major contributor to seasonal outbreaks of acute respiratory illness (ARI). The origins of locally circulating HCoV-OC43 strains and characteristics of their genetic diversity are unknown for most settings despite significance to effective HCoV control strategies. Between December 2015 and June 2016, we undertook ARI surveillance in coastal Kenya in nine outpatients and one inpatient health facility (HF). Ninety-two patient samples tested HCoV-OC43 positive and forty (43.5%) were successfully sequenced in spike (S) gene region (2,864 long, ?70%). Phylogenetic analysis confirmed co-circulation of two distinct HCoV-OC43 clades that closely clustered with genotype G (n?=?34, 85%) and genotype H (n?=?6, 15%) reference strains. Local viruses within the same clade displayed low genetic diversity yielding identical sequences in multiple HF. Furthermore, the newly sequenced Kenyan viruses showed close phylogenetic relationship to other contemporaneous sampled strains (2015-16) including those originating from distant places (e.g. USA and China). Using a genetic similarity threshold of 99.1 per cent at nucleotide level, the HCoV-OC43 strains sampled globally between 1967 and 2019 fell into nine sequence clusters. Notably, some of these clusters appeared to have become extinct, or occurred only sporadically in a few geographical areas while others persisted globally for multiple years. In conclusion, we found that HCoV-OC43 strains spread rapidly both locally and across the globe with limited genetic evolution in the spike gene. Full-genome sequences that are spatio-temporally representative are required to advance understanding of the transmission pathways of this important human respiratory pathogen.
Project description:To determine the prevalence, epidemiology and genetic diversity of human coronavirus OC43 (HCoV-OC43) among adult patients with acute respiratory infections (ARI) in Beijing,five hundred and fifty-nine nasopharyngeal swab samples were collected from adult patients with ARI in Beijing. The prevalence of HCoV-OC43 infection among these patients was assessed using two different OneStep reverse transcription polymerase chain reaction (RT-PCR) assays. The epidemiological profiles of the patients with HCoV-OC43 infection were described. Partial S and N genes of HCoV-OC43 circulating strains were sequenced followed by phylogenetic analysis and amino acid alignment. Our results showed that the prevalence of HCoV-OC43 infection was 12.52% (95% CI: 9.78-15.26%), and the epidemic peak occurred in autumn. Fifty partial S and 40 partial N fragments were obtained from these patients. Phylogenetic analysis based on neighbour-joining method showed that at least three distinct clusters (A, B, C/D) of HCoV-OC43 strains were circulating among adult patients with ARI in Beijing. In addition, some novel unique clusters (UNT) of HCoV-OC43 were found in the S- and N-based phylogenetic trees. Furthermore, consensus amino acids substitutes for each cluster were also found after alignment of partial S or N sequence coding region in this study. In conclusion, we herein describe the prevalence of HCoV-OC43 among adult patients and provide substantial evidence for the genetic diversity of HCoV-OC43 circulating in Beijing.
Project description:In this study, we report the complete genome sequence of two contemporary human coronavirus OC43 (HCoV-OC43) strains detected in 2003 and 2004, respectively. Comparative genetic analyses of the circulating strains and the prototype HCoV-OC43 strain (ATCC VR759) were performed. Remarkably, a lower than expected similarity is found between the complete genomes and more in particular between the spike genes of the BE03 and BE04 strains. This finding suggests the existence of two genetically distinct HCoV-OC43 strains, circulating in Belgium in 2003 and 2004. Spike gene sequencing of three additional 2003 and two additional 2004 HCoV-OC43 strains, and subsequent phylogenetic analysis confirm this assumption. Compared to the ATCC prototype HCoV-OC43 strain, an important amino acid substitution is present in the potential cleavage site sequence of the spike protein of all contemporary strains, restoring the N-RRXRR-C motif, associated with increased spike protein cleavability in bovine coronaviruses. We here describe specific characteristics associated with circulating HCoV-OC43 strains, and we provide substantial evidence for the genetic variability of HCoV-OC43.
Project description:Human coronavirus OC43 (HCoV-OC43) is commonly associated with respiratory tract infections in humans, with five genetically distinct genotypes (A to E) described so far. In this study, we obtained the full-length genomes of HCoV-OC43 strains from two previously unrecognized lineages identified among patients presenting with severe upper respiratory tract symptoms in a cross-sectional molecular surveillance study in Kuala Lumpur, Malaysia, between 2012 and 2013. Phylogenetic, recombination and comparative genomic analyses revealed two distinct clusters diverging from a genotype D-like common ancestor through recombination with a putative genotype A-like lineage in the non-structural protein (nsp) 10 gene. Signature amino acid substitutions and a glycine residue insertion at the N-terminal domain of the S1 subunit of the spike gene, among others, exhibited further distinction in a recombination pattern, to which these clusters were classified as genotypes F and G. The phylogeographic mapping of the global spike gene indicated that the genetically similar HCoV-OC43 genotypes F and G strains were potentially circulating in China, Japan, Thailand and Europe as early as the late 2000s. The transmission network construction based on the TN93 pairwise genetic distance revealed the emergence and persistence of multiple sub-epidemic clusters of the highly prevalent genotype D and its descendant genotypes F and G, which contributed to the spread of HCoV-OC43 in the region. Finally, a more consistent nomenclature system for non-recombinant and recombinant HCoV-OC43 lineages is proposed, taking into account genetic recombination as an important feature in HCoV evolution and classification.
Project description:Although human coronavirus OC43-OC43 (HCoV-OC43) is the coronavirus most commonly associated with human infections, little is known about its molecular epidemiology and evolution. We conducted a molecular epidemiology study to investigate different genotypes and potential recombination in HCoV-OC43. Twenty-nine HCoV-OC43 strains from nasopharyngeal aspirates, collected from 2004 to 2011, were subjected to RNA-dependent RNA polymerase (RdRp), spike, and nucleocapsid gene analysis. Phylogenetic analysis showed at least three distinct clusters of HCoV-OC43, although 10 unusual strains displayed incongruent phylogenetic positions between RdRp and spike genes. This suggested the presence of four HCoV-OC43 genotypes (A to D), with genotype D most likely arising from recombination. The complete genome sequencing of two genotype C and D strains and bootscan analysis showed recombination events between genotypes B and C in the generation of genotype D. Of the 29 strains, none belonged to the more ancient genotype A, 5 from 2004 belonged to genotype B, 15 from 2004 to 2006 belonged to genotype C, and 1 from 2004 and all 8 from 2008 to 2011 belonged to the recombinant genotype D. Molecular clock analysis using spike and nucleocapsid genes dated the most recent common ancestor of all genotypes to the 1950s, genotype B and C to the 1980s, genotype B to the 1990s, and genotype C to the late 1990s to early 2000s, while the recombinant genotype D strains were detected as early as 2004. This represents the first study to describe natural recombination in HCoV-OC43 and the evolution of different genotypes over time, leading to the emergence of novel genotype D, which is associated with pneumonia in our elderly population.
Project description: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.
Project description:Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 x 10(-4) nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.
Project description:BACKGROUND:Human coronavirus (HCoV) OC43 is the most prevalent HCoV in respiratory tract infections. Its molecular epidemiological characterization, particularly the genotyping, was poorly addressed. METHODS:The full-length spike (S), RNA-dependent RNA polymerase (RdRp), and nucleocapsid (N) genes were amplified from each respiratory sample collected from 65 HCoV-OC43-positive patients between 2005 and 2012. Genotypes were determined by phylogenetic analysis. Recombination was analyzed based on full-length viral genome sequences. Clinical manifestations of each HCoV genotype infection were compared by reviewing clinical records. RESULTS:Sixty of these 65 samples belong to genotypes B, C and D. The remaining five strains had incongruent positions in the phylogenetic trees of the S, RdRp and N genes, suggesting a novel genotype emerging, designated as genotype E. Whole genome sequencing and bootscan analysis indicated that genotype E is generated by recombination between genotypes B, C and D. Temporal analysis revealed a sequential genotype replacement of C, B, D and E over the study period with genotype D being the dominant genotype since 2007. The novel genotype E was only detected in children younger than three years suffering from lower respiratory tract infections. CONCLUSIONS:Our results suggest that HCoV-OC43 genotypes are evolving. Such genotype shift may be an adapting mechanism for HCoV-OC43 maintaining its epidemic.
Project description:Background:Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Methods:Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. Results:HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. Conclusions:In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.
Project description:The complete genome sequences of the human coronavirus OC43 (HCoV-OC43) laboratory strain from the American Type Culture Collection (ATCC), and a HCoV-OC43 clinical isolate, designated Paris, were obtained. Both genomes are 30,713 nucleotides long, excluding the poly(A) tail, and only differ by 6 nucleotides. These six mutations are scattered throughout the genome and give rise to only two amino acid substitutions: one in the spike protein gene (I958F) and the other in the nucleocapsid protein gene (V81A). Furthermore, the two variants were shown to reach the central nervous system (CNS) after intranasal inoculation in BALB/c mice, demonstrating neuroinvasive properties. Even though the ATCC strain could penetrate the CNS more effectively than the Paris 2001 isolate, these results suggest that intrinsic neuroinvasive properties already existed for the HCoV-OC43 ATCC human respiratory isolate from the 1960s before it was propagated in newborn mouse brains. It also demonstrates that the molecular structure of HCoV-OC43 is very stable in the environment (the two variants were isolated ca. 40 years apart) despite virus shedding and chances of persistence in the host. The genomes of the two HCoV-OC43 variants display 71, 53.1, and 51.2% identity with those of mouse hepatitis virus A59, severe acute respiratory syndrome human coronavirus Tor2 strain (SARS-HCoV Tor2), and human coronavirus 229E (HCoV-229E), respectively. HCoV-OC43 also possesses well-conserved motifs with regard to the genome sequence of the SARS-HCoV Tor2, especially in open reading frame 1b. These results suggest that HCoV-OC43 and SARS-HCoV may share several important functional properties and that HCoV-OC43 may be used as a model to study the biology of SARS-HCoV without the need for level three biological facilities.
Project description:Coronaviruses (CoVs) continuously threaten human health. However, to date, the evolutionary mechanisms that govern CoV strain persistence in human populations have not been fully understood. In this study, we characterized the evolution of the major antigen-spike (S) gene in the most prevalent human coronavirus (HCoV) OC43 using phylogenetic and phylodynamic analysis. Among the five known HCoV-OC43 genotypes (A to E), higher substitution rates and dN/dS values as well as more positive selection sites were detected in the S gene of genotype D, corresponding to the most dominant HCoV epidemic in recent years. Further analysis showed that the majority of substitutions were located in the S1 subunit. Among them, seven positive selection sites were chronologically traced in the temporal evolution routes of genotype D, and six were located around the critical sugar binding region in the N-terminal domain (NTD) of S protein, an important sugar binding domain of CoV. These findings suggest that the genetic drift of the S gene may play an important role in genotype persistence in human populations, providing insights into the mechanisms of HCoV-OC43 adaptive evolution.