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Circulation of genetically distinct contemporary human coronavirus OC43 strains.

ABSTRACT: 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.

SUBMITTER: Vijgen L

PROVIDER: S-EPMC7111800 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Circulation of genetically distinct contemporary human coronavirus OC43 strains.

Vijgen Leen L Keyaerts Els E Lemey Philippe P Moës Elien E Li Sandra S Vandamme Anne-Mieke AM Van Ranst Marc M

Virology 20050601 1

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 distinc ...[more]

PMID: 15914223

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Project description:Coronaviruses have been at the forefront of the news for the last 2 years. Unfortunately, SARS-CoV-2, the etiologic agent for the COVID-19 pandemic, must be manipulated in biosecurity level 3 settings, which significantly limits research. Meanwhile, several less pathogenic human coronaviruses (HCoV) exist and can be studied in much more common biosafety level 2 laboratories. Among them, HCoV-OC43 is a good surrogate candidate for SARS-CoV-2 since both are phylogenetically related human Betacoronaviruses. However, one issue has been the lack of standardized means among laboratories to propagate and titer this less virulent coronavirus. The present study probes the optimal parameters to propagate HCoV-OC43. First, testing of five different cell lines (MRC-5, Huh7.5, Vero, HCT-8, HRT-18) indicated that the physiologically relevant MRC-5 human lung cell line produced among the highest viral titers. HRT-18 may however be an interesting alternative as they are quick growing cells that also led to higher viral titers and a better tropism for various HCoV-OC43 variants. We also probed the impact of serum and temperature during viral expansion and confirmed that the normal temperature of the upper respiratory track (33 °C) improves viral yields over the typical 37 °C used to grow many other viruses. Meanwhile, we did not notice any evidence that serum concentrations significantly affected the virus but interestingly noted that the virus grew quite efficiently in a serum-free media formulation. Meanwhile sonication of viral stocks somewhat improved viral titers. Four titration methods (plaque assays, TCID50-CPE, TCID50-IFA and TCID50–IPA) were also probed using two cell lines (VeroE6 and HRT-18). In our hands, plaque assays proved unreliable and quantification of the virus by scoring CPE positive wells was significantly less sensitive than antibody-based assays (IFA and IPA). While the latter methods were equally sensitive, we favor the TCID50-IPA method since simpler, faster and cheaper than the IFA protocol. Moreover, the HRT-18 cells appeared more sensitive to quantify the virus. Perhaps most importantly, these optimized protocols routinely led to high titer viral stocks in the order of 108 TCID50/ml magnitude, which should fulfill the requirements of most experimental settings.

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Circulation of genetically distinct contemporary human coronavirus OC43 strains.

Publications

Circulation of genetically distinct contemporary human coronavirus OC43 strains.

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