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Fatal Pneumonia Associated With a Novel Genotype of Human Coronavirus OC43.

ABSTRACT: Since its first discovery in 1967, human coronavirus OC43 (HCoV-OC43) has been associated with mild self-limiting upper respiratory infections worldwide. Fatal primary pneumonia due to HCoV-OC43 is not frequently described. This study describes a case of fatal primary pneumonia associated with HCoV-OC43 in a 75-year-old patient with good past health. The viral loads of the respiratory tract specimens (bronchoalveolar lavage and endotracheal aspirate) from diagnosis to death were persistently high (3.49 × 10⁶-1.10 × 10¹⁰ copies/ml). HCoV-OC43 at a 6.46 × 10³ copies/ml level was also detected from his pleural fluid 2 days before his death. Complete genome sequencing and phylogenetic analysis showed that the present HCoV-OC43 forms a distinct cluster with three other HCoV-OC43 from United States, with a bootstrap value of 100% and sharing 99.9% nucleotide identities. Pairwise genetic distance between this cluster and other HCoV-OC43 genotypes ranged from 0.27 ± 0.02% to 1.25 ± 0.01%. In contrast, the lowest pairwise genetic distance between existing HCoV-OC43 genotypes was 0.26 ± 0.02%, suggesting that this cluster constitutes a novel HCoV-OC43 genotype, which we named genotype I. Unlike genotypes D, E, F, G, and H, no recombination event was observed for this novel genotype. Structural modeling revealed that the loop with the S1/S2 cleavage site was four amino acids longer than other HCoV-OC43, making it more exposed and accessible to protease, which may have resulted in its possible hypervirulence.

SUBMITTER: Lau SKP

PROVIDER: S-EPMC8795699 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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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Fatal Pneumonia Associated With a Novel Genotype of Human Coronavirus OC43.

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