Plaque assay for human coronavirus NL63 using human colon carcinoma cells

doi:10.1186/1743-422X-5-138

Virology Journal

Volume 5

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Methodology

Plaque assay for human coronavirus NL63 using human colon carcinoma cells

Petra Herzog¹^,3 , Christian Drosten² and Marcel A Müller²

¹Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, D-20359 Hamburg, Germany

²Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany

³Qiagen Hamburg GmbH, Königstr. 4a, D-22767 Hamburg, Germany

author email corresponding author email

Virology Journal 2008, 5:138doi:10.1186/1743-422X-5-138


Published:	12 November 2008

Abstract

Background

Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV) NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen.

Results

12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2) replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2). CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4^thday of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL.

Conclusion

CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.