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Plaque assay for human coronavirus NL63 using human colon carcinoma cells.

Herzog P, Drosten C, Müller MA - Virol. J. (2008)

Bottom Line: Coronaviruses cause a broad range of diseases in animals and humans.Human coronavirus (hCoV) NL63 is associated with up to 10% of common colds.CaCo-2 cells showed cytopathogenic effects 4 days post infection.

View Article: PubMed Central - HTML - PubMed

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

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 4th day 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.

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Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cells. 25 cm2 flasks of LLC-MK2 or CaCo-2 cells were infected at multiplicities of infection of 0.005 for 1 h, washed twice with PBS, and subsequently supplied with 10 mL DMEM. Samples were taken daily from day 0 to 7 (except day 4) and analyzed by real time RT-PCR. Error bars indicate ranges of three independent experiments.
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Figure 2: Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cells. 25 cm2 flasks of LLC-MK2 or CaCo-2 cells were infected at multiplicities of infection of 0.005 for 1 h, washed twice with PBS, and subsequently supplied with 10 mL DMEM. Samples were taken daily from day 0 to 7 (except day 4) and analyzed by real time RT-PCR. Error bars indicate ranges of three independent experiments.

Mentions: For confirmation of differential replication efficiencies, CaCo-2 and LLC-MK2 cells were infected in parallel. Both cell lines were seeded in 25 cm2 flasks, and infected at multiplicities of infection of 0.005. Samples of supernatants were taken daily from day 0 to 7 and analyzed by real time RT-PCR. As shown in Figure 2, CaCo-2 cells replicated virus more efficiently than LLC-MK2. From day 3 onward, RNA concentrations were more than 100 fold higher in CaCo-2 cells. Because of the clear CPE observed in CaCo-2 cells, these cells were tested for their utility in a cytopathogenic plaque assay.


Plaque assay for human coronavirus NL63 using human colon carcinoma cells.

Herzog P, Drosten C, Müller MA - Virol. J. (2008)

Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cells. 25 cm2 flasks of LLC-MK2 or CaCo-2 cells were infected at multiplicities of infection of 0.005 for 1 h, washed twice with PBS, and subsequently supplied with 10 mL DMEM. Samples were taken daily from day 0 to 7 (except day 4) and analyzed by real time RT-PCR. Error bars indicate ranges of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2603006&req=5

Figure 2: Growth kinetics of hCoV-NL63 on LLC-MK2 and CaCo-2 cells. 25 cm2 flasks of LLC-MK2 or CaCo-2 cells were infected at multiplicities of infection of 0.005 for 1 h, washed twice with PBS, and subsequently supplied with 10 mL DMEM. Samples were taken daily from day 0 to 7 (except day 4) and analyzed by real time RT-PCR. Error bars indicate ranges of three independent experiments.
Mentions: For confirmation of differential replication efficiencies, CaCo-2 and LLC-MK2 cells were infected in parallel. Both cell lines were seeded in 25 cm2 flasks, and infected at multiplicities of infection of 0.005. Samples of supernatants were taken daily from day 0 to 7 and analyzed by real time RT-PCR. As shown in Figure 2, CaCo-2 cells replicated virus more efficiently than LLC-MK2. From day 3 onward, RNA concentrations were more than 100 fold higher in CaCo-2 cells. Because of the clear CPE observed in CaCo-2 cells, these cells were tested for their utility in a cytopathogenic plaque assay.

Bottom Line: Coronaviruses cause a broad range of diseases in animals and humans.Human coronavirus (hCoV) NL63 is associated with up to 10% of common colds.CaCo-2 cells showed cytopathogenic effects 4 days post infection.

View Article: PubMed Central - HTML - PubMed

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

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 4th day 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.

Show MeSH
Related in: MedlinePlus