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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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Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlays. HCoV-NL63 was serially diluted on CaCo-2 cells (10e-1 until 10e-5). After 1 h of virus adsorbtion different overlays were added. After 5 days cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution. A) carboxymethyl-cellulose; B) agarose; C) Avicel.
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Figure 3: Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlays. HCoV-NL63 was serially diluted on CaCo-2 cells (10e-1 until 10e-5). After 1 h of virus adsorbtion different overlays were added. After 5 days cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution. A) carboxymethyl-cellulose; B) agarose; C) Avicel.

Mentions: Plaque assays were incubated without disturbing at 37°C and 5% CO2. Overlays were removed on day five and cells were fixed with a solution of 4% formaldehyde in PBS. After 30 min the formaldehyde solution was removed, cells were washed twice with PBS, and stained with a 0.2% crystal violet solution. As shown in Figure 3, plaques were visible with all three overlays, but staining was clearest with Avicel.


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

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

Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlays. HCoV-NL63 was serially diluted on CaCo-2 cells (10e-1 until 10e-5). After 1 h of virus adsorbtion different overlays were added. After 5 days cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution. A) carboxymethyl-cellulose; B) agarose; C) Avicel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Plaque assay for hCoV-NL63 on CaCo-2 cells using different overlays. HCoV-NL63 was serially diluted on CaCo-2 cells (10e-1 until 10e-5). After 1 h of virus adsorbtion different overlays were added. After 5 days cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet solution. A) carboxymethyl-cellulose; B) agarose; C) Avicel.
Mentions: Plaque assays were incubated without disturbing at 37°C and 5% CO2. Overlays were removed on day five and cells were fixed with a solution of 4% formaldehyde in PBS. After 30 min the formaldehyde solution was removed, cells were washed twice with PBS, and stained with a 0.2% crystal violet solution. As shown in Figure 3, plaques were visible with all three overlays, but staining was clearest with Avicel.

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