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Virology Journal
Published in: Virology Journal 1/2010

Open Access 01-12-2010 | Research

An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

Authors: Andreas Jurgeit, Stefan Moese, Pascal Roulin, Alexander Dorsch, Mark Lötzerich, Wai-Ming Lee, Urs F Greber

Published in: Virology Journal | Issue 1/2010

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Abstract

Background

Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA.

Results

Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections.

Conclusions

We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes without the risk of assay specific artifacts.
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Metadata
Title
An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections
Authors
Andreas Jurgeit
Stefan Moese
Pascal Roulin
Alexander Dorsch
Mark Lötzerich
Wai-Ming Lee
Urs F Greber
Publication date
01-12-2010
Publisher
BioMed Central
Published in
Virology Journal / Issue 1/2010
Electronic ISSN: 1743-422X
DOI
https://doi.org/10.1186/1743-422X-7-264

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