Abstract
BackgroundPicornaviruses 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.ResultsImmunocytochemical 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.ConclusionsWe 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.
Highlights
Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses
We found that the appearance of mabJ2-positive double-strand RNA (dsRNA) replication centers in Human rhinoviruses (HRV) or coxsackievirus infected cells correlated with the emergence of capsid protein epitopes and infectious virus titer, and the mabJ2 assay was applicable for prototypic high throughput, image-based small interfering RNAs (siRNAs) and small compound screens
Double-strand RNA replication centers identify HRV and coxsackievirus infected cells We first tested if the formation of dsRNA-positive replication centers can be used as an assay for infection of HeLa cells strain Ohio with HRV or CV
Summary
Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. Picornaviruses are small, non-enveloped RNA viruses with an icosahedral capsid of about 28-30 nm in diameter [7], and a single strand, plus-sense RNA genome, which is in case of enteroviruses about 7.2 to 8.45 kb [8]. Since its establishment requires de novo lipid synthesis, it may represent an anti-viral target, as suggested from work with drosophila C virus, a dicistronic virus, which is in many ways similar to picornaviruses, for example, encoding a polyprotein by a single positive-strand RNA genome, or using capindependent, internal ribosome entry site-dependent translation [19,20]
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