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

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.

The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

“…Replicating viral RNA was detected by staining double-stranded replicative intermediates and replicative forms with an anti-dsRNA antibody (47). ACBD3 and replicating viral RNA had similar distributions in the perinuclear region of cells at 4 h p.i.…”

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments

“…Previously, we had shown that the number of viral particles productively uncoated and the number of cells producing viral antigen are strongly correlated (44). This applies also for the generation of dsRNA produced as a consequence of viral replication and detected with MAb J2 (47). We thus determined the number of infected cells at 16 h postinfection (p.i.…”

“…In the experiment with nocodazole, MAb J2 at 1:3,000 (0.66 g/ml; English and Scientific Consulting, Bt. Szirák, Hungary) was applied to detect double-stranded RNA (dsRNA) stemming from replicating virus (47). Numbers of infected cells were determined using TissueQuest software (see above) and normalized to the level in the control (i.e., the percentage of cells infected in the absence of drug was set to 100%).…”

Rhinoviruses

“…Additionally, cell age after plating (<72 h), inoculum volume (relevant to the culture vessel), medium pH (6.8-7.3), and cell density were important factors for the reproducible appearance of HRV-induced plaques and for higher virus yields [ 48 -51 ]. The HRVs can grow at temperatures above 35 °C (some prefer that under certain conditions) [ 52 ], but rolling at 33 °C, preceded by a 2-4-h stationary incubation period [ 41 ], has historically provided the highest yield and fastest in vitro HRV growth [ 36 , 50 , 53 , 54 ].…”