Background
The family of picornaviridae comprises a wide variety of human and animal pathogens [
1]. Notable members of the twelve genera are the enteroviruses, such as poliovirus, the causative agent for poliomyelitis, which affected millions of people before broad vaccinations became available in the last decades. Within the picornavirus subgenera, the number of serotypes per species varies from three in the case of poliovirus up to more than one hundred for human rhinoviruses (HRV). HRVs are the main cause of common cold [
2], and for recurring infections in humans [
3]. HRV infections lead to severe exacerbations in patients with asthma or chronic obstructive pulmonary disease [
4]. HRVs comprise species A, B and C [
2]. Twelve HRVs from species A bind to the minor receptors from the low density lipoprotein (LDL) receptor family, and the other 61 A-members as well as the B-viruses bind to intercellular adhesion molecule 1 (ICAM-1) for infection [
5]. The receptor(s) for the HRV-C serotypes are unknown. The enterotropic coxsackieviruses (CV) can cause myocarditis, pancreatitis and meningitis. The hepatitis A hepatovirus is responsible for mild forms of human hepatitis. An example of a non-human picornavirus is the foot-and-mouth disease virus of the apthovirus genus, which induces lesions in cloven-hoof animals, such as cattle, swine, goat, sheep and buffalo, and is the cause for tremendous economic losses, as experienced during the last outbreak in England in 2001 [
6].
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]. The genome encodes a single polyprotein that is proteolytically processed by viral proteases into structural and non-structural proteins. The replication of picornaviruses takes place in the cytoplasm in close association with endo-membranes containing single-and multi-membrane vesicles and complex membranous structures of various sizes [
9]. Cytoplasmic membranes are well known to support the replication of plus-sense RNA viruses, for example the alphavirus Semliki Forest virus [
10‐
12], the rubivirus rubella virus [
13,
14], the enterovirus poliovirus [
15], or the flaviviruses hepatitis C, Dengue and West Nile viruses [
16‐
18], where it is referred to as
membranous web. Membrane associated replication structures are thought to protect the replicating viral RNA from anti-viral factors recognizing double-strand RNA (dsRNA), and may provide a scaffold for anchoring and locally concentrating the viral replication complexes. 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 cap-independent, internal ribosome entry site-dependent translation [
19,
20].
The replication process of viruses has been a target for classical anti-viral agents directed against proteases, polymerases or integrases in the case of human immunodeficiency syndrome viruses (HIV) or hepatitis C viruses (HCV) [reviewed in [
21]]. Enterovirus inhibitors have been developed against the HRV protease 3C [
22] or the capsid uncoating mechanism [for example, pleconaril, [
23]]. Alternative approaches against host factors that support viral replication included protein kinases involved in virus entry, such as the serine/threonine kinase PAK1 for echoviruses, adenoviruses or vaccinia virus [
24‐
28], as well as tyrosine kinases for coxsackievirus B3-RD [
29] or microbial pathogens [for a review, see [
30]]. To enhance the identification of anti-viral agents, standardized infection assays should be developed for cultured cells as a basis for high throughput screening projects.
Here we describe a simple immunofluorescence-based infection protocol to quantitatively assess infection of cultured cells with enteroviruses, using the mouse monoclonal anti-dsRNA antibody J2 [mabJ2, [
31]]. It recognizes dsRNA duplexes larger than about 40 bp and was used earlier to detect replicating HCV genomes in distinct cytoplasmic foci [
32], or RNA replication intermediates from the groundnut rosette virus RNA-dependent RNA polymerase [
31]. The cytoplasmic foci recognized by mabJ2 are similar to foci recognized by an anti-dsRNA serum in rubella virus or Semliki Forest virus-infected cells [
13,
33]. We found that the appearance of mabJ2-positive dsRNA replication centers in 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 siRNA and small compound screens.
Discussion
Comprehensive studies of the vast number of enterovirus serotypes and their cell biological mechanisms of infection are a key foundation for developing new antiviral therapies. Progress in this area has been limited by the lack of reagents to detect infection of all the serotypes, and hence it has remained difficult to stringently compare the infection mechanisms from different virus serotypes or families.
Here we present a dsRNA replication center assay that can be used to detect infections by a broad range of enteroviruses in HeLa cells, that is, five human rhinovirus and three coxsackievirus serotypes. In the case of the minor HRV serotypes HRV1A and HRV2 the assay also detected infection of primary human lung WI-38 fibroblasts. The assay is applicable for high content screening, and infection readouts are time, dose and temperature-dependent.
Importantly, our assay is compatible with siRNA screening approaches, which have received considerable attention in the last few years, due to the promise to uncover much of the so far hidden host functions that support viral infections. Recently genome wide or subgenomic screens have been published for a variety of viral pathogens, including HIV [
57‐
59], HCV [
60,
61], dengue virus [
62], West Nile virus [
63], influenza virus [
64‐
68], human papillomavirus [
69] and vaccinia virus [
70]. The multiple screens for HIV, influenza virus and HCV, however, identified only very few overlapping genes for the individual viruses. Reasons for such findings have been attributed to the biological nature of cells and viruses, including virus strain differences, cell line differences, cell context-dependent effects and redundancies of host factors. Among the technical reasons for the low levels of overlapping hits from the published screens are also the different sources and efficacies of siRNAs, which depended on the manufacturer, or whether single siRNAs or siRNA pools were used. In addition, the different hit scoring algorithms, including post-processing filters and variable accounts for toxicity and specificity, hit ranking algorithms, or consideration of hit assignment to previously known functional networks of cellular pathways can contribute to different hit lists from siRNA screens. Last but not least, the assays for infection are not standardized, that is, different types of infection assays cover variable phases of the viral replication cycle with variable efficacies and, hence, detection sensitivities and hit identifications are poorly informed.
Our data support the notion that mabJ2 detects replicating dsRNA in infected cells rather than genomic RNA from incoming virus particles. MabJ2 is hence useful to measure viral replication. We suggest that mabJ2 (or any similar antibody) can be used to detect infections of any positive-strand RNA virus that is actively replicating. It may even be used to detect dsRNA from certain DNA virus infections [
71]. These findings and the fact that mabJ2 detects dsRNA with high sensitivity in solid support based assays [
31] open a path towards standardized and reproducible infection assays, and possibly clinical diagnostics.
Our dsRNA replication assay was validated at several levels. The dsRNA readout correlated with single step growth curves, whereby the infectious titers produced per cell were similar to values reported in the literature, that is, in the range of 40 plaque forming units per cell [
47]. We have also validated the assay with two proof of concept chemical compounds known to block enterovirus infections, the capsid binding component pleconaril [
23,
72] and the 2C protein inhibitor guanidine [
50]. While pleconaril was an entry inhibitor with a half maximal inhibition time of about 25 to 30 min, guanidine blocked infection until 2 to 4 h pi, reflecting the different modes of action of these compounds. Hence, our dsRNA replication assay in the image-based high content format may prove useful also for screening of small chemical libraries against viral infections.
Conclusions
The mabJ2 RNA replication assay has proven to be a reliable procedure to study enterovirus infections on a systematic level opening new doors for comparative genomic and chemical studies. It fulfils requirements such as robustness, good signal-to-noise ratio and practical usability, making it broadly and systematically applicable for high content infection assays for enteroviruses, and possibly other plus-sense RNA viruses. The assay covers steps required for virus entry, translation and RNA replication, and can be extended to a full replication cycle assay. It is based on a commercially available mouse monoclonal antibody, which is readily accessible for both academic and commercial laboratories. The assay also offers a way to carry out mechanistic studies with many different serotypes, including emerging picornaviruses, and hence identify serotype independent requirements for picornavirus infection.
Acknowledgements
We are grateful to M. Kikkert (Molecular Virology Laboratory, Department of Medical Microbiology, Leiden, The Netherlands) for providing mabJ2 for initial experiments, Dr. T. Hyypiä for coxsackievirus strains and advice, Dr.'s C. Tapparel and L. Kaiser for advice in diagnostic sequencing and providing HRV2, 14, 37 and HeLa-Ohio cells, and Qian Feng (Department of Medical Microbiology, Radboud University Nijmegen) for comments on the manuscript.
Competing interests
The project was in part financially supported by a grant from 3-V Biosciences Inc (Zurich, Switzerland, and Menlo Park, CA, USA), the Swiss National Science Foundation, the Swiss SystemsX.ch initiative, grant InfectX and the Kanton Zurich to UFG. The funders had no role in study design, data collection and analysis or preparation of the manuscript. UFG is a founder of 3-V Biosciences, and UFG and SM are shareholders of 3-V Biosciences.
Authors' contributions
AJ set up and optimized the assay and performed all experiments documented by figures; UFG had the initial idea to test mabJ2 in high content infection screening; SM provided the Matlab code for analysis of infection experiments; AD, PR, AJ and ML designed and performed the diagnostic sequencing of HRVs and CVs; WML provided essential antibodies and protocols for virus growth, UFG & AJ wrote the manuscript.All authors have read and approved the final manuscript.