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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

Abstract

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

Introduction

Hand, foot, and mouth disease (HFMD) is a disease most commonly caused by coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) in young children. Recently across the Asia-Pacific region, there has been a significant uptick in CVA16-induced HFMD. While symptoms can be mild, severe complications can occur that affect the brain and the heart, with potential fatality1,2. At present, there are no licensed antiviral therapies or vaccinations available for CVA16, and thus there is a pressing need to develop antiviral strategies to curb future outbreaks and the associated complications.

CVA....

Protocol

NOTE: All cell culture and virus infections must be conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. The two tannin-class of small molecules chebulagic acid (CHLA) and punicalagin (PUG), that were observed to efficiently block CVA16 infection9, are used as examples of candidate inhibitory agents. For basic principles in virology techniques, virus propagation, determination of virus titer, and concepts of plaque formi.......

Representative Results

The time-of-drug-addition assay is indicated in Figure 1 and shows the influence from treatment using the small molecules CHLA and PUG on CVA16 infection either pre-viral entry (pretreatment), during viral entry (co-addition), or post-viral entry (post-infection). Both small molecules only produced marginal impact against CVA16 infectivity whether in the pretreatment of the host cells prior to viral infection (Figure 1A) or in th.......

Discussion

In this report, we described the protocols that are useful for the identification of antiviral candidates that target viral entry, in particular against the non-enveloped CVA16. The assays are designed in ways to dissect the early events during viral entry, which is helpful to clarify the mechanism(s) of action and potential target(s) of the test agents' antiviral activity. The 'time-of-drug-addition assay' permits to broadly determine the potential target of the test compounds, for instance the uninfected ho.......

Acknowledgements

The authors are grateful to Dr. Joshua Beckham at the University of Texas at Austin for technical support with molecular docking. This study was partly supported by funding from the Ministry of Science and Technology of Taiwan (MOST107-2320-B-037-002 to C.-J.L. and L.-T.L.; MOST106-2320-B-038-021 and MOST107-2320-B-038-034-MY3 to L.-T.L.).

....

Materials

NameCompanyCatalog NumberComments
4% ParaformaldehydeSigmaAL-158127-500G
Alexa 488-conjugated anti-mouse IgGInvitrogenA11029
Amphotericin BGIBCO15290-018
Anti-VP1 antibodyMerck-MilliporeMAB979Anti-Enterovirus 71 Antibody, cross-reacts with Coxsackie A16, clone 422-8D-4C-4D
Beckman Coulter CytometerBeckman CoulterFC500
CorinaMolecular Networks GmbH
Crystal violetSigmaC3886-100G
DMEMGIBCO11995-040
DMSOSigmaD5879
FBSGIBCO26140-079
FormaldehydeSigmaF8775
Graphpad PrismGraphPad
Heparin sodium saltSigmaH3393
In vitro toxicology assay kit, XTT-basedSigmaTOX2
MethylcelluloseSigmaM0512-100G
PBS pH 7.4GIBCO10010023
Penicillin-StreptomycinGIBCO15070-063
PyMolSchrödinger
UCSF ChimeraUniversity of California, San Francisco

References

  1. Legay, F., et al. Fatal coxsackievirus A-16 pneumonitis in adult. Emerging Infectious Diseases. 13, 1084-1086 (2007).
  2. Wang, C. Y., Li Lu, ., Wu, F., H, M., Lee, C. Y., Huang, L. M. Fatal coxsackievirus A16 infection. Pediatric Infectiou....

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