title: Development of a Real-Time Cell Analysing (RTCA) method as a fast and accurate screen for the selection of chikungunya virus replication inhibitors.
abstract: BACKGROUND
  The xCELLigence real-time cell analysis (RTCA) system is an established electronic cell sensor array. This system uses microelectronic biosensor technology that is verified for real-time, label-free, dynamic and non-offensive monitoring of cellular features, including detection of viral cytopathic effect (CPE). Screening viral replication inhibitors based on presence of CPE has been applied for different viruses, including chikungunya virus (CHIKV). However, most CPE-based methods, including MTT and MTS assays, do not provide information on the initiation of CPE nor the changes in reaction rate of the virus propagation over time. Therefore, in this study we developed an RTCA method as an accurate and time-based screen for antiviral compounds against CHIKV.
  
  
  METHODS
  CHIKV-infected Vero cells were used as an in vitro model to establish the suitability of the RTCA system as a quantitative analysis method based on the induction of CPE. We also performed an MTS assay as a CPE-based conventional method. Experimental assays were carried out to evaluate the optimal seeding density of the Vero cells, cytotoxicity of the tested compounds, titration of CHIKV and the antiviral activity of ribavirin, which has been reported as an effective compound against CHIKV in vitro replication.
  
  
  RESULTS
  The optimal time point for viral inoculation was 18 h after seeding the cells. We determined that the maximum non-toxic dose (MNTD) of ribavirin was 200 μg/ml for Vero cells. Regarding the dynamic monitoring of Vero cell properties during antiviral assay, approximately 34 h post-infection, the normalised Cell Index (CI) values of CHIKV-infected Vero cells started to decrease, while the vehicle controls did not show any significant changes. We also successfully showed the dose dependent manner of ribavirin as an approved in vitro inhibitor for CHIKV through our RTCA experiment.
  
  
  CONCLUSIONS
  RTCA technology could become the prevailing tool in antiviral research due to its accurate output and the opportunity to carry out quality control and technical optimisation.
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. suria_marlina@yahoo.com.
authors.forename: Suria
authors.initials: S
authors.lastname: Marlina
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. christinasmh@gmail.com.
authors.forename: Meng-Hooi
authors.initials: MH
authors.lastname: Shu
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. sazaly@um.edu.my.
authors.forename: Sazaly
authors.initials: S
authors.lastname: AbuBakar
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. keivan@um.edu.my.
authors.forename: Keivan
authors.initials: K
authors.lastname: Zandi
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D015374:Biosensing Techniques / Q000379:methods*; D002646:Chikungunya virus / Q000187:drug effects* / Q000502:physiology*; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D012254:Ribavirin / Q000494:pharmacology; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D012254:Ribavirin