Cytopathic Effect Inhibition Assay

The development of effective antiviral drugs is an important biomedical scientific achievement. After treatment, viruses that maintain latency or persistence are not specifically cleared from the body, whereas the replication can be effectively suppressed. The antiviral activity was determined by cytopathic effect (CPE) inhibition assay.

Cytopathic effect is defined by the changes in host cell morphology caused by the target infecting virus. CPE occurs when the infecting virus causes lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce. CPE inhibition assay can evaluate the ability of test articles to inhibit CPE.

Many combinations of cells and viruses can be applied, but certain pairs tend to be used most frequently, such as L929 cells with vesicular stomatitis virus (VSV), often the New Jersey strain. This cell line is sensitive to alpha, beta, and gamma interferon. For the measurement of human interferon, a wider variety of systems are used. Many researchers will use the human lung carcinoma cell line A549 due to their ease of growth and relatively high sensitivity to all forms of human interferons. These cells are often challenged with EMCV. Human alpha interferon can be assayed using the bovine kidney line MDBK challenged with VSV.

The following is a summary of an A549/EMCV Cytopathic Effect Inhibition assay for Recombinant Human Interferon Alpha:

• A flask of A549 cells will be treated with trypsin/EDTA until the cells detach. The cells can then be diluted in media and plated in 96-well plates.
• Serial two-fold dilutions of IFN samples and standards will prepared in a separate 96-well plate. Equal volumes of the dilutions will be added to the cells. One row has six wells reserved for the cell control (no IFN, no virus) and the virus control (no IFN, virus added).
• The plate will then be incubated for 18-24 hours at 37°C/5% CO₂.
• A dilution of EMCV, which has a 100% killing efficiency within 40 hours, is added to the plates, except the cell control wells that receive media only. After the completed killing (approximately 40 to 56 hours), the media will be removed from the wells, and the live cells will be fixed and stained with crystal violet solution.
• The endpoint is determined by microscopic examination to be well where 50% of the cells remain compared to the cell control (100% survival) and the virus control (0% survival).

Figure 1. CPE assay of SARS-CoV-2 in A. Vero and B. MDBK cell lines with different virus titers.
(Cihan TAŞTAN, et al. 2020)

CPE inhibition assay is inherently complex due to the metabolic state of cells, virus replication, and the ability of IFN to protect cells. Creative Diagnostics provides CPE inhibition assay service with high level of assay sensitivity and accuracy, we have rich experience serving pharmaceutical, biotechnology, contract research and academic scientists.

Our Features

• Cheap and easy to implement also when virus-specific antibodies are not available.
• Very little information on the virus itself is required.
• Compared with plaque assay method, the error is small.
• High sensitivity and accuracy.

Our Capabilities

• Cytomegalovirus (CMV)
• Dengue virus (DENV)
• Hepatitis B virus (HBV)
• Hepatitis C virus (HCV)
• Herpes simplex virus (HSV)
• Human cytomegalovirus (HCMV)
• Human immunodeficiency virus (HIV)
• Human rhinovirus (HRV)
• Influenza virus (IFV)
• Respiratory syncytial virus
• Ross River virus (RRV)
• Semliki forest virus (SFV)
• Sindbis virus (SINV)
• Vaccinia virus (VACV)
• Zika virus (ZIKA)

(Other viruses or microorganisms may be available on request)

We combine infection and analytical expertise to provide our clients with the most powerful portfolio of antiviral and antimicrobial in vitro testing services. Facing an increasing demand for new antiviral and antimicrobial compounds for the treatment of infectious diseases, Creative Diagnostics can test these compounds in vitro to determine their potential efficacy in vivo models.

Reference:

1. Cihan TAŞTAN, et al. SARS-CoV-2 isolation and propagation from Turkish COVID-19 patients. Turkish Journal of Biology. (2020) 44: 192-202.