Betacoronavirus Suspension-based Antiviral Assays – ASTM E1052 Test

Emery Pharma offers suspension-based antiviral assays using the betacoronavirus OC43 (ATCC VR-1558). Betacoronavirus OC43 is a human coronavirus that belongs to the same family as SARS-CoV-2 (the novel coronavirus that causes COVID-19) and was originally isolated from a male patient with cold-like symptoms. Like all viruses, OC43 can only replicate after it has successfully infected a host cell. The human cell lines HCT-8 (ATCC CCL-244) and MRC-5 (ATCC CCL-171) are widely used as host cells for OC43. Infected host cells will exhibit cytopathic effect (CPE), which are distinct changes in cellular structure and viability due to viral replication, within a few days post-infection.

Suspension-based antiviral assays against OC43 can be tailored to either a direct inactivation of the virus or a cell protective assay. In direct inactivation the test article directly inactivates the OC43 virus, while in the cell protective assay the host cells are pretreated with the test article, then infected with the OC43 virus to assess whether the test article protects host cells from infection.

Direct Inactivation: A solution of test article, such as an antiseptic or a drug candidate, is added to a suspension of OC43 virus to assess whether the test article can inactivate the virus and thus prevent it from subsequently infecting host cells. After a certain amount of dwell time with the virus (usually in the range of 30 seconds to a few minutes), the test article’s activity can be neutralized by the addition of a neutralizing agent. This mixture is then added to the host cell and CPE is monitored over time. Endpoint can be determined by CPE scoring by a trained microbiologist, and/or by the addition of MTS reagent and spectrophotometer reading. This type of assay can be performed according to ASTM E1052 guideline.

Cell Protective assay: The test article is applied to the host cell before infection to prevent or inhibit the ability of OC43 to infect the cells. Following this pretreatment step, the test article is then removed and the cells are challenged with OC43 virus. CPE is monitored over time and endpoint can be determined by CPE scoring by a trained microbiologist, and/or by the addition of MTS reagent and spectrophotometer reading.

In both types of assays, it is important to first determine a non-cytotoxic concentration for the test article and neutralizing agent on the host cell by performing an MTS cell cytotoxicity assay before the antiviral work. During the antiviral study, control solution(s) containing no virus will also be included so that any effect on cell morphology can be noted.

References
ASTM Standard Practice to Assess the Activity of Microbicides against Viruses in Suspension. Method E1052 – 20.