Method Development and Validation for Detecting Specific Bovine Adventitious Viruses Contaminated in Vero Cell Substrates

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Supaporn Chumpol
Sukanlayanee Chaimee
Wereyamarst Jaroenkunathum
Supaporn Phumiamorn


Background: Cell substrates are used in the production of vaccines, which must be quality controlled in accordance with World Health Organization requirements. Detection of bovine adventitious virus contaminants in cell substrates is one of the important item tested to confirm that the cell in the production are not contaminated by bovine virus. This study strengthened the laboratory’s capacity for characterization and quality control of cells so they are able to be transmitted to Thai domestic vaccine manufacturers and provide quality inspection services for Vero cells used vaccine production.

Objectives: The purpose of this study was to optimize and validate method for the detecting specific bovine adventitious viruses in Vero cell substrates. This method was used as a standard laboratory method for testing the quality of Vero cell substrates in vaccine production.

Methods: The optimal conditions of three analytical techniques including cytopathic effect (CPE), hemadsorption and immunofluorescence assay (IFA) were investigated. In addition, validation method study was performed on the parameters of specificity and limit of detection.

Results: The results of optimal conditions showed that the appropriate cell numbers of bovine turbinate cells (BT), Madin-Darby bovine kidney cells (MDBK) and African green monkey kidney cells (Vero) for CPE formation were 1x105, 4x104 and 4x104 cells/mL, respectively. CPE results were consistent with those of IFA, except REO-3 that did not cause CPE in BT cell, but the virus was detected by IFA. The hemadsorption assay was positive only by BPI-3 and BPV viruses. The developed methods were specific to five specific bovine viruses and the lowest viral titers for those bovine viruses were 0.01, 0.1, 0.1, 0.01 and 0.001 CCID50/mL, respectively.

Conclusions: The optimal conditions and validation of detecting specific bovine adventitious viruses contaminated in Vero cell substrates were studied using three methods including CPE, hemadsorption and Immunofluorescent assay. The results found that all three methods were able to detect specific bovine viral contamination and met the acceptance criteria. The standard method for detecting specific bovine adventitious viruses contaminated in Vero cell was obtained to assess the safety and purity of the Vero cell substrates used in vaccine production.

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Chumpol S, Chaimee S, Jaroenkunathum W, Phumiamorn S. Method Development and Validation for Detecting Specific Bovine Adventitious Viruses Contaminated in Vero Cell Substrates. TFDJ [Internet]. 2022 Oct. 31 [cited 2024 Apr. 19];29(3):23-3. Available from:
Research Article


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