engleski

Accuracy of conventional cell culture potency assays for mumps virus

Viral titer is an important parameter for virus characterisation in virology and vaccinology. Most frequently used methods for titer determination are the plaque assay, based on the cytopathogenic effect (CPE) in form of plaques, and the 50% cell culture infectious dose (CCID50) assay, based on detection of CPE in 50% of the infected cell cultures. In these assays, the viral titer is determined after macroscopic plaque counting or after CPE detection using light microscopy. Both processes are prone to operator’s subjectivity, which can lead to inaccurate determination of titer. The aim of this research was to compare viral titres in both assays using the two ways of titar determination: a) conventionally, using the light microscopy or macroscopic plaque counting and b) by using fluorescence microscopy. In this study, we prepared recombinant mumps viruses, MRV3 and Vdeopti-MRV3, by inserting the enhanced green fluorescent protein gene into the consensus sequence of L-Zagreb strain. We detected significant difference in results of the plaque assay, because both viruses had poorly visible plaques for macroscopic plaque counting. The proportion of these plaques can lower the viral titer for 0.3-0.4 log plaque forming units in comparison to the viral titer obtained by using fluorescence microscopy and lead to inaccurate titer determination. Viral titres in the CCID50 assays were comparable. For viruses with poorly visible plaque morphology, CCID50 assay is a better choice for titer determination.

mumps virus, plaque assay, cytopathogenic effect, 50% cell culture infectious dose (CCID50), fluorescence microscopy, viral titer

nije evidentirano