Elsevier

Vaccine

Volume 28, Issue 7, 17 February 2010, Pages 1887-1892
Vaccine

Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay

https://doi.org/10.1016/j.vaccine.2009.11.049Get rights and content

Abstract

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID50) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID50 assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID50 and plaque assay quantification results differed for different strains and were in a range of 0.66–10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID50 or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay.

Introduction

During viral infection of cell, virus replicative cycle is accompanied by a number of biochemical and morphological changes within the cell which usually culminate with cell death [1]. These morphological changes are referred to as the virus cytopathic effect (CPE). CPE may take several forms, e.g. syncytia formation, cell rounding, disorientation, swelling or shrinking, detachment from the surface, total cell lysis, etc. The form of CPE depends both on the virus and on the cells in which it is grown.

The two most commonly used methods for the determination of a virus titre (or a virus potency estimate) are plaque assay and 50% cell culture infective dose (CCID50) assay. Both of these methods are based on CPE observation after viral infection of a susceptible cell culture. The plaque method is based on the ability of infectious virions to form a plaque, a scoring event, on a confluent monolayer culture of cells covered with solid culture media. As the test is performed at limiting dilutions, a single plaque is formed as a result of infection of one cell by one virus particle. Therefore, the number of plaques equals the number of plaque forming units (PFU) in the viral suspension [2].

In contrast to plaque assay, in CCID50 assay the virus is added to cells cultured in liquid media in microtitre wells. The entire cell layer in a well is considered to represent a test unit (equivalent to a single cell in a plaque assay) [2]. The virus is added to a number of test units and each is scored as infected or noninfected. The virus titre is calculated from the proportion of noninfected units at the endpoint dilution [2].

Based on Poisson distribution of virus particles among cells for the CCID50 assay, one CCID50 unit corresponds to 0.69 PFU obtained by plaque assay [3]. Still, in a number of studies in which the correlation of the results was determined experimentally, the results significantly varied from the expected value [4], [5]. This poses a significant problem in various areas of virology as accurate and precise titre determination is critical not only in basic virus research but also in vaccine production and quality control, gene therapy, investigation of antivirals, etc.

Mumps virus (MuV), an enveloped, non-segmented, negative-stranded RNA virus [6] represents a very good example of a virus with diverse forms of CPE depending on a viral strain [4]. MuV causes a human communicable disease usually characterized by parotitis and mild nonspecific symptoms. Since the late 1960s, as the usage of live attenuated mumps vaccines became widespread, the number of mumps cases dramatically declined. Based on the nucleotide sequence of the SH gene, MuV strains are placed into genotypes named A-M [7], [8], but there is a number of strains (both wild type and vaccines) that do not fulfil the criteria for genotype designation and therefore their genotype is not specified. Specific genotypes are neither associated with differences in severity of disease, nor with attenuation status, neurotropism or neurovirulence.

Although in the research of MuV, both CCID50 and plaque assays are used with similar frequencies, in the field of vaccinology majority of manufacturers and national control authorities express the potency of MuV vaccine in CCID50 units. Very often there is a need to compare the results of MuV potency estimates obtained by the two assays. In 1994 the World Health Organization (WHO) Expert Committee on Biological standardization established the first, and so far the only, International Reference Reagent of Mumps Vaccine (Live), code 90/534 [9], which can be obtained from the National Institute for Biological Standards and Control (NIBSC, UK). This reagent contains Urabe mumps virus strain and its potency is declared as 4.6 log10 of infectious units per ampoule [9], [10]. This titre was established on the basis of the results of a collaborative study performed in 14 laboratories in nine countries, in which both quantification methods were used. The Committee accepted a proposal that the results obtained by CCID50 and plaque assay system used should be combined [9].

At the Institute of Immunology, both CCID50 and plaque methods for MuV quantification are established in concordance with the WHO recommendations [11] and current European Pharmacopoeia (cPh.Eur) requirements [12]. Over a period of years, we collected a number of various MuV strains and noticed that their CPEs extremely differ. Furthermore, we noticed that the ratio of potency estimates determined by plaque and CCID50 assays is not always 1, as was established for Urabe strain in the WHO study, nor it is 0.69 as expected from mathematic calculations.

In this paper, we present our results of comparisons of the potency estimates obtained by plaque and CCID50 assays for nine MuV strains and show that the ratio of the potency estimates obtained by the two methods differs for different MuV strains.

Section snippets

Vero cell culture and MuV strains

Vero cell culture (African green monkey kidney cells) was obtained from the European Collection of Cell Culture (UK) and maintained in Minimum Essential Medium with Hank's salts (MEM-H) (AppliChem, Germany) supplemented with 10% fetal calf serum (FCS) (Moregate, Australia) and 50 μl/ml neomycin (Gibco-BRL, USA).

MuV wild-type strains ZgA/Cro69 (genotype D) [13], ZgB/Cro69 (genotype pending) [13], 9218/Zg98 (genotype pending) [14], Du/CRO05 (genotype G) [14] and Zg/CRO06 (genotype D) [15] detected

Results and discussion

Both plaque and CCID50 assays are conventional, routinely performed virological tests for quantification of infectious virus particles. Although these methods are gold standard techniques, they are still characterized by a number of limitations and vaguenesses. In the case of MuV, an accurate and precise quantification is an important issue not only for scientific purposes but also for public health as it is a pathogen against which vaccination with live attenuated vaccines is conducted and

Conclusion

A strong intrinsic feature of MuV strains to interact specifically with Vero cell culture results in very different forms of CPE. Our results indicate that MuV quantification results for some MuV strains are almost identical regardless of whether CCID50 or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay.

The results reported here indicate that care should be taken when comparing the numbers of virions in samples obtained by these

Acknowledgments

The authors thank Dr. Ante Sabioncello for useful comments and suggestions. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, grant nos. TP-05/0021-02 (to R.M.) and 021-0212432-3123 (to M.Š.).

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