Vaccine 28 (2010) 1887–1892

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Comparisons of mumps virus potency estimates obtained by 50% cell culture

infective dose assay and plaque assay
Dubravko Forcic ∗ , Tanja Košutić-Gulija, Maja Šantak, Renata Jug, Jelena Ivancic-Jelecki,
Maja Markusic, Renata Mažuran
Molecular Biomedicine Unit, Research and Development Department, Institute of Immunology Inc., Rockefellerova 10, 10000 Zagreb, Croatia

a r t i c l e i n f o a b s t r a c t

Article history: The two most commonly used methods for the determination of a virus potency are plaque assay and
Received 1 October 2009 50% cell culture infective dose (CCID50 ) assay, both based on cytopathic effect observation. We compared
Received in revised form 7 November 2009 the potency estimates obtained by plaque and CCID50 assays for nine mumps virus strains that produce
Accepted 18 November 2009
different cytopathic effects in Vero cells. The ratios of CCID50 and plaque assay quantification results
Available online 2 December 2009
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,
Keywords:
while the potency estimates of other strains strongly depend on the choice of the assay.
Mumps virus
Cytopathic effect © 2009 Elsevier Ltd. All rights reserved.
Virus potency estimate

1. Introduction
During viral infection of cell, virus replicative cycle is accompa-
nied by a number of biochemical and morphological changes within
the cell which usually culminate with cell death [1]. These morpho-
logical 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 per-
formed 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
∗ Corresponding author. Tel.: +385 1 4684 500; fax: +385 1 4684 303.
E-mail address: dforcic@imz.hr (D. Forcic).
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 cor-
relation of the results was determined experimentally, the results
significantly varied from the expected value [4,5]. This poses a sig-
nificant 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, inves-
tigation 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 dis-
ease, 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 major-
ity of manufacturers and national control authorities express the
potency of MuV vaccine in CCID50 units. Very often there is a need to

0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2009.11.049
1888 D. Forcic et al. / Vaccine 28 (2010) 1887–1892
compare the results of MuV potency estimates obtained by the two
assays. In 1994 the World Health Organization (WHO) Expert Com-
mittee 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 Insti-
tute 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 estab-
lished 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 meth-
ods for MuV quantification are established in concordance with
the WHO recommendations [11] and current European Pharma-
copoeia (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.
2. Materials and methods
2.1. 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 !l/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 in outbreaks in Croatia were isolated on Vero cell culture
supplemented with 2% FCS and 50 !l/ml neomycin. The L-Zagreb
(L-Zg) (genotype pending) mumps vaccine strain was produced
at the Institute of Immunology, Croatia. The Jeryl Lynn 2 (JL2)
(genotype A) and the Jeryl Lynn 5 (JL5) (genotype A) MuV virus
strains were kindly provided by B. Rima (The Queen’s University of
Belfast, UK). MuV Urabe strain (genotype B; International Reference
Reagent of Mumps Vaccine (Live), code 90/534) was obtained from
the NIBSC. In our laboratory vaccine virus strains were propagated
in Vero cells supplemented with 2% FCS and 50 !l/ml neomycin for
no more than two passages. The detailed phylogenetic analysis and
relatedness of used strains was presented in our previous article
[15].
2.2. MuV plaque assay
One million Vero cells per well were seeded in six-well plates
for 48–72 h until the confluent cell monolayers were formed. An
aliquot of each virus strain was thawed and twofold serially diluted
in MEM-H with 2% FCS and neomycin. Supernatant was removed
and cells were infected with 1 ml of viral suspension. After 1 h at
35 ◦ C viral suspension was aspirated, cells were washed twice with
PBS and overlayed with 3 ml of semisolid medium consisting of
1 (v/v) 2× MEM-H with 10% FCS without phenol red and 1 (v/v)
1.5% Noble agar (Sigma, USA). Plates were incubated at 35 ◦ C in
a humidified atmosphere of 5% CO2 . After 6 days the monolayers
were stained with 1 ml of 0.05% neutral red (Sigma, USA) so that
plaques could be visualized. The cells were incubated at 35 ◦ C for
1 h. A surplus neutral red solution was removed and plates were
incubated at 35 ◦ C for the next 4 h. The virus titre was determined
by calculation of the mean number of plaques for each dilution and
multiplication by the reciprocal value of the dilution factor [11,16].
Virus dilutions that yielded 20–100 plaques in a single well were
counted. Each assay contained four replicas of each sample dilution.
The titre was expressed as the number of PFU per ml.
To ensure reproducibility of the test results, each assay included
a sample of Urabe mumps strain as the working reference prepa-
ration (in-house standard). Its potency was established in over 10
independent measurements.
The assay was considered accurate only when the differ-
ence in titre between any two replicas of the test sample was
within 0.5 log10 . Furthermore, only the assays in which the titre
of the working reference preparation was within 0.5 log10 of the
established value were regarded as valid (the tolerable intra-test
deviation of the plaque assay is 0.5 log10 [11]).
2.3. MuV CCID50 assay
The assays were performed in 96-well plates. The 1.6 × 104 Vero
cells in 0.1 ml MEM-H with 5% FCS per well were seeded. Viral
samples were serially diluted in 0.48 log10 steps (threefold dilu-
tions) from 10−1 to 10−5 in MEM-H without FCS. The 0.05 ml of
virus dilution and 0.05 ml of test medium (without FCS) were added
to Vero cells. The plates were incubated at 35 ◦ C in 5% CO2 for 10
days. After incubation period, the cells were checked for cytopathic
changes and positive wells were recorded. The titre was calculated
in CCID50 per 1 ml on the basis of the final reading using the Kärber
formula: log10 CCID50 = L − d/(S − 0.5) where L = log10 starting dilu-
tion, d = log10 dilution step, S = sum of the proportion of positive
replicate [11,12,17]. The mean CCID50 value is calculated from nine
results (three aliquots of the viral suspension were thawed, each
was used for three quantifications).
Each CCID50 testing included a sample of L-Zagreb mumps
strain as the working reference preparation (in-house standard). Its
potency was calibrated against the International Reference Reagent
of Mumps Vaccine (Live). The potency of L-Zagreb working refer-
ence preparation was determined in 46 CCID50 independent assays.
In each assay two independent virus dilution series (consisting of
six dilution steps, each of 0.48 log10 dilution factors) were pre-
pared from one thawed L-Zagreb reference preparation aliquot.
Each dilution series was added to Vero cells on a separate 96-well
plate (16 wells/virus concentration). The obtained average geo-
metric mean of the titre was 5.298 log10 CCID50 /0.5 ml, arithmetic
mean was 5.295 log10 CCID50 /0.5 ml and median was 5.305 log10
CCID50 /0.5 ml. Day-to-day assay variability was very low (coeffi-
cient of variation was 1.69%).
Like in plaque assay, the CCID50 test was considered accurate
only when the difference in titre between any two replicas of the
test sample was within 0.5 log10 . Moreover, only the assays in
which the titre of the working reference preparation was within
0.5 log10 of the established value were regarded as valid (as in
plaque assay, the tolerable intra-test deviation of CCID50 method is
0.5 log10 [11]).
3. 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 vague-
 D. Forcic et al. / Vaccine 28 (2010) 1887–1892
Fig. 1. Different morphology of CPE for different mumps viruses in the monolayers of Vero cells. (A) Vero cell monolayer infected with 9218/Zg98 or L-Zagreb. (B) Vero cell
monolayer infected with JL5 or Urabe strain. CPE for 9218/Zg98 and L-Zagreb are indicated with an arrow (A).
nesses. 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 challenged with a number of
problems, one of them being neurovirulence risk associated with
the use of certain vaccine strains.
3.1. Distinct vs. discrete MuV CPE patterns
When we infected Vero cells with nine different MuV strains,
we observed huge differences in their CPE patterns. Some strains
(namely JL2, JL5, Urabe, Du/CRO05 and Zg/CRO06) formed classic
syncytia, producing a major cytopathic effect and showing high
fusogenic activity (Fig. 1A). In contrast to them, a second group
of viruses (ZgA/Cro69, ZgB/Cro69, 9218/Zg98 and L-Zg) produced
CPE with very discrete syncytia and specific morphological changes
(cell rounding or elongation, different light reflection, irregular
cell shape) indicating a very low fusogenic activity of the viruses
(Fig. 1B).
Since syncytia development plays a key role in plaque for-
mation, two forms of plaques were also observed: (a) clear and
distinct plaques (either large or small) formed by viruses with high
fusogenic activity (Fig. 2A); or (b) diffuse, hardly distinguishable
plaques differing in size observed after infection with strains that
produced CPE with discrete syncytia (Fig. 2B).
During the optimization of the methods, we tried to find
conditions that would enable the occurrence of clearly visible
CPE/plaques for all strains. Still, in spite of changing of different
assays conditions (e.g. temperature and time of incubation, time
and type of staining used for plaque visualization), it seems that
the feature of some strains to produce hardly noticeable CPE and
diffuse plaques is their intrinsic characteristic.
It is known that MuV growth is strain- as well as host cell-
dependent [18]. Therefore it is possible that some strains would
produce different, more easily detectable CPE if another cell line
was used. There are some data indicating that CaCo-2 or B95a might
be a better substrate for in vitro propagation of MuV, although these
cell lines do not establish a uniform cell monolayer and therefore
their application as a substrate for assays based on CPE is limited
[19,20].
Fig. 2. Plaque morphology in Vero cells. (A) Vero cell monolayer infected with JL5 or Urabe. (B) Vero cell monolayer infected with 9218/Zg98 or L-Zg strain.
1889
Based on our results and experience, we think that the most
accurate quantification results would be obtained if assays’ condi-
tions were specifically adapted to each virus strain. Still, it is highly
improbable that in reality this can be implemented. As numer-
ous MuV strains are known and new ones are being isolated every
year, it is unrealistic to expect that for every strain a detailed study
regarding optimal conditions for quantification methods will be
conducted, especially in research laboratories that are not spe-
cialized and committed only to virus quantifications. As far as we
know, even the national control authorities in countries where var-
ious vaccine strains are registered do not use different methods for
different vaccine strains.
3.2. Comparisons of quantification results obtained by CCID50
and plaque assays
After both CCID50 and plaque assays were optimized to the
extent we considered best for all analyzed strains, we compared the
quantification results obtained by the two methods. We wanted to
investigate whether there is a correlation between the results that
could be applied to all strains, no matter how genetically diverse
they are.
First we performed a detailed analysis of quantification results
for the Urabe virus strain. Twelve aliquots of the same virus sus-
pension were thawed on different days, and the virus potency was
determined in 12 independent tests by both CCID50 and plaque
assays.
Average titres determined by plaque and CCID50 assays were
4.28 log10 and 4.92 log10 , respectively (Table 1). The difference
in the titre obtained by the two methods was in a range of
0.44 log10 –0.81 log10 , mean intermethod difference was 0.63 log10
(Table 1). Only in one out of 12 independent tests the titres obtained
by the two methods differed less than 0.5 log10 .
The ratio of the results obtained by CCID50 assay and plaque
assay for Urabe strain was 4.39. As we do not have the details of
the collaborative study in which the titre of the first International
Reference Reagent for Mumps Vaccine was established, we cannot
compare the difference we obtained in the potency estimates for
Urabe strain with the differences obtained in that study. Still, our
results do not support the finding that the Urabe strain titre is the
same, regardless which method is used [9]. This discrepancy points
1890 D. Forcic et al. / Vaccine 28 (2010) 1887–1892

Table 1
Potency estimates of the Urabe virus sample and comparison of results obtained by CCID50 and plaque assays.

Assay no. Titre obtained by Titre obtained by log10 of Ratio of CCID50 and plaque
plaque assay CCID50 assay inter-method assays’ quantification
(log10 PFU/ml) (log10 CCID50 units/ml) difference results

1 4.23 ± 0.04 4.99 ± 0.10 0.76 5.75

2 4.27 ± 0.05 4.79 ± 0.12 0.52 3.31
3 4.38 ± 0.08 5.04 ± 0.07 0.66 4.57
4 4.46 ± 0.06 4.90 ± 0.15 0.44 2.75
5 4.17 ± 0.12 4.98 ± 0.07 0.81 6.45
6 4.26 ± 0.07 4.85 ± 0.07 0.59 3.89
7 4.33 ± 0.04 4.97 ± 0.09 0.64 4.36
8 4.28 ± 0.01 4.88 ± 0.15 0.60 3.98
9 4.24 ± 0.07 4.88 ± 0.04 0.64 4.36
10 4.28 ± 0.06 5.0 ± 0.07 0.72 5.25
11 4.21 ± 0.07 4.71 ± 0.11 0.50 3.16
12 4.30 ± 0.04 4.98 ± 0.11 0.68 4.78

Mean ± std 4.28 ± 0.09 4.92 ± 0.12 0.63 ± 0.11 4.39 ± 1.08

to the need to specify the assay conditions when the potency data is
presented, as differences in the procedure can lead to significantly
different results.
To compare potency estimates of other eight MuV strains we
determined the titre of each by CCID50 and plaque assays in at
least three assays using aliquots of the same viral suspensions.
In contrast to Urabe strain, a higher titre determined by plaque
assay was obtained in samples of the 9218/Zg98 (0.18 log10 ) and
Du/CRO05 (0.08 log10 ) (Table 2). In all other strains an average
titre obtained by plaque assay was lower than the titre obtained
by CCID50 assay. For those seven strains, the smallest difference
between titres was found for JL5 (0.1 log10 ) and the biggest for L-Zg
(1.0 log10 ) (Table 2). Like in the Urabe strain quantification, the titre
value for L-Zg, ZgB/Cro69 and ZgA/Cro69 obtained by plaque assay
differed more than 0.5 log10 from the titre value obtain by CCID50
assay (Tables 1 and 2).
The ratios of the results obtained by CCID50 assay and plaque
assay in quantification of the nine virus samples ranged between
0.66 and 10 (Tables 1 and 2). Ratios near to theoretically expected
value of 0.69 were obtained for only two viruses (0.66 for
9218/Zg98 and 0.83 for Du/CRO05; Table 2). For other seven MuV
strains potency ratio values were significantly higher then 0.69
(Tables 1 and 2). As previously mentioned, WHO established that
the ratio for Urabe strain is 1 [9]. In our study, only JL5 and
Du/CRO05 had the ratios relatively close to 1 (1.26 and 0.83, respec-
tively; Table 2). The ratios for remaining seven strains considerably
differed from 1 (Tables 1 and 2).
In our plaque assay, we counted plaques 5 h after addition of
neutral red. In order to be certain that 5 h is enough time to allow
for optimal staining of plaques, particularly those in the process
of developing, during the optimization of the method we tried
incubating plates with neutral red for 1–24 h. We did not obtain
any statistically significant difference in the number of plaques
between 5 and 24 h postneutral red addition. Even determination
of the titre of different viruses on day 8 after infection did not result
in significant differences in titres (data not shown).
Flanagan and Baron compared CCID50 assay and plaque assay
quantifications for four MuV strains [4]. Both of their assays were
performed in BGM cell line, which is also a continuous line of
African green monkey kidney cells but unlike Vero BGM cells are
epithelial cells, not fibroblasts. The ratios they found were 0.21 for
Boston strain, 0.63 for ABC strain, 1.7 for Ricki strain and 0.66 for
Jeryl Lynn vaccine. Their results, as well as ours, indicate that gross
differences in quantification results can be obtained, depending on
the method of choice. Such high variations in potency estimates
pose the problem for the quality control of live virus vaccines.
WHO and cPh.Eur specify the minimal amount of vaccine virus that
one dose should contain. Interlaboratory variations of potency esti-
mates can be crucial when the decision whether a vaccine lot is
accepted or rejected is made.
3.3. Factors influencing the results of quantification assays
The morphology of CPE plays no role in the ratio values between
the results of the two analyzed methods. Both low and high ratio
values were found for viruses that produced discrete as well as
distinct CPE (Table 2). The strains that produce less pronounced
CPE with very discrete syncytia tend to show a higher discrepancy
between the titres obtained by the two methods (Table 2). The
inter-methods difference in titres for strains which formed clear
syncytia, producing strong cytopathic effect and showing high fuso-
genic activity was below 0.5 log10 with the exception of the Urabe
strain (Tables 1 and 2).
The phylogenetic position of a strain also had no definite effect
on the ratio of the results obtained by CCID50 assay and plaque
assay. Closely related viruses JL2 and JL5, both classified as geno-
type A strains, showed almost identical ratio (Table 2). In contrast

Table 2
Potency estimates of eight mumps virus strains’ samples and comparison of results obtained by CCID50 and plaque assays. Strains that produced discrete cytopathic effect
are indicated in bold. n = number of assays.

Virus Titre obtained by Titre obtained by log10 inter-method Ratio of CCID50 and plaque
plaque assay (log10 CCID50 assay (log10 difference assays’ quantification
PFU/ml) CCID50 units/ml) results

L-Zg 5.46 ± 0.17 (n = 5) 6.46 ± 0.14 (n = 4) 1.00 10

9218/Zg98 7.12 ± 0.06 (n = 4) 6.94 ± 0.18 (n = 3) 0.18 0.66
ZgA/Cro69 4.73 ± 0.27 (n = 3) 5.61 ± 0.14 (n = 3) 0.88 7.6
ZgB/Cro69 4.77 ± 0.16 (n = 4) 5.28 ± 0.18 (n = 3) 0.51 3.23
JL2 5.51 ± 0.12 (n = 4) 5.73 ± 0.09 (n = 3) 0.22 1.65
JL5 6.49 ± 0.17 (n = 4) 6.59 ± 0.14 (n = 3) 0.10 1.26
Du/CRO05 6.74 ± 0.23 (n = 4) 6.66 ± 0.12 (n = 4) 0.08 0.83
Zg/CRO06 5.13 ± 0.03 (n = 4) 5.53 ± 0.12 (n = 5) 0.40 2.51
 D. Forcic et al. / Vaccine 28 (2010) 1887–1892
to those two strains, ZgA/Cro69 and Zg/CRO06, both genotype D
strains, showed significantly different values (Table 2).
In our CCID50 assay viral samples were diluted in MEM-H with-
out FCS and virus suspensions were added to the wells in which the
media with 5% FCS had been previously put. For the plaque assay,
virus samples were diluted in MEM-H with 2% FCS, but the entire
supernatant of Vero monolayers had been removed before the virus
suspension was added. For both assays the dilution processes lasted
only for a few minutes and the viral suspensions were immediately
added to the cells. The primary infections of cells were performed
in both methods under similar FCS concentrations: 2.5% in CCID50
assay and 2% in plaque assay for the first hour, 5% FCS afterwards.
As the process of virus dilution was performed very quickly, it is
our opinion that the presence/absence of FCS in the diluting media
did not influence the survival of the virions in the samples.
We cannot speculate to which extent did the chosen assays’
conditions influence our quantification results. Still, this question
was indirectly addressed in 1993, in a collaborative study that
was conducted to assess the variability of potency estimates of
measles, mumps and rubella trivalent vaccines in 10 different Euro-
pean laboratories [21]. In this study, participants were asked to
use their standard assay method. Although in the report present-
ing the results of this study the methods’ details were not specified
[21], it is reasonable to assume that the methods at least slightly
differed as WHO [11] and the cPh.Eur [12] do not specify precise
test parameters and source or passage number of cell substrate.
The mumps component showed greatest variability of the three
viruses tested: the range of potency estimates between laborato-
ries varied from 2.50 log10 (obtained for (a) a vaccine containing
Jeryl Lynn mumps component and (b) for Urabe International Ref-
erence Reagent of Mumps Vaccine) to even 3.24 log10 (obtained for
a vaccine containing Rubini mumps component) [21]. Expressing
the potency of mumps vaccines relative to a standard reference
improved agreement between the laboratories but only for those
vaccines with Urabe mumps component, and not Jeryl Lynn or
Rubini [21]. Thus, if results are expressed relative to a reference
reagent, it is necessary to use the same strain of the reference
reagent and the tested viral suspension. This is not possible to
implement regularly as the only available international reference
reagent of mumps virus is established for Urabe strain. Similarly,
in 2005, Rubin et al. published a study in which potency estimates
of 12 different MuV suspensions were established in parallel at the
NIBSC and at the Food and Drug Administration (FDA) [16]. The two
laboratories used slightly different plaque assays [16]. For all 12
suspensions different potency values were obtained and the differ-
ences ranged from 0.1 to 0.92 log10 PFU/ml. One additional concern
has to be mentioned when potency estimates of MuV are dis-
cussed. Because of the neurotropic and neurovirulent properties of
MuV, testing of live mumps vaccines for neurovirulence is required
by WHO and cPh.Eur. It is recommended that the neurovirulence
test should be performed in rhesus monkeys. The total amount of
MuV given to a monkey should not be less than the amount con-
tained in the recommended single human dose of vaccine [11]. The
questions whether this test can reliably discriminate neurovirulent
from non-neurovirulent strains have been raised [22,23]. In 2000,
Rubin et al. proposed an alternative, neonatal rat model for pre-
diction of MuV neurovirulence in humans in which neonatal rats
are inoculated with 100 PFU i.c. [24]. To better evaluate the per-
formance of the neonatal rat-based neurovirulence safety test, the
above-mentioned interlaboratory collaborative study was initiated
between the FDA and the NIBSC [16] in which the neurovirulence
scores of 12 virus preparations were measured. Although there
were a few differences in the execution of the neurovirulence test
between the two laboratories, the use of different virus doses is
likely to be the major contributor to the observed interlaboratory
differences in neurovirulence scores [16]. The use of different virus
1891
doses at the two laboratories was a result of virus potency being
determined at each test site [16]. In order for neonatal rat-based
test to yield truthful comparison of neurovirulence between vari-
ous MuV strains, it is necessary to estimate potencies of all tested
strains with a unique assay that is of equal accuracy and precision
for all strains. According to our experience, defining evidence-based
precise conditions for such a test will be very difficult to achieve.
4. Conclusion
A strong intrinsic feature of MuV strains to interact specif-
ically 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 methods of quantification. We have shown that the universal
conversion factor for the results of these two methods cannot be
unequivocally specified. The relation between the results must be
determined experimentally for each MuV strain and quantification
assays’ conditions.
Acknowledgments
The authors thank Dr. Ante Sabioncello for useful comments
and suggestions. This work was supported by the Ministry of Sci-
ence, 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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