Download Improved Dengue Virus Plaque Formation on BHK21 and LLCMK

Improved Dengue Virus Plaque Formation on
BHK21 and LLCMK2 Cells: Evaluation of Some Factors
Mayling Alvarez, Rosmari Rodriguez-Roche, Lídice Bernardo, Luis Morier
and Maria G. Guzman!
Department of Virology, PAHO/WHO Collaborating Center for Viral Diseases,
"Pedro Kourí" Tropical Medicine Institute, Havana, Cuba
Abstract
Neutralizing antibodies play a key role in the prevention of dengue infection. It is important that dengue
virus plaque titration and plaque reduction neutralization tests (PRNT) be highly reproducible using
standardized methods. To evaluate factors that have influence in the PRNT for dengue viruses, neutralizing
antibodies were determined in 24 serum samples and 12 blood samples collected on filter paper,
obtained through national dengue surveillance in Cuba. The influence of pH in the overlay medium,
the percentage of ambient CO2, the use of two different cell lines and of rapid centrifugation on dengue
plaque formation were evaluated. The efficiency of the plaquing system was optimal when the overlay
medium was buffered to pH 7.5. The rapid centrifugation of the virus on confluent cells increased the
virus titres. Higher virus titres were obtained on BHK21 rather than LLCMK2 cells when the viruses were
added to cell suspension. Under optimal conditions, PRNT was highly reproducible and is
recommended for seroepidemiological and vaccine studies using either BHK21 or LLCMK2 cells. This
communication also highlights the infection of LLCMK2 cell suspensions for measuring neutralizing
antibodies.
Keywords: Dengue, plaque reduction neutralization technique, diagnosis, Cuba.
Introduction
Dengue fever (DF)/dengue haemorrhagic fever
(DHF) has re-emerged as a public health
problem worldwide[1-3]. The dengue viruses are
serologically classified into four antigenically
distinct serotypes (DENV-1, DENV-2, DENV-3
and DENV-4) and are maintained in a humanmosquito transmission cycle, with Aedes aegypti
as the main vector. Currently, dengue is
endemic in most of the tropical areas in the
world[4,5].
!
Despite the antigenic relatedness of
dengue viruses, two or more serotypes may
sequentially infect one host. Epidemiological
studies in Thailand and Cuba showed that DHF
occurs in individuals with a secondary infection.
These studies suggest that the presence of
heterotypic dengue antibodies in a sequential
secondary infection is a risk factor for
developing the severe disease[6,7].
Neutralizing antibodies to dengue virus play a
very significant role in the prevention of dengue
[email protected]; " 53-7-2020450; Fax: 53-7-2046051
Dengue Bulletin – Vol 29, 2005
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Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
infection. Non-neutralizing cross-reactive
antibodies augment dengue virus infection of
Fc receptor cells. These virus antibody
complexes bind to Fc receptors on cells via
the Fc portion of IgG, resulting in enhancement
of dengue virus infection. This phenomenon
has been called antibody-dependent
enhancement (ADE)[8,9].
The ideal vaccine against DF and DHF
must prevent infection caused by all serotypes
avoiding ADE. Ideally, life-long neutralizing
antibodies to the four serotypes should be
present[10,11]. Towards this end, technological
innovation for improved plaque formation such
as speed, accuracy, economy, ease of
performing test as well as global standardization
is urgently required.
Plaque reduction neutralization technique
(PRNT) remains the “gold standard” for the
titration of neutralizing antibodies. Several
protocols have been described; however,
factors affecting the reproducibility of dengue
plaque assays have not been fully studied[12-21].
These include the use of solid or semi-sold
substances in overlay media, the cell substrate,
serum inhibitors and pH of the overlay medium.
Taking into account the need to determine
neutralizing antibodies for seroepidemiological
and vaccine studies, we decided to validate a
reliable plaque assay system to the four dengue
serotypes. The influence of pH range in the
overlay medium and the percentage of CO2
atmosphere for cell incubation were evaluated.
Also, the usefulness of rapid centrifugation
(shell vial assay) to improve the efficiency of
virus adsorption and consequently plaque
formation was also evaluated. Finally, efficiency
for virus titration and PRNT of baby hamster
kidney cell line (BHK21) and LLCMK2 as cell
suspensions were compared.
Materials and Methods
Viruses
Table 1 shows the dengue virus strains used in
this study.
Serum specimens
A total of 24 sera obtained through the Cuban
national dengue surveillance system during
Table 1. Details of the strains utilized in the neutralization tests
P: passage, MB: mouse brain, M: mosquitoes
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Dengue Bulletin – Vol 29, 2005
Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
2000 and 12 blood samples collected on filter
paper from healthy individuals during 2003
were selected for study. The first samples were
used to test the reproducibility of PRNT on
BHK21 cells. Filter paper eluates were tested
for DENV-1 to DENV-4 neutralizing antibodies
on BHK21 and LLCMK2 cells at a 1:30 dilution,
using exactly the same methods used
previously[22]. Antibody titres obtained in both
cells were compared.
Cell culture
BHK21, clone 15 cells were grown at 37 °C in
complete medium E-MEM (Minimum Essential
Medium with Earle’s balance salts)
supplemented with 10% of heat-inactivated
foetal bovine serum, 1% penicillin/
streptomycin, 10% L-glutamine and sodium
bicarbonate for adjusting the medium to pH
7.4-7.8. LLCMK2 cells (ATCC) were kindly
provided by Sutee Yoksan (Center for Vaccine
Development, Mahidol University) and were
grown in the same conditions.
the case of BHK21, and for the LLCMK2 cells
the plaques of all the serotypes were counted
on the 7th day. After incubation, plates were
rinsed gently under tap water and fixed and
stained with a solution of naphthol blue black
and acetic acid[16].
Plaque Reduction Neutralization
Test (PRNT)
PRNT was performed on both tested cell lines.
For antibody titration, 100 ul of serum dilution
(in MEM) was incubated for 1 hour at 37 °C
with 100 ul of the virus working dilution
calculated to give 10-20 pfu/50 ul of the final
volume of virus-serum mixture. After incubation,
50 ul of virus-serum mixture was added to the
cell suspension in triplicate. The pH during the
virus adsorption was in the range 7-7.5. Plates
were incubated for 4 hours at 37 °C under CO2
atmosphere. The rest was followed as above.
Each serum dilution was tested four times.
Calculations of 50% endpoint plaque reduction
neutralization titres were made using log probit
paper by the method of Russell et al., 1967[23].
Virus titration
Plaque titration was performed on BHK21,
clone 15 and in LLCMK2 as previously described
by Morens et al[16]. For virus titration, 50 ul of
virus dilutions (101 to 106) was added to 0.5 ml
of BHK21 cell suspension (1.5 x 105 cells) in
each of three wells of a 24-well polystyrene
plate. After 4 hours’ incubation at 37 °C in
CO2 atmosphere, each well received 0.5 ml
of 3% medium viscosity carboxymethyl
cellulose – made up in Earle’s minimum
essential medium without phenol red (MEM)
with 10% heat-inactivated foetal bovine serum,
1% glutamine 2 mM and 100 U of penicillin
plus 100 ug/ml streptomycin. Infected cells
were incubated for 5–9 days in the same
conditions as above depending on the virus
serotype (7–9 days for DENV-1 and DENV-3,
5 days for DENV-2 and 6 days for DENV-4) in
Dengue Bulletin – Vol 29, 2005
Evaluation of pH of overlay
medium, CO2 atmosphere and
rapid centrifugation of virus
during cell adsorption phase
on the efficiency of dengue virus
plaque assays in BHK21 cells
(1) In order to evaluate the influence of the
pH of the nutrient overlay medium, this
was adjusted at 6.5, 7, 7.5 and 8 final pH
using a solution of 7.5% sodium
bicarbonate. Cells were incubated under
CO 2 atmosphere at 4% and the four
dengue serotypes were titrated.
(2) To test the influence of CO2 atmosphere,
the pH of the overlay medium was
adjusted to 7.5, and cells were incubated
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Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
under CO2 atmosphere both at 4 and
4.5%. Under these conditions the four
dengue serotypes were titrated.
(3) For the evaluation of the rapid
centrifugation assay, 24 well plates were
used. DENV-2/A15 strain was inoculated
on cells in suspension or monolayer and
centrifuged at 37.73 x G (1500 rpm) for
30 min at 37 °C using an ALC model PM
140/140R centrifuge. After centrifugation,
cells were incubated for 4 hours at 37 °C
and overlay media (at pH 7.5) was added.
Infected cells were incubated at 37 °C in
CO2 atmosphere at either 4 or 4.5%.
Results
Evaluation of different factors
influencing dengue virus
plaque assays in BHK21 cells
Effect of pH of overlay medium
Titres of DENV-1, 3 and 4 depended on pH of
overlay media (Table 2). The highest titres were
observed at pH 7.5. For DENV-2, titres were
relatively similar at pH values ranging from 6.5
to 7.5, pH 7.5 being the optimal.
Table 2. Effect of pH of overlay medium on
dengue viruses plaque formation
NP: no plaques
Influence of CO2 atmosphere
Considering that the highest titres of the four
dengue serotypes were observed at pH 7.5 of
the overlay medium, this pH was used to test
the influence of CO2 atmosphere. In general,
titre and plaque size were relatively unaffected
by cell incubation under 4 or 4.5% CO 2
atmosphere.
Influence of rapid centrifugation of
virus during cell adsorption phase
Table 3 shows the effect of dengue virus
plaque formation of rapid centrifugation assay
both in cell suspensions and confluent
monolayer. Rapid centrifugation on confluent
cell cultures gave the highest viral titres,
compared to the conventional method, both
at 4 and 4.5% of CO2 atmosphere.
Table 3. Effect on dengue virus plaque formation of rapid centrifugation assay
using cell suspensions and confluent monolayer at two different percentages
of CO2 atmosphere*
* pH of overlay medium was adjusted to 7.5
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Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
Table 4. Replicate 50% neutralizing antibody titres and geometric mean titres (GMT) to
DENV-1 and DENV-2 on BHK21 cells
*Each serum was tested twice at two different days. Results are presented here as number 1 to 4.
Dengue Bulletin – Vol 29, 2005
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Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
Reproducibility of dengue PRNT
assays in BHK21
To test the reproducibility of PRNT assays, sera
were tested twice on two different days (in total,
antibody titre in each serum was determined
four times). Titres less than 1/10 to DENV-3 and
-4 were obtained. Only 2 (8.3%) sera showed
greater than twofold differences in the
neutralizing antibody titre to DENV-1 (Table 4).
Comparison of BHK21 and LLCMK2
cells for virus titration
and assay of neutralizing antibody
Titres of the four dengue viruses on BHK21
and LLCMK2 cell lines using an overlay media
buffered to pH 7.5 and incubated at 37 °C in a
4% CO2 atmosphere were compared. With the
exception of DENV-3, the highest viral titres
were obtained on BHK21 cell cultures (Table
5). Viral plaques were similar in size and
morphology in both cell lines with the exception
of DENV-4 plaques that were larger in both
cultures.
Table 5. DENV-1 to -4 titres determined by
plaque assay on BHK21 and LLCMK2 cell
suspensions*
*Plaque titre (Log 10 pfu/ml)
The percentage of plaque reduction to
each serotype was determined in 12 samples
collected on filter papers tested in both cell
lines. The results were similar in 100% of the
samples (Table 6).
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Discussion
Taking into account the necessity of measuring
neutralizing antibodies during vaccine trials,
it is important to apply standardized and
validated methods. However, factors affecting
the reproducibility of dengue plaque assays
have not been fully identified. Of the factors
studied in this work, the pH of the overlay
medium was the most critical variable. The
optimal pH of the overlay medium for
obtaining clear dengue virus plaques was pH
7.5. Other investigators have reported optimal
dengue virus plaquing over a wide range of
pH values in the overlay medium (pH 6.6 to
8.6) when using LLCMK 2 cells [19]. When
DENV-3 was assayed, plaque size decreased
at pH 8.6[19]. Dieg and Watkins[24] have found
that Colorado tick fever virus plaque
production was relatively insensitive over a
range of pH of 7.1 to 8.1 while plaque
formation failed to occur at pH 7.0 or lower.
In hamster lung cell, plaque formation of
Japanese encephalitis virus was not observed
when pH of the overlay medium was under
6.6 or above 7.8.[18] Lee et al[25]. have reported
dengue plaque formation at a pH range of
7.4 to 7.6. Sodium bicarbonate concentrations
have been reported to influence plaque size
with differences two- to eight-fold differences,
especially for DENV-2, -3 and -4[19]. In our
study no significant difference in the plaque
size was observed over the range of pH values
tested, with the exception of DENV-4 which
produced large plaques at pH 7.5.
In general, titre and plaque size were
relatively unaffected by incubating infected
cells at different percentages of CO 2
atmosphere. Stim TB [19] have reported
increased sensitivity of dengue virus plaque
assays at high concentrations of sodium
bicarbonate.
Dengue Bulletin – Vol 29, 2005
Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
Table 6. Comparative plaque reduction neutralization of DENV-1 to -4 in BHK21 and LLCMK2
cells infected as cell suspension*
*Whole blood on filter papers was re-suspended at 1:30 dilution
In our laboratory, the rapid centrifugation
or shell vial assay has greatly improved the
isolation rate of some viruses. This method of
enhancing virus-cell interactions decreases the
time of the onset of cytopathic effect of many
viruses present in clinical diagnostic specimens.
It was applied to plaque assays and improved
consistency of input virus dose. For dengue
Dengue Bulletin – Vol 29, 2005
viruses, rapid centrifugation improved the
isolation rate in blood and tissues[26]. Recently,
Chingsuwanrote et al. (2004) observed a
relationship of the number of plaques and postinfection incubation time, with the longest postinfection incubation time giving the highest
number of plaques[27].
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Improved Dengue Virus Plaque Formation on BHK21 and LLCMK2 Cells
Several cell lines have been used for dengue
plaque assays but there are few published
comparisons between cell systems using
optimized conditions[12,14,15,17,27]. In the present
study, virus titres were higher in BHK21 than in
LLCMK2 cell cultures, except for DENV-3.
In summary, (i) the pH of the overlay
medium may be an important factor in obtaining
clear and large dengue plaques; (ii) the
centrifugation of virus inoculate on cells increases
virus titres; (iii) higher virus titres were obtained
in BHK21 compared with LLCMK2 cell cultures;
(iv) highly reproducible dengue PRNT titres were
obtained on BHK21 cells; and (v) using the cell
suspension method, DENV-3 resulted in higher
titres in LLCMK2 than BHK21 cells.
The current work confirms that under
optimal conditions, plaque reduction
neutralization titres can be repeated
reproducibly. Seroepidemiological and vaccine
studies can be performed satisfactorily in both
BHK21 and LLCMK2 cells.
Acknowledgements
This work was supported by TDR/WHO grant
ID No. 990904. We thank Prof. Robert Shope
of Texas University and Prof. Scott Halstead
from the Pediatric Dengue Vaccine Initiative,
Bethesda, Maryland, USA, for their useful
suggestions and comments.
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