Download Study of Biologic Attributes of Cuban Dengue 2 Virus after Serial

Original Report
Study of Biologic Attributes of Cuban Dengue 2 Virus
after Serial Passage in Primary Dog Kidney Cells
Mayling Alvarez, MSc ;* Maria G. Guzmb, PhD; * Maritza Pupo, MSc; *
Luis Morier, BcS;* Jo& Bravo, MD;* and Rosmari Rodriguez, MSc*
ABSTRACT
Objective: The serial passage of dengue viruses in primary dog
kidney (PDK) cells has resulted in selection of attenuated
viruses, However, the molecular changes responsible for loss
of virulence are not well characterized. This article describes the
isolation and biologic attributes of one dengue 2 virulent strain
as a first step to allow the study of determinants
of virulence
at the molecular level.
Methods: A15 dengue 2 Cuban strain was isolated from the
viremic plasma of a patient with uncomplicated
dengue fever
during the 1981 epidemic. This was then subjected to serial
passage in PDK cells. Viruses resulting from several PDK passages were compared to the parent strain for plaque size and
temperature
sensitivity, neurovirulence
in newborn mice, and
cytopathogenic
effects on LLC-MK, and C6/36-HT cell lines.
Results: Al5 dengue 2 Cuban strain was successfully propagated in PDK cells. Primary dog kidney 52 to 53 viruses exhibited several biologic
attributes,
such as small plaques,
temperature
sensitivity, reduced mouse neurovirulence,
and
cytopathic effect in permissive cell lines.
Conclusions:
These results represent the first step to allow
attenuation of this strain of dengue 2 virus.
Key Words: attenuation,
vaccine
Int J Infect Dis 2001;
dengue,
dengue
hemorrhagic
fever;
5:35-39.
Demographic and societal changes decreasing resources
for vector-borne infectious disease prevention and control, and changes in public health policy have all contributed to increased epidemic dengue activity, the
development of hyperendemicity, and the emergence of
dengue fever (DF) and dengue hemorrhagic fever (DHF)
*Department of Virology,
edses,
Address
Institute,
Havana,
Tropical
Medicine
PAHO/WHO
Collaborator
Center
Institute
of Havana, Cuba.
correspondence
to Prof. Maria
Autopista
Novia de1 Mediodia,
Cuba. E-mail: [email protected].
for Viral Dis-
G. GuzmPn, Tropical
Medicine
Km 6, apdo 601, Marianao
13,
epidemics. Theoretically, prevention could be achieved
either through vector control or through immunization.’
Vector control is the only preventive method available
at present, but it is expensive, has only a temporary benefit, and has been ineffective in controlling disease when
not maintained. Prevention through vaccination is an
important objective; however, in spite of multiple studies,
no effective vaccine is available, and more basic studies
are needed.’
Early attempts to develop dengue vaccines focused
on the attenuation of the virus by passage in mice and
cell cultures. Specifically, the serial passage of dengue 1,
2, and 4 in primary dog kidney (PDK) cells has resulted
in selection of attenuated viruses.“” This approach has
produced a number of dengue virus strains that have various degrees of reduced virulence for humans compared
to the parent virus strains from which they were derived.
The nucleotide sequence of parent dengue 2 virus with
an attenuated strain derived by 53 serial passages in PDK
cells revealed 53 nucleotide
changes resulting in
27 amino acid alterations scattered throughout
the
genome.’ However, because it is not easy to determine
which changes are actually responsible for loss of virulence and which may be unimportant, it would be useful to compare the sequences of different strains at
different PDK passage levels.’
One of the most serious DHF/dengue shock syndrome (DSS) epidemics occurred in Cuba in 1981, where
a dengue 2 strain caused an outbreak resulting in 344,203
DF and DHF cases. Of these, 10,3 12 were classified as
severe and very severe. There were 158 fatalities (101 in
children) in only 4 months.8 Twenty-two dengue 2 isolates were obtained from DF and DHF cases.“The
genomic study of four of these showed them to be similar to those from southeast Asia and specifically the New
Guinea C and 16681 dengue 2 strains.“‘-‘*
To investigate virulence markers at the molecular
level, the Al5 dengue 2 Cuban strain was serially passaged in PDK cells. This article describes the isolation
and biologic attributes of the Al5 passages in PDK cells
as a first step to allow further study of the determinants
of virulence at the nucleotide and amino acid levels.
35
36
International
MATERIALS
Journal
of Infectious
Diseases / Volume 5, Number 1
AND METHODS
Virus Strain
A15 dengue 2 Cuban strain was obtained from the
viremic plasma of a patient with uncomplicated DF contracted during the 1981 epidemic.13 The strain had been
passaged four times in suckling mouse brain before passage in PDK cells. This strain constituted the parent virus.
A 10% suspension in phosphate buffered saline (PBS) was
used as starting material for inoculation of tissue culture
for attenuation studies. For serial passages, 0.5 mL of undiluted virus from the previous passage was inoculated in
PDK cells grown in 25-cm2 plastic bottles with a confluent monolayer. After 1 hour of adsorption at 37°C supplemented
medium was added and cultures were
incubated at 33°C for 7 days. The serial passage and the
propagation of seed viruses were done under Good Laboratory Practices conditions. lja
Cell Cultures
The source of PDK cells was frozen primary beagle kidney cells from a colony raised and maintained under specific pathogen-free conditions, obtained from CEMPALAB,
Cuba. Individual ampules were thawed when needed and
grown and maintained in Dulbecco medium with 10%
heat-inactivated fetal bovine serum (HFBS). One lot of
cells derived from the kidneys of four dogs was used
throughout the study. I4
Aedes albopictw (C6/36-HI) and LLC-MK, (CCL-7;
American type collection) cell cultures were grown in
Eagle’s minimal medium supplemented with HFBS, 1%
nonessential amino acids, and 1% 200 mM glutamine solution. Maintenance medium was identical to the growth
medium but supplemented with 2% of HFBS.Cells grown
in plastic tubes, were inoculated at two multiplicities of
infection (ml = 0.01 and m, = 0.0001) with parent and
PDK 20, 40, 52 to 55 viruses. Following adsorption for
30 minutes, 1 mL of supplemented medium was added.
Cells were kept at 33” and 37°C respectively (depending
on the cell culture), and observed daily for viral cytopathic effect (CPE).
bovine serum. Plates were incubated for 5 days in the
same conditions as above. The medium was discarded by
inverting plates. The plates were then rinsed gently under
tap water, fured, and stained using a solution of naphtol
blue-black and acetic acid. For temperature sensitivity studies, inoculated cells were incubated both at 37°C and at
39.2”C. Each experiment was repeated at least twice.
Immunofluorescence
An indirect immunofluorescence
test was used to detect
virus antigen in inoculated PDK cells using dengue 2-specific monoclonal antibody donated by Dr. D. Gubler from
Centers for Disease Control and Prevention (Atlanta, Georgia, USA).
Animal
Inoculation
One- to 2-day-old suckling mice were inoculated intracerebrally with 0.02-mL volumes of diluted viruses. The
mice were observed for sickness and death for 21 days.
For the purposes of this calculation, mice surviving over
2 1 days were scored as dying on day 2 1. The mouse 50
lethal dose (LD50) endpoints were determined by the
Reed-Muench method.lb Deaths observed in the first 24
to 48 hours were not considered.
Statistical Analysis
Student’s t-test, Spearman correlation, and variance analysis were used.
RESULTS
Sixty serial passages were obtained from Al5 dengue 2
Cuban strain on PDK cells. At different passage levels in
PDK cells, approximately 15 mL of virus seed was prepared. Characteristics of the selectants are shown in
Tables 1 to 4.
Immunofluorescence
was positive in all tested viral
passages, allowing the study of viral replication.
Moderate growth (circa 102-lo3 plaque-forming units
(PFU)/mL) occurred with all PDK viruses. Parent virus
Virus Assays
Tttrations
Viral plaque assay to test the infectivity of viral passages,
to identify the plaque size, and to identify temperature
sensitivity were performed in baby hamster kidney (BHK21, clone 15) cell line, as previously described.15 Briefly,
virus dilution (50 p,L) was added to 0.5 mL of BHK-21
suspension (1.5 X lo5 cells) in each of three wells of a
24-well polystyrene plate. After 4 hours of incubation at
37°C in 5% CO,, each well received 0.5 mL of 3% mediumviscosity carboxymethyl cellulose, made up Earle’s minimum essential medium with 10% heat-inactivated fetal
Table
PDK
Passage
4* (parent
1.
Viral Plaque
Size of Some
Plaque
Level
Mean
virus)
20
40
52
53
54
55
P < 0.01; *4 passages
in suckling
mouse
Passages
Size (mm)
Standard
Deviation
0.394
1.520
1.780
1.660
0.210
1.529
1.164
0.856
1.032
0.400
0.401
0.246
0.384
brain.
0.280
Cuban Dengue 2 Virus /Alvarez et al
Table
PDK Passage
4* (parent
20
40
52
53
54
55
P < 0.01; PFU = plaque-forming
Effect
of Temperature
37°C
Level
virus)
2.
2.10
2.40
3.40
1.16
6.60
7.60
1.16
x
X
x
x
x
X
x
units; *4 passages
on Viral Infectivity
105
IO3
103
lo3
102
10"
lo3
0.86 x 102
1.00 x 10
1.20 x 103
0.13 x 10
0.98 x 10
1.30 x 10
0.13 x 10
in suckling
mouse
Table
4* (parent
20
40
52
53
54
55
Level
virus)
P < 0.05: PFU = plaque-forming
3.
Suckling
x
x
x
x
x
X
x
units: *4 passages
Mouse
105
103
103
103
103
lo*
103
60.0
59.0
64.7
89.0
86.0
83.0
89.0
Conventional techniques for attenuation of viruses, such
as passage of virus at low temperature or passage in heterologous tissues continues to have a role in the development of live-virus vaccines.” In the particular case of
dengue, propagation in tissue culture has yielded viruses
that have been demonstrated to have attenuated virulence in humans.‘8-2” Differential propagation has also
been shown to affect the tropism of dengue viruses for
various cell lines in vitro, temperature sensitivity, plaque
size, cytopathic effects in vitro, and mouse neurovirulence.*r These biologic markers have been reported as
important for viral attenuation; however, owing to the
lack of a good animal model for dengue infection, the
distinction between nonattenuated and attenuated viruses
must be determined empirically, using these biologic
markers.**
The development of DHF has been related to more
virulent strains, however, until now only nonspecific
nucleotide or amino acid changes have been determined.2”-25 It is possible that if a more virulent strain
could be attenuated, it would be possible to identify and
characterize the factors influencing virulence and, as a
consequence, enhanced immunogenic&y.
In agreement with observations made by other
authors,4-6 serial passage of the Al5 dengue 2 Cuban
strain in PDK cells, resulted in several stable changes,
such as temperature of restricted replication, small plaque
size, extension of survival in suckling mice, and reduced
Neurovirulence
1046'
102.74
lOSO
102O8
lo'g'
102'3
10'85
mouse
0.400
0.410
0.431
0.110
0.140
0.170
0.110
Percentage
DISCUSSION
LD50
in suckling
Reduction
brain.
37°C
2.1
1.1
3.4
1.2
1.0
3.2
1.9
Passages
PFU 39.2W37”C
39.2"C
showed the highest titer (lo5 PFU/mL) and PDK 53 to 54
the lowest (lo* PFU/mL). Stability of viral titers was
observed.
Plaque size diminished from 1.5 mm in the parent
virus to between 0.8 and 1.1 mm at PDK 53 to 55 viruses
(P < 0.01). After PDK 55 virus passage, an increase in the
plaque size was observed (data not shown). Table 1
shows the means and standard deviations of plaque size
at different passages.
Viral infectivity of some PDK passages was compared
with the parent virus grown at 37°C and 39.2”C (see
Table 2). A gradual decrease of infectivity was observed
at PDK 52 to 55 (r = -0.75; P < 0.01). The parent virus
showed some restriction of growth at 39.2”C. In a separate test, 40°C was found to be nonpermissive for plaque
formation by the parent virus.
Primary dog kidney cultures inoculated with serially
passaged viruses were observed daily for CPE; PDK 53
to 55 viruses showed almost no CPE in LLC-MK, and
C6/3GHT cell lines at both multiplicities tested, being
more evident at the lowest. Parental virus produced complete CPE in 4 to 5 days.
An increase in PDK passage level resulted in a gradual lengthening of the mean interval to the day of death
after intracerebral inoculation into suckling mice (see
Table 3). Also, PDK 52 and 55 showed a 90 to 97% reduction of neurovirulence (r = -0.78; P < 0.05).
Table 4 shows a summary of biologic properties of
dengue 2 (A15) at different passage levels in PDK cell
culture.
PDK Passage
of Some
37
brain,
of Some
Passages
LD5O/PFU37”C
0.190
0.490
0.290
0.091
0.105
0.220
0.030
Reduction
Percentage
81.0
51 .o
71.0
90.9
89.5
78.0
97.0
International Journal of Infectious Diseases / Volume 5, Number 1
38
Table
4.
Summary
of Biologic
Properties
of Cuban
Dengue
2 Virus
at Different
Passage
Levels
in Primary
PDK Passage
EGologic
Parameter
Parent
Temperature
sensitivity
at 39.2%
PDk plaque size (mmj
LLC-MK,
CPE
C6/36-HT
CPE
Suckling
mouse neurovirulence
reduction
CPE = cytopathic
Kidney
Cell Culture
Levels
20
40
52
53
54
55
1.5
+
1.5
+
1.4
+
+
1.5
-
+
1 .l
-
+
1
-
EA
5’1
7’1
90
a9
+
0.8
78
-
percentage
Dog
97
effect.
cytopathic effect in two permissive cell lines. These biologic markers have been used to evaluate changes in biologic properties
that may be suitable for empirical
assessment. A decrease in viral titer was also observed,
probably demonstrating the nonpermissive action of PDK
cells to dengue virus. These findings are consistent with
previous observations and suggest that the biologic attributes of the Cuban strain were changed during PDK 53 to
55 passages.
Recently, molecular characterization of dengue virus
attenuation was attempted by comparison of sequences
of the parental virus and a derived vaccine strain prepared by serial passage in PDK cellsz6 The degree of
attenuation appeared to increase with PDK passage level,
and multiple mutations probably contributed to this high
level of attenuation. In this study, multiple nucleotide
changes in various regions of the dengue virus genome,
some of which resulted in amino acid changes, have been
found in the vaccine strain. It remains to be defined
which mutations are responsible for attenuation and
which are not.27 In another study, the authors demonstrated profound changes, including decreased growth in
LLC-MK, and C6/36-HT cells, of deletions engineered into
the 5’ NC region of dengue 4 virus variants.”
The comparison of other viruses, of different temporal and geographic origins but with the same cell history of passage could allow the definition of which
nucleotide and amino acid changes are persistent. These
results are important
in relation to the attenuation
process and, consequently, vaccine development. They
will be used in molecular comparison studies of Al5
Cuban attenuated strain with the parent virus.
ACKNOWLEDGMENTS
The expertise of Dr. Paul Kane in the preparation
script is acknowledged.
of this manu-
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