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Previous Article | Next Article 
Journal of Virology, April 2000, p. 3011-3019, Vol. 74, No. 7
Center for Vaccine Development, Institute of
Science and Technology for Development, Mahidol University at Salaya,
Nakhonpathom 73170, Thailand,1 and
Division of Vector-Borne Infectious Diseases, National Center
for Infectious Diseases, Centers for Disease Control and Prevention,
U.S. Department of Health and Human Services, Fort Collins,
Colorado 805222
Received 26 July 1999/Accepted 27 December 1999
The genome of a candidate dengue type 2 (DEN-2) vaccine virus,
strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides.
Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK2 cells, growth rate in C6/36 cells,
and neurovirulence in newborn mice. All of the viruses replicated to
peak titers of 107.3 PFU/ml or greater in
LLC-MK2 cells. The crippled replication of PDK-53 virus in
C6/36 cells and its attenuation for mice were determined primarily by
the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of
PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val
mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the
small-plaque phenotype of PDK-53 virus. Reversions at two or three of
these loci in PDK-53 virus were required to reconstitute the phenotypic
characteristics of the parental 16681 virus. The prM-29 locus had
little or no effect on viral phenotype. Sequence analyses showed that
PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.
Dengue (DEN) viruses belong to the
genus Flavivirus (family Flaviviridae)
(66). Flaviviruses contain a single-stranded, positive-sense
RNA genome of approximately 11 kb. The RNA genome is capped at the 5'
end and lacks a 3'-terminal poly(A) tail. The gene order is 5'
noncoding region (5'NC)-capsid-premembrane/membrane (prM/M)-envelope
(E)-nonstructural protein 1 (NS1)-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'NC. Three structural and
seven nonstructural proteins are co- and posttranslationally processed
from the polyprotein precursor by both virus-specified and cellular
proteases (14, 66).
DEN viruses are classified antigenically as four serotypes (DEN-1,
DEN-2, DEN-3, and DEN-4). Infection with any of these serotypes can
produce clinical illness, ranging from a nonspecific febrile syndrome
to severe and fatal dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS). An immunopathological response following secondary infection
of humans with a heterologous serotype of DEN virus may be one of the
risk factors for the more severe forms of the disease (25, 29, 48,
50). Transmission of DEN virus has increased during the past 10 years because of the worldwide distribution of Aedes aegypti
mosquitoes in the tropics and subtropics. Currently, an estimated 100 million cases of dengue fever occur each year, and DHF is a leading
cause of death and hospitalization of children in many countries of
Asia and South America (27).
An effective vaccine against DEN virus is currently not available. Live
attenuated vaccine candidates for all four serotypes have been
developed by serial passage of wild-type viruses in primary dog kidney
(PDK) cells or other cell types at Mahidol University, Bangkok,
Thailand (70). The Mahidol candidate DEN-2 vaccine virus,
strain PDK-53, was derived by passaging the wild-type DEN-2 16681 virus
53 times in PDK cells. The PDK-53 virus has several biological markers
associated with attenuation, including temperature sensitivity, small
plaque size, loss of neurovirulence for suckling mice, and decreased
incidence of viremia in monkeys (70). In one report, all 7 monkeys challenged with DEN-2 16681 virus, but only 2 of 10 challenged
with PDK-53 virus, had detectable viremia (viral titers not reported)
(70). In another report, PDK-53 virus was minimally
neurotropic for rhesus monkeys, and no viremia was detected (by the
direct plaque method in LLC-MK2 cells) in monkeys
challenged intramuscularly, intracerebrally, and intraspinally with
either 16681 or PDK-53 virus (1). DEN-2 16681 virus itself
was only weakly neurovirulent in these monkeys (1). None of
the monkeys had detectable prechallenge neutralizing antibodies against
any of the four DEN virus serotypes (1). Clinical trials of
the candidate PDK-53 virus vaccine have shown that it is safe and
immunogenic in humans (4, 6, 65) and induces DEN
virus-specific T-cell memory responses in human vaccinees (19). To prevent the possible occurrence of DHF/DSS in
vaccinees, a tetravalent vaccine is needed to provide immunity against
all four serotypes of the virus.
We previously identified nine nucleotide mutations (three silent)
between the genomes of the DEN-2 16681 and PDK-53 viruses (32). One C-to-T (16681-to-PDK-53) nucleotide mutation
occurs at genome nucleotide position (nt) 57 in the 5'NC region. Five nucleotide mutations encode amino acid substitutions Asp-to-Val at
prM-29, Gly-to-Asp at NS1-53, Leu-to-Phe at NS2A-181, Glu-to-Val at
NS3-250, and Gly-to-Ala at NS4A-75. The candidate PDK-53 virus vaccine
has a mixed genotype at genome nt 5270: a significant portion
(approximately 29%) of the virus population encodes the NS3-250-Glu
that is present in the parental DEN-2 16681 virus. In the present
study, we investigated several genetic loci as possible determinants of
in vitro and in vivo markers of attenuation in the candidate PDK-53
virus vaccine by analyzing a panel of recombinant DEN-2 16681/PDK-53
viruses. These viruses were engineered by recombination of full
genome-length cDNA clones derived from both DEN-2 16681 and PDK-53
viruses (32).
Viruses and cell cultures.
The parental DEN-2 16681 virus,
several intermediate PDK passages (PDK-5, -10, -14, -35, and -45) of
16681 virus, recombinant 16681/PDK-53 viruses, and the genetically
characterized LLC-MK2-1 passage (32) of the
candidate PDK-53 vaccine virus were investigated.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Attenuation Markers of a Candidate Dengue Type 2 Vaccine Virus, Strain 16681 (PDK-53), Are Defined by Mutations in the
5' Noncoding Region and Nonstructural Proteins 1 and 3
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Construction of recombinant DEN-2 16681/PDK-53 viruses. During the genetic validation of clone-derived DEN-2 viruses in the present study, we discovered two cDNA cloning errors, nt 6665 A-to-G (NS4A-97 Tyr-to-Cys) and nt 8840 A-to-G (NS5-424 Glu-to-Gly), in the previously reported PDK-53 virus-specific pD2/IC-130Vc-K (NS3-250-Val variant) clone (32). These defects were corrected in a newly derived PDK-53 virus-specific (NS3-250-Val variant) clone, pD2/IC-VV45R.
In preliminary studies, we derived recombinant 16681/PDK-53 viruses, each containing PDK-53 virus-specific gene regions within the genetic background of 16681 virus, to investigate the genetic loci involved in the attenuation markers of PDK-53 virus (unpublished data). Analyses of these viruses indicated that the PDK-53 mutation at nt 57 in the 5'NC region (5'NC-57) and the amino acid mutations at NS1-53 (analyzed in a linked manner with the NS2A-181 mutation) and NS3-250 were likely determinants of the PDK-53 virus-specific phenotype (unpublished data). The prM-29 mutation appeared to have little effect. Because the 5'NC mutation occurred in a possible stem structure, and the NS1 and NS3 mutations occurred at loci that are conserved among various flaviviruses (analyses not shown), we decided to investigate these three genetic loci individually and in all possible combinations. We constructed 14 recombinant pD2/IC-16681/PDK-53 plasmids (Table 1) by exchanging cDNA fragments between pD2/IC-30P-A (16681 clone) and pD2/IC-VV45R (PDK-53 clone) at restriction enzyme sites SstI (preceding the T7 promoter), SalI (nt 165), SphI (nt 1380), SpeI (nt 2370 and 3579), KpnI (nt 4493), XhoI (nt 5426), and XbaI (3' end of the clone). All recombinant plasmids were grown in Escherichia coli XLI-Blue and were linearized at the unique XbaI site engineered at the 3' terminus of the cDNA. BHK-21 cells were transfected with transcribed viral RNA by the method of Liljeström et al. (37) as described previously (32).
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Characterization of the replication phenotypes of recombinant
16681/PDK-53 viruses.
Viruses were analyzed for plaque size,
temperature sensitivity, and replication in LLC-MK2 and
C6/36 cells. Plaque sizes were evaluated after 9 days of incubation
under agarose in LLC-MK2 cell monolayers grown in six-well
plates (32). Viral growth curves were performed in
75-cm2 flasks of LLC-MK2 or C6/36 cells
inoculated at multiplicity of infection (MOI) of approximately 0.001 PFU/cell. After adsorption at 37°C for 2 h, 30 ml of DMEM
(LLC-MK2 cells) or overlay nutrient medium (C6/36 cells)
containing penicillin-streptomycin and 5% FBS was added, and the
cultures were incubated in 5% CO2 at 37 or 29°C,
respectively. Aliquots of culture medium were removed at 48-h
intervals, adjusted to 12.5% FBS, and stored at 
80°C prior to
virus titration.
Mouse neurovirulence assay. Litters of newborn, outbred white ICR mice were inoculated intracranially with 104 PFU of virus in a volume of 30 µl. Mice were individually weighed once a week and were observed for paralysis or death for 35 days.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Plaque phenotypes of recombinant 16681/PDK-53 viruses.
Mean
diameters of virus plaques (n = 12) at 9 days after
infection under agarose overlay in LLC-MK2 cells are shown
in Fig. 1A. The largest plaques (3.2 to
3.4 mm, mean diameter) were produced by the wild-type 16681 virus, its
clone-derived 30P-A virus, and the recombinant P513 virus, which
contained the 5'NC, NS1, and NS3 16681 virus-specific loci in the VV45R
(PDK-53) virus genetic background (Table 1). These three 16681 virus-specific loci within the PDK-53 virus genetic background were
sufficient to reconstitute the large-plaque phenotype of the 16681 virus (Fig. 1A).
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Replication of recombinant 16681/PDK-53 viruses in LLC-MK2 and C6/36 cells. In a single experiment, all of the viruses in this study replicated well in LLC-MK2 cells, reaching peak titers of 107.3 to 107.9 PFU/ml at 6 to 8 days after infection (Fig. 1B; Pp, P5p, Vp, and V5p viruses not shown). PDK-53 virus and its clone-derived VV45R virus replicated more slowly than the other viruses during the first 4 days after infection (data not shown). To determine temperature sensitivities, virus-infected LLC-MK2 cells were incubated at 37 or 38.7°C in two to five experiments. Temperature sensitivity (defined here as a 90% or greater reduction in virus titer at 38.7°C) scores were determined at 8 days after infection. All of the viruses exhibited some degree of temperature sensitivity under these conditions. In individual experiments, wild-type 16681 virus showed 75 to 80% titer reduction at 38.7°C. Virus V1 and cognate virus pair P3 (V51), which showed 84 to 86% average reductions in titer, were slightly more temperature sensitive than 16681 virus. However, only PDK-53, VV45R, V513, P5p, Pp, and the cognate recombinant viruses V13 and P5, all of which contained both the NS1-53-Asp and NS3-250-Val PDK-53 virus-specific loci, reproducibly showed 90 to 97% average reduction in titer at 38.7°C (Fig. 1B and data not shown).
The 16681 virus and its clone-derived 30P-A virus replicated to average peak titers of 108.6 to 108.8 PFU/ml at 12 days after infection in two independent growth curve experiments in C6/36 cells (Fig. 1C). The replication of PDK-53 virus (peak titer of 104.5 PFU/ml) and its clone-derived VV45R (peak titer of 104.6 PFU/ml) virus was approximately 15,000-fold less efficient in C6/36 cells. The 16681 virus-specific 5'NC and NS1 loci within the PDK-53 background of P51 virus fully reconstituted the replication efficacy to that of wild-type 16681 virus (Fig. 1C). Conversely, the PDK-53 virus-specific 5'NC and NS1 loci within the 16681 virus background of V51 virus were sufficient to establish the crippled replication phenotype of PDK-53 virus. Recombinant cognate virus pairs V5 (P13) and V1 (P53), which contained the PDK-53 virus-specific 5'NC region or NS1 locus, respectively, replicated to average peak titers of 105.9 to 106.7 PFU/ml. Although the average peak titer of V53 virus was about 40-fold greater than that of V5 virus in C6/36 cells, the peak titers of P513, P13, P53, V3, V13, V513, and P3 viruses were very similar to those of P51, P1, P5, 30P-A, V1, V51, and VV45R viruses, respectively. These data indicated that the NS3-250 locus had little or no observable effect on replication in C6/36 cells (Fig. 1C). Vp and 16681 viruses had nearly equal average peak titers in C6/36 cells, as did P5 and P5p viruses. Pp and V5p viruses produced average peak titers that were slightly higher (8- and 40-fold, respectively) than those of PDK-53 and V5 viruses, respectively (data not shown). The prM-29 locus appeared to have little or no effect on viral replication in C6/36 cells.Neurovirulence of recombinant 16681/PDK-53 viruses in newborn
mice.
To investigate the neurovirulence of the recombinant
viruses, two litters of newborn white ICR mice, eight mice per litter, were infected intracranially with 104 PFU of virus. The
DEN-2 16681 virus and its clone-derived 30P-A virus cause 50 to 100%
mortality in these mice (32). Average survival times (AST)
for mice succumbing to challenge with 16681 or 30P-A virus ranged from
15.2 to 16.8 days in various experiments. Mice were weighed
individually every 7 days after infection. A single mouse died by day 1 after infection, presumably as a result of inoculation trauma, in each
of the P53 and Vp groups (Table 2). These
two mice were excluded from the analyses. There were no fatalities and
no weight loss in the control, diluent-inoculated group (not shown in
Table 2).
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Evolution of mutations in the DEN-2 PDK-53 virus vaccine
strain.
We analyzed intermediate passages PDK-5, -10, -14, -35, and -45 of the 16681 virus to determine the accrual of the nine
nucleotide mutations in the PDK-53 virus vaccine strain. Amplicons were
amplified directly from genomic mRNA extracted from the viral seed by
RT-PCR. Automated sequencing of small genomic regions, which contained the nine relevant loci, was performed by using appropriate primers. The
nucleotide residues identified at each of the nine loci for these
viruses are shown in Table 3. The
NS2A-181 Leu-to-Phe mutation and the silent mutations at E-37, NS3-342,
and NS5-334 appeared by passage PDK-5 and were the predominant moieties
by passage PDK-10 (NS2A-181), PDK-14 (E-373, NS3-342), or PDK-35
(NS5-334). 5'-NC-57 C-to-T, prM-29 Asp-to-Val, NS1-53 Gly-to-Asp, and
NS4A-75 Gly-to-Ala mutations occurred by passage PDK-35. The 5'-NC-57-T mutation was predominant at passage PDK-35, while the other listed mutations became predominant by passage PDK-45. The NS3-250 Glu-to-Val mutation appeared by passage PDK-45 and is not fully mutated to the
virus-specific Val in the current PDK-53 virus vaccine candidate (Table
3). Approximately 29% of the viral population in the PDK-53 virus
vaccine contains NS3-250-Glu (32). The PDK-45 virus was genetically equivalent to the PDK-53 virus vaccine. In the present study, we did not attempt to determine the relative proportions of the
two nucleotides at the mixed genetic loci shown in Table 3.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Amino acid substitutions in the viral E protein, as well as other viral proteins, are associated with attenuation or virulence in flaviviruses (3, 8, 13, 28, 30, 43, 53, 63). It is apparent that the potential repertoire of attenuating mutations in the flavivirus genome is quite extensive. Mutations that occur between other parent/attenuated flaviviruses, such as yellow fever Asibi/17D viruses (28), Japanese encephalitis SA14/SA14-14-2 viruses (53), and DEN-1 Western Pacific 74/45AZ5 PDK-0/45AZ5 PDK-27 viruses (59), do not coincide with each other or with the mutations identified between the DEN-2 16681/PDK-53 viruses (32). The prM-29 Asp-to-Val substitution is the only amino acid mutation that occurs in the structural proteins of the candidate DEN-2 PDK-53 virus vaccine, while the other four amino acid mutations occur in nonstructural genes (32). Depending on the particular recombinant 16681/PDK-53 virus investigated in the current and earlier pilot (unpublished data) studies, the prM-29 locus had minor or no effect on plaque size, replication in LLC-MK2 or C6/36 cells, or mouse neurovirulence. Neither the prM-29-Asp nor the NS2A-181-Leu residue of DEN-2 16681 virus is conserved among flaviviruses. The NS4A-75-Gly is conserved only among the four serotypes of DEN virus. The latter two loci were not specifically investigated in this study. In contrast, the NS1-53-Gly residue is highly conserved among the aligned amino acid sequences of the NS1 proteins of nearly all flaviviruses reported to date, and the NS3-250-Glu is conserved among the mosquito-borne flaviviruses other than yellow fever virus (aligned data not shown). Such sequence conservation probably reflects important functionality of these two loci in the NS1 and NS3 proteins of flaviviruses.
All of the viruses studied here replicated to peak titers of 107.3 PFU/ml or greater in LLC-MK2 cells by 6 to 8 days after infection. However, the three 5'-NC-57 C-to-T, NS1-53 Gly-to-Asp, and NS3-250 Glu-to-Val mutations all contributed individually, as well as synergistically in all combinations, to the small-plaque phenotype of PDK-53 virus. These results indicated that all three mutations affected viral replication in LLC-MK2 cells. Limited spread of viruses among LLC-MK2 cells under agarose might be expected to enhance small differences in replication efficiency. In multiple experiments in our laboratory, PDK-53 and VV45R viruses, which produce small plaques, have clearly and consistently shown lower replication rates during the first 4 to 6 days after infection of LLC-MK2 cells. However, we could not clearly identify a correlation between plaque size under agarose and early replication rate for the other viruses in the single growth curve experiment shown in Fig. 1B. All four of the Mahidol candidate DEN virus vaccines are temperature sensitive to some extent in LLC-MK2 cells (70). In five independent experiments in the present study, the slight temperature sensitivity of PDK-53 virus in LLC-MK2 cells, relative to 16681 virus, appeared to result from synergy between the NS1-53-Asp and NS3-250-Val mutations. We previously reported that 130Vc-K virus, a clone-derived homolog of PDK-53 virus (NS3-250-Val variant), was more temperature sensitive than PDK-53 virus itself (32). In the present study, we identified and corrected two amino acid errors in the pD2/IC-130Vc-K clone to derive pD2/IC-VV45R. The degree of temperature sensitivity of VV45R virus was similar to that of PDK-53 virus. Both 130Vc-K (32) and VV45R viruses replicated very inefficiently in C6/36 cells, as did PDK-53 virus.
Replication of PDK-53 virus is greatly reduced in orally fed A. aegypti mosquitoes (31) and in A. albopictus C6/36 cells (32) relative to its parental 16681 virus. The PDK-53 virus is not transmitted by A. aegypti mosquitoes (31). We demonstrated in this study that the low efficiency of PDK-53 viral replication in C6/36 cells resulted from the 5'NC-57 C-to-T and NS1-53 Gly-to-Asp mutations. Each of these mutations engineered into the 16681 virus-specific background (V5 and V1 viruses) resulted in a 250-fold or greater decrease in peak titer relative to that of 16681 virus. The synergistic effect of both mutations in V51 virus resulted in an approximately 200-fold further decrease in peak titer in C6/36 cells. Low infectivity for mosquitoes reduces the potential for secondary transmission of vaccine viruses and may be a useful biological marker of viral attenuation (2, 31, 46, 61). Low infectivity for mosquitoes has been demonstrated for the 17D and French neurotropic yellow fever vaccine viruses (20, 69), attenuated Japanese encephalitis virus (17), and the candidate DEN-2 PR-159/S-1 virus vaccine (2, 46).
Although determining fine distinctions among viruses of low to moderate virulence in an insensitive mouse model is difficult, attenuation of PDK-53 virus for newborn mice was due primarily to synergy between the 5'NC-57-T and NS1-53-Asp loci. Except for P53 virus, all of the viruses containing the NS1-53-Asp locus of PDK-53 virus were attenuated or nearly attenuated for newborn mice. The NS3-250 locus did not appear to contribute significantly to mouse neurovirulence phenotype, except for P53 virus (relative to the nearly attenuated phenotype of P5 virus).
The in vitro phenotypic markers of small plaque size, temperature sensitivity, and reduced level of replication in C6/36 cells all showed positive correlations with the attenuated phenotype of PDK-53 virus in mice. The NS1-53 locus affected the phenotypic traits of plaque size, temperature sensitivity, replication in C6/36 cells, and mouse neurovirulence. The 5'NC-57 locus affected plaque size, replication in C6/36 cells, and mouse neurovirulence. The NS3-250 locus influenced plaque size and temperature sensitivity, and possibly affected mouse virulence to a minor extent. These three genetic loci defined nearly completely the tested markers of attenuation for PDK-53 virus in order of importance NS1-53-Asp > 5'NC-57-T > NS3-250-Val. These loci probably contribute to the attenuated phenotype of PDK-53 virus in humans and nonhuman primates, but we cannot exclude contributions of the prM-29, NS2A-181, and NS4A-75 loci in these hosts. The relative effects of these mutations might be expected to differ in different cell types and hosts. This seems clear for viruses containing the PDK-53 virus-specific 5'NC-57-T and NS1-53-Val loci. These viruses clearly showed different replication efficiencies in LLC-MK2 and C6/36 cells (Fig. 1B and C). Processing of flavivirus proteins, including NS1 (41), is known to differ in mammalian and mosquito cells.
The N terminus of NS3 possesses serine protease activity (16), while the C terminus exhibits RNA helicase and RNA triphosphatase activities (67). The protease activity of NS3, which resides within the amino-terminal 180 amino acids of the protein, appears to depend on an interaction with NS2B (10, 23, 24). The NS1 protein is a glycoprotein that is present on the cell surface of, and secreted from, flavivirus-infected cells. NS1-specific antibodies are present in the serum of flavivirus-infected individuals, and protection of animals immunized with NS1 protein or passively immunized with anti-NS1 monoclonal antibody has been reported (64). Recent data indicate that the NS1 protein functions in the replication of viral RNA, probably before or during minus-strand synthesis (38). A single Arg-to-Ala mutation at NS1-299 of yellow fever virus resulted in a temperature-sensitive defect in RNA accumulation that delayed virus production (52). Elimination of the first or both glycosylation sites of the yellow fever NS1 decreased RNA accumulation and decreased mouse neurovirulence (51). The mutations in the NS1 and NS3 proteins of DEN-2 PDK-53 virus probably impair the functions of these nonstructural proteins during viral replication. These two mutations resulted in a demonstrable decrease in the replication efficiency of the temperature-sensitive viruses in this study, whose NS1 and NS3 proteins apparently were unable to establish or maintain their fully functional, wild-type conformation at the higher temperature.
The 5'NC region of flaviviruses and other positive-stranded viruses has the potential to form secondary structures that are functionally important for viral replication (11, 12, 40, 62). Mutations in the 5'NC region might affect the function of the positive-sense RNA strand as well as the function of its complement in the negative-sense strand during replication. Determinants of attenuation have been demonstrated to lie within the 5'NC region of the Sabin polio vaccine viruses (22, 40) and Venezuelan equine encephalitis virus (33). The 5'NC-57-T mutation in DEN-2 PDK-53 virus occurs within the context of a possible 6-mer stem structure (36) of 16681 viral RNA consisting of genome nt 11 to 16 (5'-CUACGU-3') and 56 to 61 (5'-ACGUAG-3'; underlined nucleotide is U in PDK-53 virus). The replicative importance of short stem structures in RNA molecules may be inferred by identification of pairs of compensatory nucleotide mutations that maintain the stem (44, 54, 56). Two pairs of such stem-preserving, compensatory substitutions occur at nt 11 to 16 (5'-CUGUGU-3') and 55 to 60 (5'-ACACAG-3') in the 5'NC region of DEN-4 814669 virus (71).
Infectious cDNA clones have been used to study the genetic basis of attenuation in a number of viruses, including poliovirus (5, 39, 40, 42, 55, 60, 68), Venezuelan equine encephalitis virus (21, 33), and Japanese encephalitis virus (63). Construction of an infectious clone of DEN-4 virus (35) has led to an understanding of genetic markers of neurovirulence in mice (9) as well as important functions of the 5'NC and 3'NC regions on growth of this virus in cell culture (12, 45). An understanding of the genetic basis of attenuation for the promising DEN-2 PDK-53 vaccine candidate should improve the prospect of constructing safe and reliable attenuated vaccines for DEN virus. The PDK-53 virus replicates well in mammalian cell culture, and its genotype was stable after at least eight passages in PDK cells (Table 3) and another passage in LLC-MK2 cells (32). The phenotypic markers of PDK-53 virus appear to remain stable after sequential passage in human vaccinees and A. aegypti mosquitoes (31). The present study demonstrated that complete reversion of PDK-53 virus to the phenotypic characteristics of the parental 16681 virus required at least two reverse mutations at the 5'NC-57 and NS1-53 loci. Reversion at the NS3-250 locus was also required to reconstitute the large-plaque phenotype, and possibly the mouse virulence phenotype, of 16681 virus.
Chimeric viruses expressing structural genes of DEN-1, DEN-2, DEN-3, or tick-borne encephalitis virus have been engineered successfully in the genetic background of DEN-4 virus (7, 8, 18, 34, 57, 58). Chimeric viruses expressing the prM and E genes of Japanese encephalitis virus within the genetic background of yellow fever 17D virus have also been reported (15, 26, 49). The restriction of the major genetic loci affecting the markers of attenuation to nonstructural regions of PDK-53 virus makes the infectious clone of this virus an attractive candidate for the development of chimeric viruses that express the structural proteins of DEN-1, DEN-3, DEN-4, or other flaviviruses. The attenuated genetic background of PDK-53 virus, which has been characterized in detail in this study, may permit the engineering of chimeric vaccine candidates which express the structural genes of wild-type, heterologous flaviviruses, assuming that the attenuated phenotypic markers of DEN-2 PDK-53 virus are retained in the chimeric viruses. We have recently engineered chimeric DEN-2/DEN-1 viruses that express the structural genes of DEN-1 16007 virus or its candidate DEN-1 PDK-13 virus vaccine derivative within the genetic background of DEN-2 PDK-53 virus to investigate the feasibility of such chimeras (unpublished data).
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ACKNOWLEDGMENTS |
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We are grateful to the Biotechnology Core Facility Branch, Centers for Disease Control and Prevention (Atlanta, Ga.), for supplying the synthetic oligonucleotide primers used in this study. We thank Barry Miller and Mary Crabtree for providing protocols for plaque titration of viruses. We thank Richard Tsuchiya for running sequence reactions on the Prism 377 DNA sequencer.
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FOOTNOTES |
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* Corresponding author. Mailing address: Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522. Phone: (970) 221-6494. Fax: (970) 221-6476. E-mail: rmk1{at}cdc.gov.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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