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Journal of Virology, October 2001, p. 9731-9740, Vol. 75, No. 20
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.20.9731-9740.2001

Chemical Mutagenesis of Dengue Virus Type 4 Yields Mutant Viruses Which Are Temperature Sensitive in Vero Cells or Human Liver Cells and Attenuated in Mice

Joseph E. Blaney Jr.,* Daniel H. Johnson, Cai-Yen Firestone, Christopher T. Hanson, Brian R. Murphy, and Stephen S. Whitehead

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received 1 May 2001/Accepted 24 July 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2ADelta 30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2ADelta 30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Delta 30) in the 3' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2ADelta 30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5' and 3' UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Delta 30 deletion, thereby creating rDEN4Delta 30-4995, a recombinant virus which is ts and more attenuated than rDEN4Delta 30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The mosquito-borne dengue (DEN) viruses (serotypes 1 to 4 [DEN1 to -4]) are members of the Flavivirus genus and contain a single-stranded positive-sense RNA genome of approximately 10,600 nucleotides (nt) (43). The genome organization of DEN viruses is 5'-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3' (where UTR is untranslated region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural) (10, 48). A single viral polypeptide is cotranslationally processed by viral and cellular proteases, generating three structural proteins (C, M, and E) and seven NS proteins. The disease burden associated with DEN virus infection has increased over the past several decades in tropical and semitropical countries. Annually, there are an estimated 50 to 100 million cases of DEN fever (DF) and 500,000 cases of the more severe and potentially lethal DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS) (19).

The site of viral replication in DEN virus-infected humans and the pathogeneses of DF and DHF/DSS are still incompletely understood (26). In humans, DEN virus infects lymphocytes (32, 55), macrophages (21, 52), dendritic cells (34, 63), and hepatocytes (36, 39). The liver is clearly involved in DEN virus infection of humans, as indicated by the occurrence of transient elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the sera of the majority of DEN virus-infected patients and by the presence of hepatomegaly in some patients (29, 31, 42, 60). DEN virus antigen-positive hepatocytes are seen surrounding areas of necrosis in the livers of patients with fatal cases (13, 25), and DEN virus sequences were identified in such cases using reverse transcription-PCR (RT-PCR) (50). Of potential importance to the etiology of severe DEN virus infection, three studies have demonstrated that the mean levels of ALT and AST were significantly increased in the sera of patients with DHF/DSS compared to those in patients with DF (29, 42, 60).

A vaccine for DEN viruses is not presently licensed. Since previous infection with one DEN virus serotype can increase the risk for DHF/DSS during infection with a different serotype (8, 22, 54), it is clear that a DEN virus vaccine will need to protect against each of the four DEN virus serotypes, namely, DEN1, DEN2, DEN3, and DEN4. Several strategies are being actively pursued in the development of a live attenuated tetravalent DEN virus vaccine (2, 4, 20, 24, 30). Recently, Durbin et al. demonstrated that a live attenuated DEN4 vaccine candidate, 2ADelta 30, was attenuated and immunogenic in a group of 20 human volunteers (15). This recombinant DEN4 virus contains a 30-nt deletion (Delta 30) in the 3' UTR that removes nt 10,478 to 10,507 and was restricted in replication in rhesus monkeys. Levels of viremia in humans were low or undetectable, and virus recovered from the vaccinees retained the Delta 30 mutation. An asymptomatic rash was reported in 50% of patients. The only laboratory abnormality observed was an asymptomatic, transient rise in the ALT levels in the sera of 5 of 20 vaccinees. Elevated serum ALT and AST levels have also been observed in clinical trials of other DEN virus vaccine candidates (17, 18, 30, 59). All 2ADelta 30 vaccinees developed a neutralizing antibody response to DEN4 virus (mean titer, 1:580) in their sera. Importantly, 2ADelta 30 was not transmitted to mosquitoes fed on vaccinees and has restricted growth properties in mosquitoes (56). The presence of rash and elevated ALT levels suggests that the 2ADelta 30 vaccine candidate is slightly underattenuated in humans. Because of the desirable properties conferred by the Delta 30 mutation, chimeric vaccine candidates which contain the structural genes of DEN1, -2, and -3 and the attenuated DEN4 vector bearing the genetically stable Delta 30 mutation are being constructed.

Although the initial findings suggest the utility of the 2ADelta 30 vaccine candidate, many previous attempts to develop live attenuated DEN virus vaccines have yielded vaccine candidates that were either over- or underattenuated in humans (5, 17, 27, 30, 40). Therefore, we have begun to develop a menu of point mutations which confer temperature-sensitive (ts) and attenuation (att) phenotypes upon DEN4. It is anticipated that these mutations should be able to attenuate DEN4 viruses to various degrees and therefore will be useful in fine-tuning the level of attenuation of vaccine candidates such as 2ADelta 30. Addition of such mutations to 2ADelta 30 or to other novel DEN4 vaccine candidates should result in the generation of a vaccine candidate that exhibits a satisfactory balance between attenuation and immunogenicity for humans.

In the present study, chemical mutagenesis of DEN4 has been used to identify point mutations which confer a ts phenotype in Vero cells, since such ts viruses are often attenuated in humans. Additionally, because of the reported involvement of the liver in natural DEN virus infection and the elevated ALT levels in a subset of 2ADelta 30 vaccinees, mutagenized DEN4 viruses were also evaluated for temperature sensitivity in HuH-7 liver cells, which were derived from a human hepatoma (45). Here we describe the identification of 20 DEN4 ts mutant viruses, each of which replicates efficiently in Vero cells (the proposed substrate for vaccine manufacture) and each of which is attenuated in mice. Finally, the feasibility of modifying the att phenotype of the 2ADelta 30 vaccine candidate by introduction of a point mutation in NS3 is demonstrated.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cells and viruses. World Health Organization Vero cells (African green monkey kidney cells) were maintained in modified essential medium (MEM; Life Technologies, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS; Summit Biotechnologies, Fort Collins, Colo.), 2 mM L-glutamine (Life Technologies), and 0.05 mg of gentamicin (Life Technologies) per ml. HuH-7 cells (human hepatoma cells) (45) were maintained in Dulbecco's MEM-F-12 (Life Technologies) supplemented with 10% FBS, 1 mM L-glutamine, and 0.05 mg of gentamicin per ml. C6/36 cells (Aedes albopictus mosquito cells) were maintained in complete MEM as described above and supplemented with 2 mM nonessential amino acids (Life Technologies).

The wild-type (wt) DEN4 2A virus was derived from a cDNA clone of DEN4 strain 814669 (Dominica, 1981) (37). A DEN4 vaccine candidate, 2ADelta 30, contains a 30-nt deletion in the 3' UTR which removes nt 10,478 to 10,507 (41). As previously described, cDNA clones p4 and p4Delta 30 were derived from the 2A cDNA clone by the introduction or removal of translationally silent restriction enzyme sites to facilitate subsequent construction of recombinant DEN4 (rDEN4) cDNA clones (15). The cDNA clones p4 and p4Delta 30 were used previously to generate the recombinant viruses rDEN4 and rDEN4Delta 30, respectively. The GenBank accession number for rDEN4 is AF326825, and that for rDEN4Delta 30 is AF326827.

Chemical mutagenesis of DEN4. Confluent monolayers of Vero cells were infected with wt DEN4 2A at a multiplicity of infection (MOI) of 0.01 and incubated for 2 h at 32°C. Infected cells were then overlaid with MEM supplemented with 2% FBS and 5-fluorouracil (5-FU; Sigma, St. Louis, Mo.) at concentrations ranging from 10 mM to 10 nM. After incubation at 32°C for 5 days, cell culture medium was harvested, clarified by centrifugation, and frozen at -70°C. Clarified supernatants were then assayed for virus titer by plaque titration in Vero cells. Serial 10-fold dilutions of the clarified supernatant were prepared in Opti-MEM I (Life Technologies) and inoculated onto confluent Vero cell monolayers in 24-well plates. After incubation at 35°C for 2 h, monolayers were overlaid with 0.8% methylcellulose (EM Science, Gibbstown, N. J.) in Opti-MEM I supplemented with 2% FBS, gentamicin, and L-glutamine. Following incubation at 35°C for 5 days, plaques were observed by immunoperoxidase staining. Vero cell monolayers were fixed in 80% methanol for 30 min and washed for 10 min with antibody buffer which consists of 3.5% (wt/vol) nonfat dry milk (Nestle, Solon, Ohio) in phosphate-buffered saline. Cells were then incubated for 1 h at 37°C with an anti-DEN4 rabbit polyclonal antibody preparation (50% plaque reduction neutralization titer of >1:2,000) diluted 1:1,000 in antibody buffer. After one wash with antibody buffer, cells were incubated for 1 h with peroxidase-labeled goat-anti-rabbit immunoglobulin G (KPL, Gaithersburg, Md.) diluted 1:500 in antibody buffer. Monolayers were washed with phosphate-buffered saline, allowed to dry briefly, and overlaid with a peroxidase substrate (KPL), and plaques were counted.

Virus yields in cultures treated with 1 mM 5-FU were reduced 100-fold compared to those of untreated cultures, and the virus suspension from the 1 mM 5-FU-treated culture was terminally diluted to derive clones for phenotypic characterization. Briefly, 96-well plates of Vero cells were inoculated with the 5-FU-treated virus at an MOI that yielded 10 or fewer virus-positive wells per plate. After a 5-day incubation at 35°C, tissue culture media from the 96-well plates were removed and temporarily stored at 4°C and the virus-positive cell monolayers were identified by immunoperoxidase staining. Virus from each positive well was transferred to confluent Vero cell monolayers in 12-well plates for amplification. Cell culture medium was harvested from individual wells 5 or 6 days later, clarified by centrifugation, aliquoted to 96-deep-well polypropylene plates (Beckman, Fullerton, Calif.), and frozen at -70°C. A total of 1,248 virus clones were prepared from the 1 mM 5-FU-treated cultures. Two virus clones, 2A-1 and 2A-13, without a ts phenotype, were generated in the same manner from non-5-FU-treated control cultures passaged in parallel and served as control viruses with a wt phenotype.

Screening of clones for ts and att phenotypes. The 1,248 virus clones were screened for temperature sensitivity by assessing virus replication at 35 and 39°C in Vero and HuH-7 cells. Cell monolayers in 96-well plates were inoculated with serial 10-fold dilutions of virus in L-15 medium (Quality Biologicals, Gaithersburg, Md.) supplemented with 2% FBS, L-glutamine, and gentamicin. Cells were incubated at the temperatures indicated above for 5 days in temperature-controlled water baths, and the presence of virus was determined by immunoperoxidase staining as described above. Virus clones which demonstrated a 100-fold or greater reduction in titer at 39°C were terminally diluted an additional two times and amplified in Vero cells. The efficiencies of plaque formation (EOP) at the permissive and restrictive temperatures of each triply biologically cloned virus suspension were determined as follows. Plaque titration in Vero and HuH-7 cells was performed as described above except that virus-infected monolayers were overlaid with 0.8% methylcellulose in L-15 medium supplemented with 5% FBS, gentamicin, and L-glutamine. After incubation of replicate plates for 5 days at 35, 37, 38, or 39°C in temperature-controlled water baths, plaques were visualized by immunoperoxidase staining and counted.

The replication of DEN4 5-FU ts mutant viruses was evaluated in Swiss Webster suckling mice (Taconic Farms, Germantown, N.Y.). All animal experiments were carried out in accordance with the regulations and guidelines of the National Institutes of Health. Groups of six 1-week-old mice were inoculated intracerebrally with 104 PFU of virus diluted in 30 µl of Opti-MEM 1. Five days later, mice were sacrificed and brains were removed and individually homogenized in a 10% suspension of phosphate-buffered Hanks' balanced salt solution containing 7.5% sucrose, 5 mM sodium glutamate, 0.05 mg of ciprofloxacin per ml, 0.06 mg of clindamycin per ml, and 0.0025 mg of amphotericin B per ml. Clarified supernatants were frozen at -70°C, and subsequently the virus titer was determined by titration in Vero cells. Plaques were stained by the immunoperoxidase method described above.

Sequence analysis of viral genomes. The nucleotide sequence of the 5-FU-mutagenized DEN4 viruses was determined as described previously (15). Briefly, genomic viral RNAs were isolated from virus clones with a QIAamp viral RNA mini kit (Qiagen, Valencia, Calif.), and RT was performed using the SuperScript First Strand Synthesis System for RT-PCR (Life Technologies) and random hexamer primers or gene-specific primers. Advantage cDNA polymerase (Clontech, Palo Alto, Calif.) was used to generate overlapping PCR fragments of approximately 2,000 nt, which were purified by the HighPure PCR Product Purification System (Roche Diagnostics, Indianapolis, Ind.). DEN4 virus-specific primers were used in BigDye terminator cycle sequencing reactions (Applied Biosystems, Foster City, Calif.), and reactions were analyzed on a model 3100 genetic analyzer (Applied Biosystems). Primers were designed to sequence both strands of the PCR product, from which consensus sequences were assembled.

The nucleotide sequence of the 5' and 3' regions of the viral genome were determined as described above after circularization of the RNA genome. The 5' cap nucleoside of the viral RNA was excised using tobacco acid pyrophosphatase (Epicentre Technologies, Madison, Wis.), and the genome was circularized with RNA ligase (Epicentre Technologies). An RT-PCR fragment which overlapped the ligation junction (5' and 3' ends) was generated and sequenced as described above.

Generation of recombinant DEN4 viruses. The mutation at nt position 4995 in NS3 was introduced into the p4 cDNA construct by site-directed mutagenesis as follows. The StuI-BstBI (nt 3619 to 5072) fragment of p4 was subcloned into a modified pUC119 vector. The Uright-arrowC mutation at nt position 4995 was engineered by site-directed mutagenesis into the p4 fragment and cloned back into the p4 cDNA construct, and the presence of the mutation was confirmed by sequence analysis. The Delta 30 mutation was introduced into the 3' UTR of the p4-4995 cDNA clone by replacing the MluI-KpnI fragment with that derived from the p4Delta 30 cDNA clone, and the presence of the deletion was confirmed by sequence analysis. Full-length RNA transcripts were prepared from the above-described cDNA clones by in vitro transcription reactions with SP6 RNA polymerase (New England Biolabs, Beverly, Mass.) and purified with an RNeasy mini kit (Qiagen) as previously described (15).

For transfection of C6/36 cells, RNA transcripts were combined with DOTAP liposomal transfection reagent (Roche) in HEPES-buffered saline (pH 7.6) and added to cell monolayers in six-well plates. After incubation at 32°C for 12 to 18 h, cell culture media were removed and replaced with MEM supplemented with 5% FBS, L-glutamine, gentamicin, and nonessential amino acids. Cell monolayers were incubated for an additional 5 to 7 days, and cell culture media were harvested, clarified by centrifugation, and assayed for the presence of virus by plaque titration in Vero cells. Recovered viruses were terminally diluted twice as described above, and virus suspensions for further analysis were prepared in Vero cells.

Nucleotide sequence accession number. The nucleotide sequence of parental virus DEN4 2A has been assigned GenBank accession number AF375822.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In vitro and in vivo replication of wt DEN4 and DEN4Delta 30. The levels of replication of both wt DEN4 2A virus and the vaccine candidate, 2ADelta 30, were evaluated in Vero (monkey kidney) and HuH-7 (human hepatoma) cells, the latter of which has recently been found to efficiently support the replication of DEN2 virus (36). The patterns of replication of wt DEN4 2A virus and 2ADelta 30 were similar in the two cell lines. Viral titers from cultures infected with 2ADelta 30 at an MOI of 0.01 were slightly reduced compared to that of wt DEN4 2A virus at 72 h, but at later time points their levels of replication were equivalent (data not shown). The efficient replication of both DEN4 viruses in each cell line indicated that these continuous lines of cells would be useful for characterization of the temperature sensitivity of the 1,248 potential mutant viruses.

The level of replication of DEN4 virus administered intracerebrally to Swiss Webster mice was first determined to assess whether mice could be used to efficiently evaluate and quantitate the att phenotype of a large set of mutant viruses. Since the susceptibility of mice to DEN infection is age dependent (11, 12), mice aged 7 to 21 days were infected with 2A-13, rDEN4, or rDEN4Delta 30 virus and, after 5 days, the brain of each mouse was removed. The level of viral replication was quantitated by plaque assay (Table 1). The results indicated that the two wt DEN4 viruses and the rDEN4Delta 30 virus vaccine candidate replicated to high titers (>106.0 PFU/g) in 7-day-old mice. Virus replication in the brains of 14- or 21-day-old mice was significantly reduced compared to that in 7-day-old mice. These results demonstrated the feasibility of using 7-day-old mice to screen a large set of mutant viruses. In addition, the high levels of replication of the wt and vaccine candidate viruses permit quantitation of the magnitude of the restriction of replication specified by an attenuating mutation over a 10,000-fold range.

                              
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  		TABLE 1.   Susceptibility of mice to intracerebral DEN4 virus infection is age dependenta

Generation and in vitro characterization of DEN4 5-FU mutant viruses. A panel of 1,248 DEN4 virus clones was generated from a 5-FU-mutagenized suspension of wt DEN4 2A virus as described in Materials and Methods (Fig. 1). Each clone was tested in Vero and HuH-7 cells for temperature sensitivity at 39°C, and putative ts mutant viruses were subjected to two additional rounds of biological cloning by terminal dilution. The ts phenotype of each triply cloned virus population was examined in more detail by determining its EOP at the permissive temperature (35°C) and at various restrictive temperatures (Table 2). wt virus (clone 2A-13) lacking a ts and att phenotype was passaged in a manner identical to that used for the ts mutant viruses and served as the control virus to which each of the ts mutant viruses was directly compared for both the ts and att phenotypes.


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  		FIG. 1.   Generation of ts DEN4 viruses by 5-FU chemical mutagenesis. DEN4 virus 2A was derived from a cDNA clone of DEN4 virus strain 814669 (Dominica, 1981). ts phenotypes were determined by EOP in the indicated cells as described in Materials and Methods.

                              
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  		TABLE 2.   ts and mouse brain att phenotypes of 5-FU mutant DEN4 viruses

Thirteen 5-FU mutant viruses which have a ts phenotype in both Vero and HuH-7 cells were identified, and seven mutants were ts only in HuH-7 cells (Table 2). Mutant viruses which were ts in Vero cells but not in HuH-7 cells were not identified. Temperature sensitivity was defined as a >= 2.5- or >= 3.5-log10 PFU/ml reduction in virus titer in Vero or HuH-7 cells, respectively, at the temperatures indicated in Table 2 compared to the titer achieved at the permissive temperature of 35°C. wt DEN4 2A virus was found to have approximately a 0.5- or 1.5-log10 PFU/ml reduction in virus titer in Vero or HuH-7 cells at 39°C, respectively. The Delta 30 deletion did not confer a ts phenotype in Vero or HuH-7 cells and produced only a slight reduction in virus titer (2.2 log10 PFU/ml) at 39°C in HuH-7 cells, which was less than 10-fold greater than the reduction of wt DEN4 2A virus (1.4 log10 PFU/ml) at 39°C. Several 5-FU mutant viruses had a >10,000-fold reduction in virus titer at 39°C in both Vero and HuH-7 cells. A complete shutoff in viral replication at 39°C in HuH-7 cells was observed for five virus clones which were not ts in Vero cells (clones 571, 605, 631, 967, and 992). Mutations that selectively restrict replication in HuH-7 liver cells may be particularly useful in controlling the replication of DEN virus vaccine candidates in the livers of vaccinees.

Replication of DEN4 5-FU mutant viruses in suckling mice. The level of replication of each of the 20 ts mutant viruses in mouse brain was determined as a preliminary determinant of in vivo growth properties (Table 2). The titers obtained were compared to those of the two wt viruses, the 2A-13 and rDEN4 viruses, and to that of the 2ADelta 30 mutant, which conferred only a 0.5-log10-PFU/g reduction in mean virus titer compared to the titers of the wt controls. The observed reduction in the level of rDEN4Delta 30 virus replication was consistent among 11 separate experiments. Interestingly, the rDEN4Delta 30 virus, which was attenuated in both rhesus monkeys and humans (15), was only slightly restricted in replication in mouse brain. The panel of 20 ts mutant viruses was similarly evaluated, and their levels of virus replication were compared to that of the wt 2A-13 virus passaged in parallel (Table 2). Various levels of restriction of replication ranging from a 10-fold (clone 473) to a >6,000-fold (clone 686) reduction were observed among the mutants. Mutant viruses with ts phenotypes in both Vero and HuH-7 cells, as well as in HuH-7 cells alone, were found to have significant att phenotypes. Five of 13 mutant viruses with ts phenotypes in both Vero and HuH-7 cells and 5 of 7 mutant viruses with ts phenotypes in HuH-7 cells alone had a >100-fold reduction in virus replication in the mouse brain. There appeared to be no direct correlation between the magnitude of the reduction in replication at the restrictive temperature in vitro and the level of attenuation in vivo. The similar levels of temperature sensitivity and replication of the rDEN4 wt virus and clone 2A-13 in mouse brain indicated that the observed differences in level of replication between the ts mutants and clone 2A-13 were not simply a function of passage in Vero cells but reflect the sequence differences between these viruses.

Sequence analysis of DEN4 5-FU mutant viruses. To determine the genetic basis of the observed ts and att phenotypes, the complete nucleotide sequences of each ts mutant virus and of clone 2A-13 were determined and are summarized in Table 3 (ts in Vero and HuH-7 cells) and Table 4 (ts only in HuH-7 cells).

                              
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  		TABLE 3.   Nucleotide and amino acid differences of the 5-FU mutant viruses that are ts in both Vero and HuH-7 cells


                              
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  		TABLE 4.   Nucleotide and amino acid differences of the 5-FU mutant viruses which are ts only in HuH-7 cells

The only type of mutations identified in the 20 mutant viruses were nucleotide substitutions (no deletions or insertions occurred), and these were present in each of the coding regions except C and NS4A as well as in the 5' and 3' UTRs. Three mutant viruses (clones 239, 489, and 773) contained only a single missense point mutation in NS3 at nt position 4995, resulting in a Ser-to-Pro amino acid change at residue 1632. For mutant virus 773, this was the sole mutation present (Table 3). The translationally silent mutations identified in coding regions are not considered to be significant. The 17 additional mutant viruses had multiple mutations (two to five) in a coding region or in UTR which could potentially confer the observed ts or att phenotype. Five of the 17 mutant viruses with multiple mutations (clones 473, 718, 759, 816, and 938) also encoded the point mutation at nt position 4995. The 4995 mutation was found only in mutant viruses with a ts phenotype in both Vero and HuH-7 cells. Sequence analysis indicated that 10 mutant viruses which were ts in Vero and HuH-7 cells and 3 mutants which were ts only in HuH-7 cells contained mutations only in their NS genes and/or their 5' or 3' UTR. These mutations are especially suitable for inclusion in chimeric DEN virus vaccine candidates whose structural genes are derived from a DEN1, DEN2, or DEN3 virus serotype and whose remaining coding and noncoding regions come from an attenuated DEN4 vector.

The presence of a point mutation at nt position 4995 in eight separate mutant viruses was described above. Five additional point mutations were also represented in multiple viruses, including nucleotide changes at position 1455 in E; at positions 7162, 7163, and 7564 in NS4B; and at position 10275 in the 3' UTR (Table 5). The significance of the occurrence of these "sister" mutations in multiple viruses remains undefined. Interestingly, the wt virus passaged in parallel 2A-13, contained only a single mutation at nt position 7163 (Aright-arrowC; Leuright-arrowPhe) in NS4B, which was also observed in three 5-FU mutant viruses.

                              
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  		TABLE 5.   Mutations which are represented in multiple 5-FU mutant DEN4 viruses

Introduction of a ts mutation into rDEN4 and rDEN4Delta 30 viruses. The presence of a single nucleotide substitution (Uright-arrowC mutation at nt position 4995 in NS3) in three separate mutant viruses (clones 239, 489, and 773) strongly suggested that this mutation specified the ts and att phenotypes in each of the three viruses. This mutation was cloned into the p4 and p4Delta 30 cDNA constructs, and recombinant viruses were recovered and designated rDEN4-4995 and rDEN4Delta 30-4995, respectively. These recombinant viruses were tested for ts and att phenotypes as described above (Table 6). As expected, introduction of a mutation at nt 4995 into the rDEN4 wt virus resulted in a significant ts phenotype at 39°C in both Vero and HuH-7 cells. rDEN4-4995 virus grew to nearly wild-type levels at the permissive temperature, 35°C, in both cell types but demonstrated a >10,000-fold reduction at 39°C (shutoff temperature) in both Vero and HuH-7 cells. The addition of the mutation at nt 4995 to rDEN4Delta 30 virus yielded a recombinant virus, rDEN4Delta 30-4995, that exhibits the same level of temperature sensitivity as rDEN4-4995 virus (Table 6).

                              
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  		TABLE 6.   Addition of the ts mutation at nt 4995 to rDEN4Delta 30 virus confers a ts phenotype and further attenuates its replication in suckling mouse brain

The rDEN4 viruses encoding the mutation at nt 4995 were tested for replication in the brains of suckling mice (Table 6). The mutation at nt 4995 conferred an att phenotype upon both rDEN4 and rDEN4Delta 30 viruses. There was an approximately 1,000-fold reduction in virus replication compared to that of wt virus. The combination of the point mutation at nt 4995 and the Delta 30 deletion did not appear to result in an additive reduction of virus replication. These results confirm that the point mutation at nt 4995 indeed specifies a ts and an att phenotype. Importantly, the feasibility of modifying in vitro and in vivo phenotypes of the rDEN4Delta 30 virus vaccine candidate by introduction of additional mutations was also demonstrated.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We are currently preparing a tetravalent, live attenuated DEN virus vaccine using rDEN4Delta 30 virus as the DEN4 component and three antigenic chimeric viruses expressing the structural proteins (C, prM, and E) of DEN1, DEN2, and DEN3 from the attenuated rDEN4Delta 30 virus vector (15). DEN4 virus containing the Delta 30 mutation in the 3' UTR manifests restricted replication in humans while retaining immunogenicity (15). Since 2ADelta 30 retains a low level of residual virulence for humans despite this restricted replication, the present study was initiated to generate additional attenuating mutations that could further attenuate rDEN4Delta 30 virus and be incorporated into any of the three antigenic chimeric viruses as needed. It has been demonstrated that ts mutants of many viruses (49, 53, 62), including DEN virus (5, 16), exhibit restricted replication in vivo. We have generated a panel of 20 ts DEN4 mutant viruses, determined their genomic sequences, and assessed their in vivo attenuation phenotypes. The 20 ts DEN4 mutant viruses were generated by growth in the presence of 5-FU and were first selected for viability in Vero cells to ensure that the mutant viruses grow efficiently in these cells, since Vero cells are the substrate planned for use in the manufacture of these vaccines.

Two classes of mutant viruses were obtained: those ts in both Vero and HuH-7 cells (n = 13) and those ts only in HuH-7 cells (n = 7). The viruses exhibited a range in their level of temperature sensitivity manifested by a 100- to a 1,000,000-fold reduction in replication at the restrictive temperature of 39°C. Since the 2ADelta 30 vaccine candidate retains a low level of virulence for the liver and since other findings support the ability of DEN viruses to infect hepatocytes (36, 38) and cause liver pathology (13, 25), we sought to develop mutations that would selectively restrict replication of DEN4 virus in liver cells. Toward this end, we identified seven mutant viruses which have an HuH-7 cell-specific ts phenotype. The mutations present in these viruses are the first reported for DEN viruses that confer restricted replication in liver cells and may be helpful in limiting virus replication and pathology in the livers of vaccine recipients. The contribution of single mutations identified in the HuH-7 cell-specific ts viruses to the observed phenotypes is presently being assessed by introduction of the individual mutations into recombinant DEN4 viruses.

Recent evidence has indicated that the magnitude of viremia in DEN virus-infected patients positively correlates with disease severity; i.e., the higher the titer of viremia, the more severe the disease (44, 58). This indicates that mutations that significantly restrict replication of vaccine candidates in vivo are the foundation of a safe and attenuated vaccine. Evaluation of DEN virus vaccine candidates for in vivo attenuation is complicated by the lack of a suitable animal model that accurately mimics the disease caused by DEN viruses in humans. In the absence of such a model, the replication of the 5-FU mutant viruses in the brains of 7-day-old Swiss Webster mice was evaluated as a means to identify a preliminary in vivo att phenotype, since this animal model is well suited for the evaluation of a large set of mutant viruses. Each of the 20 ts mutant viruses exhibited an att phenotype manifesting a 10- to 6,000-fold reduction in replication in the brains of mice compared to the level of replication in DEN4 wt virus (Table 2). This finding suggests that there is a correlation between the presence of the ts phenotype in vitro and attenuation of the mutant virus in vivo, confirming the utility of selecting viruses with this marker as vaccine candidates. However, there was no correlation between the level of temperature sensitivity in vitro and the level of restriction in vivo.

In past studies, Sabin observed a dissociation between mouse neurovirulence and attenuation in humans by generating an effective live attenuated virus vaccine against DEN virus by passage of virus in mouse brain. This research actually resulted in a highly mouse-neurotropic DEN virus which, paradoxically, was significantly attenuated in humans (51). Despite this, attenuation for the suckling mouse brain has been reported for other live attenuated DEN virus vaccine candidates, including the DEN2 virus PDK-53 vaccine strain, which is nonlethal in mice, and the DEN2 virus PR-159/S-1 vaccine strain, which was significantly attenuated compared to its parental wild-type virus (5, 9, 16, 27). The 2ADelta 30 vaccine virus was found to be only mildly restricted in the brains of suckling mice, which further illustrates the complicated interpretation of growth properties in this animal model. Despite the imperfect correlation between reduced replication of dengue viruses in mouse brain and attenuation in humans, this animal model has permitted a preliminary investigation of the in vivo growth properties of a large set of mutant viruses, and several current dengue virus vaccine strains are known to be attenuated in the mouse brain. In the future, selected 5-FU mutant viruses or recombinant viruses bearing one or more of these mutations will be tested for replication in rhesus monkeys, which has been reported to be predictive of attenuation for humans (27). Recently, murine models of DEN virus infection have been developed using SCID mice transplanted with human macrophages (35) or liver cell lines (1), but these mice have not as yet been used to assess att phenotypes of candidate vaccine viruses. We are currently evaluating the replication of selected 5-FU mutant viruses in SCID mice transplanted with HuH-7 cells.

The chemical mutagenesis and sequence analysis of DEN4 viruses described here has resulted in the identification of a large number of point mutations resulting in amino acid substitutions in all genes except C and NS4A as well as point mutations in the 5' and 3' UTRs (Tables 3 and 4). This approach of whole-genome mutagenesis has the benefit of identifying mutations dispersed throughout the entire genome. Ten 5-FU mutant viruses which were ts in Vero and HuH-7 cells and three viruses which were selectively ts in HuH-7 cells contained only mutations outside of the genes encoding the structural proteins, i.e., in the 5' and 3' UTRs or NS genes. These mutations along with the Delta 30 deletion in the 3' UTR are particularly suited for inclusion in an antigenic chimeric vaccine virus which consists of an attenuated DEN4 genetic background bearing the wild-type structural genes (C, prM, and E) of another DEN virus serotype. Use of this strategy has several advantages. Each antigenic chimeric virus possesses structural proteins from a wt virus along with attenuating mutations in its UTRs or NS genes and should maintain its infectivity for humans (24), which is mediated largely by the E protein. Therefore, each vaccine component should prove to be sufficiently immunogenic, with wt E protein efficiently inducing neutralizing antibodies against each individual DEN virus. In addition, the replicative machinery of the tetravalent vaccine strains would share the same attenuating mutations in the NS genes or in the UTRs and should attenuate each vaccine component to similar degrees and thereby minimize interference or complementation among the four vaccine viruses.

Sequence analysis of DEN viruses (6, 33, 47) and yellow fever viruses (14, 23) previously generated by serial passage in tissue culture has identified mutations distributed throughout much of the genome, a pattern similar to that observed in the present study. Recent analysis of the DEN2 virus PDK-53 vaccine strain has identified important mutations involved in attenuation which are located in nonstructural regions, including the 5' UTR, NS1, and NS3 (9). This DEN2 virus vaccine strain has been used to generate a chimeric virus with C, prM, and E genes from DEN1 (24). In separate studies, the sequence of the DEN1 virus vaccine strain 45AZ5 PDK-27 was determined and compared to those of parental viruses, but the mutations responsible for attenuation have not yet been identified (47).

Several amino acid substitutions were identified in more than one ts 5-FU mutant virus (Table 5). Lee et al. have previously reported finding repeated (sister) mutations in the E and prM proteins of separate DEN3 virus clones after serial passage in Vero cells (33). A mutation (Kright-arrowN) identified in the E protein at amino acid position 202 in a single DEN3 virus passage series was also found in our 5-FU mutant virus 1012 (Kright-arrowE). However, Lee et al. sequenced only the structural genes of the Vero cell-passaged viruses. The determination of the complete genomic sequence of 21 DEN4 viruses in the present study provided an opportunity to identify mutations in the NS genes and the UTRs resulting from passage in Vero cells. Identical mutations observed among multiple sister 5-FU mutant viruses, each of which had several passages in Vero cells, may represent adaptive changes that confer an increased efficiency of DEN4 replication in Vero cells. Such mutations would be potentially beneficial for inclusion in a live attenuated DEN virus vaccine by increasing the yield of vaccine virus during manufacture. Interestingly, three distinct amino acid substitutions were found in the NS4B genes of several 5-FU mutant viruses. The exact function of this gene is unknown, but previous studies of live attenuated yellow fever vaccines (28, 61) and Japanese encephalitis vaccines (46) have identified mutations in NS4B associated with att phenotypes.

The mutation at nt position 4995 of NS3 (S1632P) was present as the only significant mutation identified in three 5-FU mutant viruses (clones 239, 489, and 773). This mutation was introduced into a recombinant DEN4 virus and found to confer a ts and att phenotype (Table 6). These observations clearly identify the mutation at nt 4995 as an attenuating mutation and suggest its utility for further attenuation of rDEN4Delta 30 virus. Analysis of a sequence alignment (10) of the four DEN viruses indicated that the Ser at amino acid position 1632 is conserved in DEN1 and DEN2 but that DEN3 contains an Asn at this position, indicating that the mutation may also be useful in modifying the phenotypes of the other DEN virus serotypes. The NS3 protein is 618 amino acids in length and contains both serine protease and helicase activities (3, 7, 57). The nt 4995 mutation results in a change at amino acid position 158 in NS3, which is located in the N-terminal region containing the protease domain. Amino-acid position 158 is located 2 amino acid residues away from an NS3 conserved region designated homology box 4. This domain has been identified in members of the flavivirus family and is believed to be a critical determinant of NS3 protease substrate specificity (3, 7). However, the exact mechanism which results in the phenotype associated with the nt 4995 mutation has not yet been identified. The nt 4995 mutation was identified in eight 5-FU mutant viruses, suggesting that the stability of this mutation during replication in Vero cells would be an advantage during vaccine manufacture.

We are currently determining the contributions of individual 5-FU mutations to the observed phenotypes by introduction of the mutations into recombinant DEN4 viruses as was demonstrated for the nt 4995 mutation in this paper. In addition, combination of individual mutations with each other or with the Delta 30 mutation is being used to further modify the att phenotype of DEN4 virus candidate vaccines. The introduction of the nt 4995 mutation into rDEN4Delta 30 virus rendered the rDEN4Delta 30-4995 double mutant virus ts and 1,000-fold more attenuated for mouse brain replication than rDEN4Delta 30 virus. This observation has demonstrated that it is feasible to modify both in vitro and in vivo phenotypes of this vaccine candidate. Once the mutations responsible for the HuH-7 cell-specific ts phenotype are identified as described above and introduced into the rDEN4Delta 30 virus vaccine candidate, we will be able to determine if these mutations attenuate the rDEN4Delta 30 virus vaccine for the livers of humans. A menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant DEN vaccine viruses and in increasing our understanding of the pathogenesis of DEN virus is being assembled.


    FOOTNOTES

* Corresponding author. Mailing address: LID, NIAID, NIH, Bldg. 7, Room 100, 7 Center Dr. MSC 0720, Bethesda, MD 20892. Phone: (301) 402-7751. Fax: (301) 496-8312. E-mail: jblaney{at}niaid.nih.gov.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, October 2001, p. 9731-9740, Vol. 75, No. 20
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.20.9731-9740.2001


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