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Short Communication |
1 Department of Anatomy and Center for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
2 Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, US Department of Health and Human Services, Public Health Service, Fort Collins, CO 80522, USA
Correspondence
Vijittra Leardkamolkarn
scvlk{at}mahidol.ac.th
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ABSTRACT |
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MAIN TEXT |
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). An effective vaccine against dengue is not yet available.
The DEN virus genome is a single-stranded, positive-sense RNA that is approximately 11 kb in length. It contains a type I cap at the 5' end, but lacks a 3'-end poly(A) tail. The genome organization is 5' non-coding region (5'NCR)–capsid protein–premembrane (prM)/membrane protein–envelope (E) protein–non-structural protein 1 (NS1)–NS2A–NS2B–NS3–NS4A–NS4B–NS5–3'NCR (Lindenbach & Rice, 2003). The 5'NCRs of flaviviruses and several other positive-strand RNA viruses are predicted to form functional secondary RNA structures (Brinton & Dispoto, 1988). These structures can interact with cellular or viral proteins to regulate viral RNA translation (Ali & Siddiqui, 1995), RNA transcription and packaging (Guesdon et al., 2001; Kuhn et al., 2002). 5'NCR sequences may also interact with downstream sequences, particularly 3'NCR sequences, in the viral genome during virus replication (Alvarez et al., 2005; You et al., 2001). The functional significance of the 5'NCR of DEN-4 virus has been demonstrated by the attenuating nature of mutations in this region, including changes that disrupt predicted base pairing (Cahour et al., 1995).
A C57U mutation in the 96 nt long 5'NCR of the candidate DEN-2 PDK-53 vaccine virus has been associated with viral attenuation (Butrapet et al., 2000; Kinney et al., 1997). The phenotypic markers of attenuation of the PDK-53 virus, which was derived from wild-type DEN-2 16681 virus, are encoded by the three mutations NS1-Gly1668153AspPDK-53, 5'NCR-C57U and NS3-Glu250Val, in order of dominance of effect on virus replication in vitro and attenuation of neurovirulence for newborn mice (Butrapet et al., 2000). The 5'NCR-C57U mutation contributes to the PDK-53 viral phenotypes of small plaque size, decreased replication in C6/36 cells and attenuation of neurovirulence for mice (Butrapet et al., 2000). The 5'NCR-57 locus lies within a predicted 6 bp stem structure consisting of nt 11–16 and 56–61 (Butrapet et al., 2000). This stem structure is one of several stems in a larger predicted 5' structure, designated SLA and consisting of nt 1–70, which has recently been shown to be recognized by the viral RNA polymerase (NS5) and to participate in cyclization of the genome through interaction with the 3'-end sequence, thereby promoting minus-strand RNA synthesis in D2/IC-30P-A clone-derived DEN-2 16681 virus (Filomatori et al., 2006). Presumably, the C57U mutation impairs this function in PDK-53 virus.
Serial in vitro passage of chimeric DEN viruses expressing the prM/E gene region of DEN-1, -2, -3 or -4 virus in the genetic background of PDK-53 virus has demonstrated high genetic stability of the NS1-53-Asp and NS3-250-Val loci. However, these chimeric viruses, as well as the PDK-53 virus itself, showed varying propensities for 5'NCR-U57C reverse mutation during serial passage. The extent of reversion in these viruses was recently examined quantitatively (Butrapet et al., 2006). The present study was undertaken to determine whether other mutations near nt 57 in the 5'NCR could attenuate wild-type DEN-2 16681 virus. Such mutations could prove useful in designing live-attenuated DEN vaccine viruses. We engineered 11 different mutations, each containing one or more point substitution and/or deletion (Table 1), into the 5'NCR of the DEN-2 16681 virus-specific infectious cDNA clone pD2/IC-30P-A by using standard in vitro mutagenesis procedures. Deletion or multiple point-substitution mutations in the 5'NCR of viable, attenuated mutant viruses should resist reversion to wild-type sequence. We considered the planned double C57U+G58C mutation, with predicted greater destabilization of the stem structure, as a potentially more attenuating and reversion-resistant locus in a viable, clone-derived virus. Several other single point mutations were engineered to begin to examine the uniqueness of the 5'NCR-C57U attenuating effect. DEN-2 16681 variants were derived by transfection of LLC-MK2 cells with viral genomic RNA transcribed from plasmid DNA as described previously (Kinney et al., 1997). RNA transcripts of the single point-mutated plasmids pD2/IC-55, -57, -60 and -69, as well as the double mutant pD2/IC-5758, yielded fully infectious viruses, as indicated by the expression of immunofluorescence assay (IFA)-detectable DEN-2 viral antigen in 100 % of cells in the transfected cell culture following 4–7 days incubation (Table 1). In our non-optimized transfection experiments with pD2/IC-30P-A clone-transcribed RNA, IFA-detectable DEN-2 viral antigen was detected routinely in a minor fraction of cells in the transfected culture during the first 2 days after electroporation (unpublished data), and the extent of IFA positivity has been a reliable indicator of clone-derived viral viability in our laboratory. The pD2/IC-V5 clone and D2/IC-V5 virus reported previously (Butrapet et al., 2000) were utilized in the current study and, for clarity, their designations are changed here to clone pD2/IC-57 and D2/IC-57 virus, respectively. Rescued viruses were amplified once more in LLC-MK2 cells, and the complete genomes (except for the 5'- and 3'-terminal primer target sequences) of the resulting virus seeds were sequenced following cDNA amplification by RT-PCR. The single and double deletion-mutant plasmids pD2/IC-d57 and pD2/IC-d5758 produced crippled, replication-deficient viruses that resulted in IFA-detectable DEN-2 antigen in approximately 5 and 10 % of transfected cells in the cultures, respectively, in four separate experiments. The remaining five mutations engineered into plasmids pD2/IC-d62/63 (deletion of nt 62, substitution at nt 63), -5760, -5661, -d5759 and -d5661 were considered lethal, because they failed to yield infectious viruses in five attempts. These results indicated that mutations involving certain 1 or 2 nt substitutions between nt 54 and 70 were well tolerated and resulted in viruses of high infectivity. However, all of the engineered deletion mutations, as well as those substitutions involving more than 2 nt, failed to yield infectious viruses. The results agree with a previous report showing that multiple contiguous deletions between nt 50 and 72 in the 5'NCR of DEN-4 virus are fatal for the virus (Cahour et al., 1995).
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). At 9 days after infection, the mean plaque diameters (Table 2) of D2/IC-30P-A virus and D2/IC-69 virus were equivalent (2.9–3.0 mm) (P>0.05, Student’s t-test). D2/IC-55 and -60 viruses produced significantly smaller plaques with a mean diameter of 2.5 mm (P<0.05), which was similar to the plaque size reported previously for D2/IC-57 virus (Butrapet et al., 2000). The D2/IC-5758 virus had a statistically significantly larger mean plaque size than the D2/IC-30P-A virus (P<0.05). All of these viruses produced plaques that were much larger than the pinpoint (<1 mm) plaques of the candidate DEN-2 PDK-53 vaccine virus included in the same assays (data not shown). All three of the 5'NCR-C57U, NS1-Gly53Asp and NS3-Glu250Val mutations have been shown to contribute independently, and additively, to the reduced plaque size of PDK-53 virus (Butrapet et al., 2000). The D2/IC-d57 and -d5758 viruses failed to produce plaques, even by 2 weeks post-infection (data not shown).
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; Miller & Mitchell, 1986). Viruses D2/IC-30P-A, D2/IC-55, -57, -60, -5758 and -69 replicated well to high peak titres of 106.7–107.4 p.f.u. ml–1 at 10–12 days after infection of LLC-MK2 cells (Fig. 1a). D2/IC-60 virus replicated more slowly than the other viruses between days 2 and 8 after infection, but attained a similarly high titre by days 10–12. The clone-derived viruses showed variable replication in C6/36 cells (Fig. 1b). D2/IC-30P-A, -60, -5758 and -69 viruses replicated to peak titres of 107.3–107.8 p.f.u. ml–1 at day 8 after infection, although replication of D2/IC-60 virus appeared to be slightly delayed in these cells as well. D2/IC-57 and -55 viruses exhibited restricted growth that was characterized by 100- and 10-fold decreases in peak titres, respectively, relative to the replication of D2/IC-30P-A virus in C6/36 cells. The peak titres of these two viruses also occurred at day 8 after infection. DEN-2 PDK-53 virus replicated to 103.3 p.f.u. ml–1 after 8 days growth in C6/36 cells (data not shown). The severely crippled replicative phenotype of the DEN-2 PDK-53 virus in C6/36 cells is caused by the 5'NCR-C57U and NS1-Gly53Asp mutations, both of which have been shown to inhibit virus replication independently, as well as additively when both mutations were engineered together into the DEN-2 16681 virus genome (Butrapet et al., 2000).
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; Huang et al., 2000, 2003; Kinney et al., 1997). Attenuation of neurovirulence for ICR mice is caused by the NS1-Gly53Asp and 5'NCR-C57U mutations (Butrapet et al., 2000). More recently, the NS3-Glu250Val mutation was also found to contribute to the attenuated phenotype of PDK-53 virus in the more DEN-2 virus-sensitive Swiss Webster mouse model (Huang et al., 2005). In the present study, the mutant 5'NCR viruses were tested for neurovirulence in newborn ICR mice. In two separate experiments, groups of 16 mice were inoculated i.c. with 104 p.f.u. clone-derived D2/IC virus and observed for 35 days for morbidity, paralysis and/or death. All mice surviving for 21 days after infection were weighed individually on that day. The pooled results (32 mice per group) from the two experiments are shown in Table 2
. Previously published data for D2/IC-57 virus (n=16 mice) are shown for comparison (Butrapet et al., 2000). The D2/IC-30P-A and D2/IC-60 viruses resulted in 84.37 % mortality, with a mean survival time (MST) of 13.6 and 15.8 days, respectively. Because we previously identified only 18.75 % mortality (MST of 21.7 days) in newborn ICR mice challenged i.c. with D2/IC-57 virus, the 87.5 %, wild-type level of mortality in mice challenged with D2/IC-5758 virus was unexpected (Table 2). Mice succumbing to challenge with this virus had the lowest MST (9.9 days), and the survivors at 21 days exhibited the lowest mean body mass (9.8 g), of all mouse groups tested. Viruses D2/IC-55 and D2/IC-69 showed lower levels of mortality of 50 and 31.25 % (MSTs of 16.8 and 19.2 days), respectively, compared with D2/IC-30P-A virus. At 21 days after i.c. infection, the surviving mice in all of the virus-challenged groups exhibited a significantly lower mean body mass than the diluent-inoculated control mice (P<0.001, Student’s t-test).
Even though the ICR mouse model of neurovirulence for DEN-2 16681 virus is fairly insensitive, the D2/IC-69 virus appeared to be partially attenuated for neurovirulence, although it exhibited the robust replication phenotype of the D2/IC-30P-A virus in terms of plaque size and replication in LLC-MK2 and C6/36 cells. The A69U mutation is located at the leading end of the first stem (nt 4–9/64–69), situated at the base of the predicted large SLA structure (Filomatori et al., 2006). This A69U substitution has been identified in wild-type DEN-2 virus and was reported to constitute a significant genetic marker in differentiating between South-East Asian DEN-2 strains and DEN-2 strains of the American genotype (Leitmeyer et al., 1999). Given the apparent partial attenuation of mouse neurovirulence afforded by the A60U mutation and the robust replication of the D2/IC-69 virus in both mammalian (LLC-MK2) and mosquito (C6/36) cell cultures, it might be fruitful to investigate a combination of this mutation with another attenuating 5'NCR mutation, such as the C57U mutation, in a potentially attenuated double mutant that might resist complete genetic reversion in the 5'NCR more readily during viral passage. D2/IC-57 virus exhibited 50 % morbidity in ICR mice. Although it is difficult to interpret such a result in the ICR model, it is possible that D2/IC-55 is slightly attenuated for neurovirulence. This virus did exhibit decreased replicative ability in C6/36 cells (although not to the extent of D2/IC-55 virus) and smaller plaque size, relative to D2/IC-30P-A virus. The A55G mutation might be another possible candidate for inclusion in an attenuated virus containing a double mutation in the 5'NCR. The D2/IC-60 virus was reproducibly less robust during the first 6–8 days of replication in LLC-MK2 cells in two independent experiments, but replicated well in C6/36 cells and was fully neurovirulent for mice.
As we were unable to derive infectious viruses from the plasmid constructs containing one or more deletion mutation or more than two nucleotide substitutions, a defect in viral protein synthesis that might have prevented virus replication was suspected. The capacity of the RNA mutants to direct protein synthesis was examined in the rabbit reticulocyte lysate system in the presence of [35S]Met. The translation products were analysed by SDS-PAGE (10 % gels) and translation efficiencies were determined by measuring incorporation of acid-precipitated [35S]Met. All of the RNA mutants were translated efficiently to variable extents, with [35S]Met incorporation ranging from 0.28- to 2.13-fold relative to the pD2/IC-30P-A RNA transcript (data not shown). The results obtained failed to show an obvious impact of mutation on translation efficiency. Nevertheless, this finding is in agreement with a recent study demonstrating that the 5'NCR SLA structure functions in minus-strand RNA synthesis, rather than RNA translation (Filomatori et al., 2006).
Interestingly, the D2/IC-5758 mutant, which was expected to be less neurovirulent in mice than the D2/IC-V5 mutant, yielded unexpected results. RNA secondary-structure prediction of the 5'NCR of each mutant DEN-2 virus was performed with the Mfold program (Zuker, 2003). The predicted stem structure formed by nt 11–16 and 56–61 was more perturbed in the D2/IC-5758 mutant than in the D2/IC-57 virus, as expected by the incorporation of the G58C mutation in addition to the partially attenuating 5'NCR-C57U mutation in this stem structure (data not shown). Rather than exhibiting more pronounced attenuation markers, the D2/IC-5758 mutant replicated as well as the wild-type virus in both LLC-MK2 and C6/36 cell cultures, exhibited a large plaque size and appeared to be more neurovirulent for newborn ICR mice than the wild-type 16681 (D2/IC-30P-A) virus. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5'NCR-C57U mutation. This observation is particularly relevant for candidate live-attenuated, chimeric DEN virus and other flavivirus vaccines based on the genetic background of the attenuated DEN-2 PDK-53 virus. Genetic analyses of such vaccine virus seeds would be able to verify the apparent absence of potentially compensatory secondary mutations in the 5'NCR, as well as a low level or absence of reversion at the 5'NCR-57U locus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Huang, C. Y., Silengo, S. J., Whiteman, M. C. & Kinney, R. M. (2005). Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 79, 7300–7310.
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Received 9 August 2006;
accepted 17 February 2007.
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, D2/IC-30P-A; 
, D2/IC-57; x, D2/IC-55; *, D2/IC-60; 
, D2/IC-5758; 
, D2/IC-69.