- Email: [email protected]
The causes and consequences of genetic variation in dengue virus
REVIEWS
The causes and consequences of genetic variation in dengue virus Eddie C. Holmes and S. Susanna Burch
T
he recent outbreak of Despite the fact that dengue is one of the of variants – genotypes – to be encephalitis in New most prevalent viral infections of humans, identified6. York, which was caused the mechanisms responsible for its A number of factors underby infection with a West Nilepathogenesis remain uncertain. pin this biodiversity. It is obvilike virus, provided a timely Evolutionary studies of dengue virus have ous that dengue virus is highly reminder that arthropod-borne revealed that its genetic diversity is mutagenic, as RNA-dependent viruses are not just a burden increasing. This, coupled with evidence RNA polymerases are thought to the health of developing that viral strains could naturally differ in to produce approximately one nations. In the future, this virulence, suggests that in the future we error per round of genome might also be true of dengue, a might be exposed to viruses with an replication7. However, it is positive-strand RNA flavivirus expanded range of pathogenic properties. equally apparent that the closely related to West Nile overall base substitution rate E.C. Holmes* and S.S. Burch are in the Dept of virus that is transmitted by the in dengue is less than that Zoology, University of Oxford, South Parks Road, domesticated Aedes aegypti observed in other RNA viruses Oxford, UK OX1 3PS. mosquito. It is estimated that replicated using the same *tel: 144 1865 271282, at least 50 million people each enzyme8, which suggests that fax: 144 1865 310447, e-mail: [email protected] year suffer dengue infection, its rate of replication per with some 2.5 billion people year might be somewhat lower living in at-risk areas1. Although most dengue infec- or that the virus is subject to stronger functional tions are relatively mild [manifesting as classical dengue constraints. fever (DF)] or even sub-clinical, the virus appears to In addition to mutation, there is now evidence that be increasingly associated with more serious clinical genetic diversity in dengue virus might also be generoutcomes in the form of dengue haemorrhagic fever ated by recombination, probably by a ‘copy-choice’ (DHF) and dengue shock syndrome (DSS), both of mechanism in which the RNA polymerase switches, which have much higher case-fatality rates (1–5%). mid-replication, between the genomes of two viruses The emergence of DHF and DSS is particularly ap- from one serotype that have infected the same cell parent in Latin America, where the number of cases and formed a hybrid RNA molecule9. At present, it is per year doubled between 1995 and 1998 (Ref. 1). uncertain how common recombination is in dengue, Against this background of rising incidence, and although this process has now been found to occur in without an effective vaccine, dengue virus research a wide range of RNA viruses10. It is also unclear has intensified. However, some outstanding ques- whether recombination could ever take place betions remain, including the respective contributions tween viruses from different serotypes, although the of differences in host genetic susceptibility, antibody- extensive sequence divergence between them makes dependent enhancement (ADE; a complex immuno- recombination at this level less likely. Indeed, the fact pathological phenomenon triggered by successive in- that putative intra-serotype recombinants are easily fection with different viral serotypes2,3) and variation identifiable suggests that recombination is relatively in virulence among viral strains4 to DHF and DSS rare compared with mutation (a high recombination pathogenesis. In this review, we concern ourselves rate would cause more sequence scrambling). Howwith the hypothesis that epidemics of DHF and DSS ever, given that the rate at which viral genetic diversity are caused by the circulation of viral strains with in- is generated will be a major factor in determining creased virulence, and discuss how recent advances in how dengue virus might respond to future vacour understanding of the evolutionary biology of cination programs, and that recombination produces dengue virus put this theory in a new light. more variation than is available to RNA viruses that are strictly clonal, it is crucial we confirm that recomThe evolutionary genetics of dengue virus bination does take place in vivo and measure its rate Like many RNA viruses, dengue exhibits substantial relative to that of mutation. genetic diversity, most notably in the existence of four Another way in which genetic variation can enter distinct serotypes (DEN-1–4), which are no more populations is via migration (gene flow), and there is similar to each other than some different ‘species’ of now plenty of data indicating that dengue virus is flavivirus5. Genetic diversity is much more restricted often transported large distances by hosts and vectors within each serotype but is still sufficient for clusters – serotypes and genotypes can therefore have broad 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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geographical distributions6. Of course, greater mixing of strains increases the opportunity for recombination and, hence, for the generation of biological novelty. In the face of such rampant gene flow the question that arises is why the virus exists as four distinct serotypes at all? At present, there is insufficient evidence to determine if the serotypes represent four viral populations that were geographically separated initially and which have only recently come together. Alternatively, they could be the outcomes of independent cross-species transfer events from the various monkey species which harbour dengue, or they could have evolved together for an extended period because of the mutual benefits ADE brings to their persistence11. It is also possible that the extent of genetic variation in dengue is increasing simply because the size and density of the human host population has also risen, thereby providing more opportunities for viral transmission8. As human populations will doubtless grow further in the future, this hypothesis predicts that we might also expect the same of dengue, at least until the advent of suitable control measures. One evolutionary implication of such an increase in viral population size is that it will allow natural selection to act with greater potency because more mutations will be available and a greater possibility of transmission means there is less stochastic loss of advantageous mutations. It is therefore important to ask whether there is any evidence for the selection of dengue strains with particular properties, such as increased infectiousness or different cell tropisms, or even for its action on particular regions within the viral genome, perhaps stimulated by the need to evade the host immune response? The limited data currently available suggest that natural selection, such as that driven by immune pressure, is a rather weak force and that genetic drift controls the evolutionary fate of mutants8. However, given the obvious potential for natural selection to shape the genetic structure of populations, particularly with an expanded range of viral strains to work with, this is clearly an area that merits further study. Implications for disease? The burgeoning biodiversity of dengue virus could mean little if strain variation plays only a minor role in disease. Indeed, there is a great deal of evidence to suggest that ADE is a key factor in DHF and DSS pathogenesis, most notably from epidemiological studies that reveal a higher incidence of severe disease in secondary, compared with primary, viral infections12,13. Yet, despite this, there are still cases of DHF and DSS that cannot be adequately explained by ADE, particularly in confirmed cases of primary infection14–16, and where the epidemic situation would be expected to lead to a high number of secondary infections but where severe dengue is rare17,18. Such epidemiological observations suggest that strains of dengue virus with a wide variety of pathogenic properties exist in nature, although there is patently a need for long-term prospective studies of dengue infection within communities.
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Although sketchy at best, tentative genetic support for differences in virulence among strains is available. For example, it has been noted19 that strains within serotypes ‘differed markedly in their reactions with dengue antisera’, and there is some evidence that DHF-associated strains of DEN-2 might crossreact with more of a panel of DEN-4 monoclonal antibodies than DEN-2 strains associated with classical DF (Refs 12,20). This suggests that some dengue strains from the same serotype could cause more severe disease than others because they have a greater ability to be enhanced by heterotypic antibodies. More recent analyses have identified mutations in the dengue virus genome that are associated with changes in virulence, either by comparing original and attenuated strains21,22, or by using chimeric viruses or cDNA infectious clones22–24. Some studies have even described the apparent segregation of viruses causing mild and severe disease in nature25,26. However, as these have generally utilized only small fragments of the viral genome, often without reliable clinical data, in reality they tell us little about the contribution of strain variation to dengue pathogenesis. The most consistent argument for a strain basis to DHF and DSS has been put forward by Rico-Hesse and colleagues6,27. Briefly, they proposed that a lowvirulence strain of DEN-2 has circulated in Latin America since the late 1960s and that epidemics of DHF and DSS did not occur in this region until the arrival of a strain of higher pathogenicity originating from South East Asia, where serious dengue disease is more common; this suggestion is supported by a recent epidemiological study of a clinically mild DEN-2 infection in Peru18. The complete genome sequences of the putative high (Asian) and low (American) virulence strains have now been compared and candidate mutations – those that differed consistently between the two sets of strains – have been identified28. Interestingly, although distinguishing mutations are scattered throughout the genome, some occur in regions known to affect pathogenicity in vitro, such as the 39-UTR (untranslated region), which is fundamental to viral replication, translation and assembly, and also in the envelope protein, which stimulates neutralizing antibodies and induces cell-mediated immune responses, as well as being the site of attachment for cellular proteins. The 39-untranslated regions (UTRs) of the American strains are characterized by a set of point mutations and a deletion, which, together, are reported to alter radically RNA secondary structure; additionally, an amino acid replacement at position 390 in the envelope protein is suggested to have possibly altered the strength of virus binding to host cells28, a proposal given added weight by the observation that other amino acid changes at this residue affect neurovirulence in mice21. Unfortunately, the lack of a suitable animal model for dengue makes it difficult to confirm whether these and other characteristic mutations really do determine virulence, either in isolation or synergistically, or whether they have accumulated simply because of the phylogenetic isolation of the Latin American strains. Despite this
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Viruses with an expanded range of pathogenic properties?
Mutation Recombination Gene flow More susceptible hosts Monkey reservoir population?
Genetic diversity Selection of strains for increased transmittability (high viraemia) and virulence? trends in Microbiology
Fig. 1. The processes that have caused an increase in the genetic diversity of dengue virus and two possible evolutionary consequences of this increase.
caveat, whole-genome analyses clearly take the study of dengue pathogenesis to a new level and provide a valuable set of testable hypotheses. Dengue virulence in perspective Studies of the evolutionary genetics of dengue virus suggest that its populations are becoming increasingly diverse, either via elevated rates of recombination and gene flow, or simply because the number and density of human hosts is rising. Of equal importance is the fact that a reservoir of dengue virus exists in Asian and African monkeys, about which little constructive work has been done, but whose long period of independent evolution will doubtless have generated viral strains with very different phenotypic properties. Taken together, these observations suggest that the conditions necessary for the emergence of new and potentially pathogenic dengue strains are in place: even if the hypothesis that strains of dengue virus differ in virulence is unproven on present data, it is increasingly likely to be true of the strains that appear in the future (Fig. 1). Might this increase in genetic diversity change the overall virulence of dengue virus? Although predictions in this area should be made with caution, it is possible that an increased diversity and mixing of viral strains will lead to increased competition among them, selecting those which attain the highest levels of viraemia, and hence maximum infectiousness. If, as has been suggested,
Questions for future research • Do particular dengue strains (serotypes or genotypes) have greater capacity for ADE than others? • Does viral load play a direct role in the pathogenesis of DHF and DSS? • Do differences in cell tropism among strains play a role in pathogenesis? • What is the rate of dengue virus recombination in vivo? Can recombination occur between viruses from different serotypes? • Does immune selection play a role in structuring genetic variation in dengue virus? Is there evidence that particular strains have higher fitness in vivo than others? • What is the extent of genetic diversity in the monkey species that harbour dengue viruses? Do these viruses have different phenotypic features from those that infect humans?
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high viraemia is a factor in generating severe dengue disease4, then the result of this evolutionary process would indeed be a net increase in virulence. The need to control the spread of dengue is perhaps more important than ever. Acknowledgements We thank the Wellcome Trust and Royal Society for financial support as well as Dr E.A. Gould and two referees for constructive comments.
References 1 WHO (1998) Dengue and dengue haemorrhagic fever. WHO Fact Sheet 117, WHO, Geneva 2 Halstead, S.B. (1988) Pathogenesis of dengue: challenges to molecular biology. Science 239, 476–481 3 Bielefeldt-Ohmann, H. (1997) Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol. 5, 409–412 4 Rothman, A.L. (1997) Viral pathogenesis of infection. In Dengue and Dengue Hemorrhagic Fever (Gubler, D.J. and Kuno, G., eds), pp. 245–271, CABI 5 Kuno, G. (1998) Phylogeny of the genus Flavivirus. J. Virol. 72, 73–83 6 Rico-Hesse, R. (1990) Molecular evolution and distribution of dengue viruses type-1 and type-2 in nature. Virology 174, 479–493 7 Drake, J.W. (1993) Rates of spontaneous mutation among RNA viruses. Proc. Natl. Acad. Sci. U. S. A. 90, 4171–4175 8 Zanotto, P.M. et al. (1996) Population dynamics of flaviviruses revealed by molecular phylogenies. Proc. Natl. Acad. Sci. U. S. A. 93, 548–553 9 Worobey, M. et al. (1999) Widespread intra-serotype recombination in natural populations of dengue virus. Proc. Natl. Acad. Sci. U. S. A. 96, 7352–7357 10 Worobey, M. and Holmes, E.C. (1999) Evolutionary aspects of recombination in RNA viruses. J. Gen. Virol. 80, 2535–2544 11 Ferguson, N. et al. (1999) The effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens. Proc. Natl. Acad. Sci. U. S. A. 96, 790–794 12 Burke, D.S. et al. (1988) A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38, 172–180 13 Thein, S. et al. (1997) Risk factors in dengue shock syndrome. Am. J. Trop. Med. Hyg. 56, 566–572 14 Scott, R.M. et al. (1976) Shock syndrome in primary dengue infections. Am. J. Trop. Med. Hyg. 25, 866–874 15 Gubler, D.J. et al. (1978) Epidemiologic, clinical, and virological observations on dengue in the kingdom of Tonga. Am. J. Trop. Med. Hyg. 27, 581–589 16 Glaziou, P. et al. (1992) Dengue fever and dengue shock syndrome in French Polynesia. Southeast Asian J. Trop. Med. Pub. Health 3, 531–532 17 Moreau, J-P. (1973) An epidemic of dengue on Tahiti associated with hemorrhagic manifestations. Am. J. Trop. Med. Hyg. 22, 237–241 18 Watts, D.M. et al. (1999) Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever. Lancet 354, 1431–1434 19 Halstead, S.B. et al. (1970) Observations related to pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host. Yale J. Biol. Med. 42, 276–292
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20 Morens, D.M. and Halstead, S.B. (1987) Disease severityrelated antigenic differences in dengue 2 strains detected by dengue 4 monoclonal antibodies. J. Med. Virol. 22, 169–174 21 Sanchez, I.J. and Ruiz, B.H. (1996) A single nucleotide change in the E protein gene of dengue virus 2 Mexican strain affects neurovirulence in mice. J. Gen. Virol. 77, 2541–2545 22 Kinney, R.M. et al. (1997) Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300–308 23 Bray, M. et al. (1998) Genetic determinants responsible for acquisition of dengue type 2 virus mouse neurovirulence. J. Virol. 72, 1647–1651
24 Gualano, R.C. et al. (1998) Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA. J. Gen. Virol. 9, 437–446 25 Chow, V.T.K. et al. (1994) Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from Southeast Asia. Intervirology 37, 252–258 26 Lanciotti, R.S. (1997) Molecular evolution and phylogeny of dengue-4 viruses. J. Gen. Virol. 78, 2279–2286 27 Rico-Hesse, R. et al. (1997) Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230, 244–251 28 Leitmeyer, K.C. et al. (1999) Dengue virus structural differences that correlate with pathogenesis. J. Virol. 73, 4738–4747
The origin of prokaryotic C2H2 zinc finger regulators Naima Bouhouche, Michael Syvanen and Clarence I. Kado
T
ranscriptional regulators C2H2 zinc finger bearing proteins are a of the virC and virD operons3, bearing a C2H2-type, large superfamily of nucleic acid binding and increased expression of or Kruppel-type, zinc proteins, which constitute a major subset the ipt gene in the T-DNA finger were thought to be conof eukaryotic transcription factors. (Ref. 2). fined to eukaryotes; however, Although originally thought to occur only recent studies have demonin eukaryotes, a novel C2H2 zinc finger Ros homologues strated their presence in transcription factor, Ros, which regulates Sequence analysis has identiprokaryotes. The basic strucboth prokaryotic and eukaryotic fied a number of Ros homoture of the C2H2 zinc finger promoters has been found in bacteria. logues, primarily among memcomprises a short peptide loop Phylogenically, Ros is distantly related to bers of the Rhizobiaceae with a small b-hairpin, followed eukaryotic zinc finger regulators. (Table 1). RosAR from by an a-helix held in place by a Agrobacterium radiobacter N. Bouhouche and C.I. Kado* are in the Davis zinc ion. In the folded state, regulates the expression of the Crown Gall Group, University of California, One the a-helix makes direct conglycosyl transferase gene exoY, Shields Avenue, Davis, CA 95616, USA; M. Syvanen tact with the major groove of which is involved in exois in the Dept of Medical Microbiology and the DNA (Ref. 1). The first polysaccharide synthesis5. MuImmunology, School of Medicine, University of California, Davis, CA 95616, USA. prokaryotic C2H2-type zinc tational inactivation of rosAR *tel: 11 530 752 0325, finger transcription-regulatory results in the elevated expresfax: 11 530 752 5674, protein, Ros, was discovered sion of exoY. In Rhizobium e-mail: [email protected] in Agrobacterium tumefaciens2. etli, RosR regulates the exRos is a 15.5-kDa protein, enpression of cell-surface comcoded by the A. tumefaciens chromosomal gene ros, ponents, competitive growth in the rhizosphere and which represses the transcription of virulence genes efficiency in nodulating Phaseolus vulgaris6. Similarly, and an oncogene located on the large Ti plasmid pres- in Rhizobium meliloti (emended as Sinorhizobium ent in this plant-tumour-inducing Gram-negative bac- meliloti), the Ros homologue MucR regulates the terium. Specifically, Ros regulates the expression of biosynthesis of succinoglycan (EPS I) and galactogluthe virulence genes of the virC and virD operons3,4, can (EPS II) (Ref. 7), exopolysaccharides that are the products of which are involved in processing the required for nodule invasion. MucR represses tranoncogene-bearing region (T-DNA) of the Ti plasmid scription of the gene encoding EPS II and activates for horizontal gene transfer from A. tumefaciens cells transcription of the gene encoding EPS I. Additionally, to plant cells. Ros also regulates expression of the an open reading frame (ORF2) upstream of syrB in oncogene ipt (Ref. 2), which encodes the isopentenyl S. meliloti was found to be similar to Ros and MucR transferase required for the biosynthesis of cytokinin (Ref. 8). As shown in Table 1, these Ros homologues in the host plant. Mutations in ros result in upregulation have relatively high sequence identity. 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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The causes and consequences of genetic variation in dengue virus Eddie C. Holmes and S. Susanna Burch
T
he recent outbreak of Despite the fact that dengue is one of the of variants – genotypes – to be encephalitis in New most prevalent viral infections of humans, identified6. York, which was caused the mechanisms responsible for its A number of factors underby infection with a West Nilepathogenesis remain uncertain. pin this biodiversity. It is obvilike virus, provided a timely Evolutionary studies of dengue virus have ous that dengue virus is highly reminder that arthropod-borne revealed that its genetic diversity is mutagenic, as RNA-dependent viruses are not just a burden increasing. This, coupled with evidence RNA polymerases are thought to the health of developing that viral strains could naturally differ in to produce approximately one nations. In the future, this virulence, suggests that in the future we error per round of genome might also be true of dengue, a might be exposed to viruses with an replication7. However, it is positive-strand RNA flavivirus expanded range of pathogenic properties. equally apparent that the closely related to West Nile overall base substitution rate E.C. Holmes* and S.S. Burch are in the Dept of virus that is transmitted by the in dengue is less than that Zoology, University of Oxford, South Parks Road, domesticated Aedes aegypti observed in other RNA viruses Oxford, UK OX1 3PS. mosquito. It is estimated that replicated using the same *tel: 144 1865 271282, at least 50 million people each enzyme8, which suggests that fax: 144 1865 310447, e-mail: [email protected] year suffer dengue infection, its rate of replication per with some 2.5 billion people year might be somewhat lower living in at-risk areas1. Although most dengue infec- or that the virus is subject to stronger functional tions are relatively mild [manifesting as classical dengue constraints. fever (DF)] or even sub-clinical, the virus appears to In addition to mutation, there is now evidence that be increasingly associated with more serious clinical genetic diversity in dengue virus might also be generoutcomes in the form of dengue haemorrhagic fever ated by recombination, probably by a ‘copy-choice’ (DHF) and dengue shock syndrome (DSS), both of mechanism in which the RNA polymerase switches, which have much higher case-fatality rates (1–5%). mid-replication, between the genomes of two viruses The emergence of DHF and DSS is particularly ap- from one serotype that have infected the same cell parent in Latin America, where the number of cases and formed a hybrid RNA molecule9. At present, it is per year doubled between 1995 and 1998 (Ref. 1). uncertain how common recombination is in dengue, Against this background of rising incidence, and although this process has now been found to occur in without an effective vaccine, dengue virus research a wide range of RNA viruses10. It is also unclear has intensified. However, some outstanding ques- whether recombination could ever take place betions remain, including the respective contributions tween viruses from different serotypes, although the of differences in host genetic susceptibility, antibody- extensive sequence divergence between them makes dependent enhancement (ADE; a complex immuno- recombination at this level less likely. Indeed, the fact pathological phenomenon triggered by successive in- that putative intra-serotype recombinants are easily fection with different viral serotypes2,3) and variation identifiable suggests that recombination is relatively in virulence among viral strains4 to DHF and DSS rare compared with mutation (a high recombination pathogenesis. In this review, we concern ourselves rate would cause more sequence scrambling). Howwith the hypothesis that epidemics of DHF and DSS ever, given that the rate at which viral genetic diversity are caused by the circulation of viral strains with in- is generated will be a major factor in determining creased virulence, and discuss how recent advances in how dengue virus might respond to future vacour understanding of the evolutionary biology of cination programs, and that recombination produces dengue virus put this theory in a new light. more variation than is available to RNA viruses that are strictly clonal, it is crucial we confirm that recomThe evolutionary genetics of dengue virus bination does take place in vivo and measure its rate Like many RNA viruses, dengue exhibits substantial relative to that of mutation. genetic diversity, most notably in the existence of four Another way in which genetic variation can enter distinct serotypes (DEN-1–4), which are no more populations is via migration (gene flow), and there is similar to each other than some different ‘species’ of now plenty of data indicating that dengue virus is flavivirus5. Genetic diversity is much more restricted often transported large distances by hosts and vectors within each serotype but is still sufficient for clusters – serotypes and genotypes can therefore have broad 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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geographical distributions6. Of course, greater mixing of strains increases the opportunity for recombination and, hence, for the generation of biological novelty. In the face of such rampant gene flow the question that arises is why the virus exists as four distinct serotypes at all? At present, there is insufficient evidence to determine if the serotypes represent four viral populations that were geographically separated initially and which have only recently come together. Alternatively, they could be the outcomes of independent cross-species transfer events from the various monkey species which harbour dengue, or they could have evolved together for an extended period because of the mutual benefits ADE brings to their persistence11. It is also possible that the extent of genetic variation in dengue is increasing simply because the size and density of the human host population has also risen, thereby providing more opportunities for viral transmission8. As human populations will doubtless grow further in the future, this hypothesis predicts that we might also expect the same of dengue, at least until the advent of suitable control measures. One evolutionary implication of such an increase in viral population size is that it will allow natural selection to act with greater potency because more mutations will be available and a greater possibility of transmission means there is less stochastic loss of advantageous mutations. It is therefore important to ask whether there is any evidence for the selection of dengue strains with particular properties, such as increased infectiousness or different cell tropisms, or even for its action on particular regions within the viral genome, perhaps stimulated by the need to evade the host immune response? The limited data currently available suggest that natural selection, such as that driven by immune pressure, is a rather weak force and that genetic drift controls the evolutionary fate of mutants8. However, given the obvious potential for natural selection to shape the genetic structure of populations, particularly with an expanded range of viral strains to work with, this is clearly an area that merits further study. Implications for disease? The burgeoning biodiversity of dengue virus could mean little if strain variation plays only a minor role in disease. Indeed, there is a great deal of evidence to suggest that ADE is a key factor in DHF and DSS pathogenesis, most notably from epidemiological studies that reveal a higher incidence of severe disease in secondary, compared with primary, viral infections12,13. Yet, despite this, there are still cases of DHF and DSS that cannot be adequately explained by ADE, particularly in confirmed cases of primary infection14–16, and where the epidemic situation would be expected to lead to a high number of secondary infections but where severe dengue is rare17,18. Such epidemiological observations suggest that strains of dengue virus with a wide variety of pathogenic properties exist in nature, although there is patently a need for long-term prospective studies of dengue infection within communities.
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Although sketchy at best, tentative genetic support for differences in virulence among strains is available. For example, it has been noted19 that strains within serotypes ‘differed markedly in their reactions with dengue antisera’, and there is some evidence that DHF-associated strains of DEN-2 might crossreact with more of a panel of DEN-4 monoclonal antibodies than DEN-2 strains associated with classical DF (Refs 12,20). This suggests that some dengue strains from the same serotype could cause more severe disease than others because they have a greater ability to be enhanced by heterotypic antibodies. More recent analyses have identified mutations in the dengue virus genome that are associated with changes in virulence, either by comparing original and attenuated strains21,22, or by using chimeric viruses or cDNA infectious clones22–24. Some studies have even described the apparent segregation of viruses causing mild and severe disease in nature25,26. However, as these have generally utilized only small fragments of the viral genome, often without reliable clinical data, in reality they tell us little about the contribution of strain variation to dengue pathogenesis. The most consistent argument for a strain basis to DHF and DSS has been put forward by Rico-Hesse and colleagues6,27. Briefly, they proposed that a lowvirulence strain of DEN-2 has circulated in Latin America since the late 1960s and that epidemics of DHF and DSS did not occur in this region until the arrival of a strain of higher pathogenicity originating from South East Asia, where serious dengue disease is more common; this suggestion is supported by a recent epidemiological study of a clinically mild DEN-2 infection in Peru18. The complete genome sequences of the putative high (Asian) and low (American) virulence strains have now been compared and candidate mutations – those that differed consistently between the two sets of strains – have been identified28. Interestingly, although distinguishing mutations are scattered throughout the genome, some occur in regions known to affect pathogenicity in vitro, such as the 39-UTR (untranslated region), which is fundamental to viral replication, translation and assembly, and also in the envelope protein, which stimulates neutralizing antibodies and induces cell-mediated immune responses, as well as being the site of attachment for cellular proteins. The 39-untranslated regions (UTRs) of the American strains are characterized by a set of point mutations and a deletion, which, together, are reported to alter radically RNA secondary structure; additionally, an amino acid replacement at position 390 in the envelope protein is suggested to have possibly altered the strength of virus binding to host cells28, a proposal given added weight by the observation that other amino acid changes at this residue affect neurovirulence in mice21. Unfortunately, the lack of a suitable animal model for dengue makes it difficult to confirm whether these and other characteristic mutations really do determine virulence, either in isolation or synergistically, or whether they have accumulated simply because of the phylogenetic isolation of the Latin American strains. Despite this
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Viruses with an expanded range of pathogenic properties?
Mutation Recombination Gene flow More susceptible hosts Monkey reservoir population?
Genetic diversity Selection of strains for increased transmittability (high viraemia) and virulence? trends in Microbiology
Fig. 1. The processes that have caused an increase in the genetic diversity of dengue virus and two possible evolutionary consequences of this increase.
caveat, whole-genome analyses clearly take the study of dengue pathogenesis to a new level and provide a valuable set of testable hypotheses. Dengue virulence in perspective Studies of the evolutionary genetics of dengue virus suggest that its populations are becoming increasingly diverse, either via elevated rates of recombination and gene flow, or simply because the number and density of human hosts is rising. Of equal importance is the fact that a reservoir of dengue virus exists in Asian and African monkeys, about which little constructive work has been done, but whose long period of independent evolution will doubtless have generated viral strains with very different phenotypic properties. Taken together, these observations suggest that the conditions necessary for the emergence of new and potentially pathogenic dengue strains are in place: even if the hypothesis that strains of dengue virus differ in virulence is unproven on present data, it is increasingly likely to be true of the strains that appear in the future (Fig. 1). Might this increase in genetic diversity change the overall virulence of dengue virus? Although predictions in this area should be made with caution, it is possible that an increased diversity and mixing of viral strains will lead to increased competition among them, selecting those which attain the highest levels of viraemia, and hence maximum infectiousness. If, as has been suggested,
Questions for future research • Do particular dengue strains (serotypes or genotypes) have greater capacity for ADE than others? • Does viral load play a direct role in the pathogenesis of DHF and DSS? • Do differences in cell tropism among strains play a role in pathogenesis? • What is the rate of dengue virus recombination in vivo? Can recombination occur between viruses from different serotypes? • Does immune selection play a role in structuring genetic variation in dengue virus? Is there evidence that particular strains have higher fitness in vivo than others? • What is the extent of genetic diversity in the monkey species that harbour dengue viruses? Do these viruses have different phenotypic features from those that infect humans?
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high viraemia is a factor in generating severe dengue disease4, then the result of this evolutionary process would indeed be a net increase in virulence. The need to control the spread of dengue is perhaps more important than ever. Acknowledgements We thank the Wellcome Trust and Royal Society for financial support as well as Dr E.A. Gould and two referees for constructive comments.
References 1 WHO (1998) Dengue and dengue haemorrhagic fever. WHO Fact Sheet 117, WHO, Geneva 2 Halstead, S.B. (1988) Pathogenesis of dengue: challenges to molecular biology. Science 239, 476–481 3 Bielefeldt-Ohmann, H. (1997) Pathogenesis of dengue virus diseases: missing pieces in the jigsaw. Trends Microbiol. 5, 409–412 4 Rothman, A.L. (1997) Viral pathogenesis of infection. In Dengue and Dengue Hemorrhagic Fever (Gubler, D.J. and Kuno, G., eds), pp. 245–271, CABI 5 Kuno, G. (1998) Phylogeny of the genus Flavivirus. J. Virol. 72, 73–83 6 Rico-Hesse, R. (1990) Molecular evolution and distribution of dengue viruses type-1 and type-2 in nature. Virology 174, 479–493 7 Drake, J.W. (1993) Rates of spontaneous mutation among RNA viruses. Proc. Natl. Acad. Sci. U. S. A. 90, 4171–4175 8 Zanotto, P.M. et al. (1996) Population dynamics of flaviviruses revealed by molecular phylogenies. Proc. Natl. Acad. Sci. U. S. A. 93, 548–553 9 Worobey, M. et al. (1999) Widespread intra-serotype recombination in natural populations of dengue virus. Proc. Natl. Acad. Sci. U. S. A. 96, 7352–7357 10 Worobey, M. and Holmes, E.C. (1999) Evolutionary aspects of recombination in RNA viruses. J. Gen. Virol. 80, 2535–2544 11 Ferguson, N. et al. (1999) The effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens. Proc. Natl. Acad. Sci. U. S. A. 96, 790–794 12 Burke, D.S. et al. (1988) A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38, 172–180 13 Thein, S. et al. (1997) Risk factors in dengue shock syndrome. Am. J. Trop. Med. Hyg. 56, 566–572 14 Scott, R.M. et al. (1976) Shock syndrome in primary dengue infections. Am. J. Trop. Med. Hyg. 25, 866–874 15 Gubler, D.J. et al. (1978) Epidemiologic, clinical, and virological observations on dengue in the kingdom of Tonga. Am. J. Trop. Med. Hyg. 27, 581–589 16 Glaziou, P. et al. (1992) Dengue fever and dengue shock syndrome in French Polynesia. Southeast Asian J. Trop. Med. Pub. Health 3, 531–532 17 Moreau, J-P. (1973) An epidemic of dengue on Tahiti associated with hemorrhagic manifestations. Am. J. Trop. Med. Hyg. 22, 237–241 18 Watts, D.M. et al. (1999) Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever. Lancet 354, 1431–1434 19 Halstead, S.B. et al. (1970) Observations related to pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host. Yale J. Biol. Med. 42, 276–292
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20 Morens, D.M. and Halstead, S.B. (1987) Disease severityrelated antigenic differences in dengue 2 strains detected by dengue 4 monoclonal antibodies. J. Med. Virol. 22, 169–174 21 Sanchez, I.J. and Ruiz, B.H. (1996) A single nucleotide change in the E protein gene of dengue virus 2 Mexican strain affects neurovirulence in mice. J. Gen. Virol. 77, 2541–2545 22 Kinney, R.M. et al. (1997) Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300–308 23 Bray, M. et al. (1998) Genetic determinants responsible for acquisition of dengue type 2 virus mouse neurovirulence. J. Virol. 72, 1647–1651
24 Gualano, R.C. et al. (1998) Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA. J. Gen. Virol. 9, 437–446 25 Chow, V.T.K. et al. (1994) Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from Southeast Asia. Intervirology 37, 252–258 26 Lanciotti, R.S. (1997) Molecular evolution and phylogeny of dengue-4 viruses. J. Gen. Virol. 78, 2279–2286 27 Rico-Hesse, R. et al. (1997) Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230, 244–251 28 Leitmeyer, K.C. et al. (1999) Dengue virus structural differences that correlate with pathogenesis. J. Virol. 73, 4738–4747
The origin of prokaryotic C2H2 zinc finger regulators Naima Bouhouche, Michael Syvanen and Clarence I. Kado
T
ranscriptional regulators C2H2 zinc finger bearing proteins are a of the virC and virD operons3, bearing a C2H2-type, large superfamily of nucleic acid binding and increased expression of or Kruppel-type, zinc proteins, which constitute a major subset the ipt gene in the T-DNA finger were thought to be conof eukaryotic transcription factors. (Ref. 2). fined to eukaryotes; however, Although originally thought to occur only recent studies have demonin eukaryotes, a novel C2H2 zinc finger Ros homologues strated their presence in transcription factor, Ros, which regulates Sequence analysis has identiprokaryotes. The basic strucboth prokaryotic and eukaryotic fied a number of Ros homoture of the C2H2 zinc finger promoters has been found in bacteria. logues, primarily among memcomprises a short peptide loop Phylogenically, Ros is distantly related to bers of the Rhizobiaceae with a small b-hairpin, followed eukaryotic zinc finger regulators. (Table 1). RosAR from by an a-helix held in place by a Agrobacterium radiobacter N. Bouhouche and C.I. Kado* are in the Davis zinc ion. In the folded state, regulates the expression of the Crown Gall Group, University of California, One the a-helix makes direct conglycosyl transferase gene exoY, Shields Avenue, Davis, CA 95616, USA; M. Syvanen tact with the major groove of which is involved in exois in the Dept of Medical Microbiology and the DNA (Ref. 1). The first polysaccharide synthesis5. MuImmunology, School of Medicine, University of California, Davis, CA 95616, USA. prokaryotic C2H2-type zinc tational inactivation of rosAR *tel: 11 530 752 0325, finger transcription-regulatory results in the elevated expresfax: 11 530 752 5674, protein, Ros, was discovered sion of exoY. In Rhizobium e-mail: [email protected] in Agrobacterium tumefaciens2. etli, RosR regulates the exRos is a 15.5-kDa protein, enpression of cell-surface comcoded by the A. tumefaciens chromosomal gene ros, ponents, competitive growth in the rhizosphere and which represses the transcription of virulence genes efficiency in nodulating Phaseolus vulgaris6. Similarly, and an oncogene located on the large Ti plasmid pres- in Rhizobium meliloti (emended as Sinorhizobium ent in this plant-tumour-inducing Gram-negative bac- meliloti), the Ros homologue MucR regulates the terium. Specifically, Ros regulates the expression of biosynthesis of succinoglycan (EPS I) and galactogluthe virulence genes of the virC and virD operons3,4, can (EPS II) (Ref. 7), exopolysaccharides that are the products of which are involved in processing the required for nodule invasion. MucR represses tranoncogene-bearing region (T-DNA) of the Ti plasmid scription of the gene encoding EPS II and activates for horizontal gene transfer from A. tumefaciens cells transcription of the gene encoding EPS I. Additionally, to plant cells. Ros also regulates expression of the an open reading frame (ORF2) upstream of syrB in oncogene ipt (Ref. 2), which encodes the isopentenyl S. meliloti was found to be similar to Ros and MucR transferase required for the biosynthesis of cytokinin (Ref. 8). As shown in Table 1, these Ros homologues in the host plant. Mutations in ros result in upregulation have relatively high sequence identity. 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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