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The Lancet 2000; 355:1902-1903
DOI:10.1016/S0140-6736(00)02303-5
Do escape mutants explain rapid increases in dengue casefatality rates within epidemics?
Prof Mar?a G Guzm?n
a 
, Prof Gustavo Kouri a and Prof Scott B Halstead b
Summary
During the Cuban dengue epidemics of 1981 and 1997, significant monthly Increases were observed in the proportion of total cases that presented as dengue haemorrhagic fever or dengue shock syndrome (DHF/DSS), and in case-fatality rates for both dengue fever and DHF/DSS. We believe that theses increases can be explained by the hypothesis that some of the population of antibodies against dengue 1 virus raised after natural primary infections react with ?neutralisation? determinants found on dengue 2 viruses. These heterotypic antibodies do not prevent secondary dengue 2 infections, but serve to down-regulate the disease to mild illness or symptomless infections. A population of dengue 2 viruses that replicates in dengue-1-immune hosts escape heterotypic neutralisation. When inoculated into a new dengue-1-immune host, these viruses are free to interact with the more abundant infection-enhancing antibodies to produce severe disease.
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During the 1981 and 1997 dengue epidemics in Cuba, two or more of the following quantitative measures of disease severity increased month by month: mortality due to dengue, the proportion of dengue cases that presented as dengue haemorrhagic fever or dengue shock syndrome (DHF/DSS), and the proportion of DHF/DSS cases resulting in death (table).1,2 Each of these epidemics was caused by dengue 2 viruses of the southeast Asian genotype in people previously infected by dengue 1 virus during its only appearance in Cuba (during 1977-79).3,4 Extensive clinical and epidemiological studies have been done on both epidemics.3,4
Table. Monthly increases in disease severity during dengue epidemics in Cuba
Because these increases in severity were seen during two epidemics, they are not likely to be merely artefacts of the reporting process. So what might be the underlying mechanism? A common explanation for rapid phenotypic change in a microorganism is the selection of neutralisation-escape mutants.5?8 Neutralisation-escape mutants of the type 3 poliomyelitis virus have been seen after the administration of trivalent oral polio vaccine to partly immune individuals; these mutants presumably arose after just one replicative cycle.5 Neutralisation-escape mutants in polio-vaccine viruses are biologically important because limited aminoacid changes also result in reversion to strains expressing wild-type virulence.5,6 Analogous mechanisms have been postulated for the selection of variant polio viruses in naturally immune populations.7 Antigenic drift in influenza might also be explained by this mechanism, since neutralisation-escape mutants can be readily selected with polyclonal mouse immune serum.8
In dengue, a likely mechanism for selection of neutralisation-escape mutants is the occurrence of heterotypic dengue neutralising antibodies after a primary dengue infection. Experiments in human volunteers suggest that such antibodies even provide some protection against challenge with a heterotypic dengue virus. Symptomatic dengue-2-virus infections did not occur until 3 or more months after dengue-1-virus infections in cross-challenged adult volunteers.' The epidemiological setting for the 1981 and 1997 epidemics recapitulates this experiment. Dengue 2 viruses were transmitted in populations in which a high proportion of individuals were monotypically immune to dengue 1 infection.1 Each dengue-2-virus infection in an individual immune to dengue 1 virus could be expected to select viruses that ?escape? from heterotypic neutralising antibodies. The progeny of this selectant, when inoculated into a dengue-1-immune host, would be expected to interact readily with antibodies that enhance infection and disease. Kliks and colleagues10 have shown that heterotypic neutralising antibodies play a central role in downregulating secondary dengue infections. When serum samples were tested at low dilutions, heterotypic neutralising antibodies were powerful predictors of protection against severe secondary dengue-2-virus infection. A similar phenomen has been described for the passively acquired dengue antibodies that modify primary dengue-2-virus infections in infants.11 With the passage of time after infection, antibodies are known to show progressively maturation affinity.12 This phenomenon and its corollary?decreased heterotypic reactivity?could explain why DHF/DSS case-fatality rates were higher in 1997 than in 1981 (unpublished observations).
Assessment of the appearance of neutralisationescape mutants during the 1997 outbreak should be possible, as should the reproduction of the phenomenon in vitro. We isolated several dengue 2 viruses early in the 1997 outbreak, as well as more strains later in the epidemic. Full-length sequencing of these isolates from viraemic serum samples and frozen autopsy tissues might reveal consistent genomic changes over the course of the epidemic. We are currently using these serum samples in in-vitro studies to select and sequence dengue 2 escape mutants.
Obviously, dengue escape mutants would not be expected to occur solely between dengue 1 antibodies and dengue 2 viruses. In southeast Asia, all four dengue viruses co-circulate.13 The hyperendemic circulation of multiple dengue serotypes has coincided with the emergence of DHF/DSS. Of interest, each of the four dengue serotypes circulate as more than one genotype.14 The twin survival advantages of neutralisation escape plus the push of antibody-dependent infection-enhancement may underlie the genetic and epidemiological complexity that contributes to periodic DHF/DSS mega-epidemics.15 As is well established for other viruses, the study of escape mutants could lead to a comprehensive understanding of structure and function in the dengue group.16
We thank Jose Bravo for statistical analysis.
<!--start simple-tail=-->References
1. Kouri GP, Guzm?n MG, Bravo JR. Why dengue haemorrhagic fever in Cuba? II: an integral analysis. Trans R Soc Trop Med Hyg 1987; 81: 821-823. Abstract | Full Text | PDF (163 KB) | MEDLINE | CrossRef
2. Valdes L, Guzm?n MG, Kour? G, et al. La epidemiologia del dengue en Cuba en 1997. Rev Panam Salud Publica 1999; 6: 16-25. Abstract | Full Text | PDF (57 KB)
3. Guzm?n MG, Kour? G, Bravo J, Soler M, Vazquez S, Morier L. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiological study. Am J Trop Med Hyg 1990; 42: 179-184(in press).. Abstract | Full Text | PDF (72 KB) | MEDLINE
4. Guzm?n MG, Kour? G, Valdes L, et al. Epidemiological studies on dengue in Santiago de Cuba. Am J Epidemiol 1997;.
5. Ogra PL, Faden HS, Abraham R, Duffy LC, Sun M, Minor RD. Effect of prior immunity on the shedding of virulent revertant virus in the feces after oral immunization with live-attenuated poliovirus vaccines. J Infect Dis 1991; 164: 191-194. Abstract | Full Text | PDF (57 KB) | MEDLINE
6. Minor PD. Attenuation and reversion of the Sabin vaccine strains of poliovirus. Dev Biol Stand 1993; 78: 17-26. Abstract | Full Text | PDF (57 KB) | MEDLINE
7. Huovilainen A, Hovi T, Kinnunen L, Takkinen K, Ferguson M, Minor P. Evolution of poliovirus during an outbreak: sequential type 3 poliovirus isolates from several persons show shifts of neutralization determinants. J Gen Virol 1987; 68: 1373-1379. Abstract | Full Text | PDF (57 KB)
8. Lambkin R, McLain L, Jones SE, Aldridge SL, Dimmock NJ. Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism of antigenic drift. J Gen Virol 1994; 75: 3493-3502. Abstract | Full Text | PDF (57 KB)
10. Kliks S, Nisalak A, Brandt WE, et al. Antibody dependent enhancement of dengue virus in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 1989; 40: 444-451. Abstract | Full Text | PDF (213 KB) | MEDLINE
11. Kliks S, Nisalak A, Brandt WE, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 1988; 38: 411-419. Abstract | Full Text | PDF (213 KB) | MEDLINE
12. Bottiger JB, Jensen IP. Maturation of rubella IgG avidity over time after acute rubella infection. Clin Diagn Virol 1997; 8: 105-111. Abstract | Full Text | PDF (532 KB) | CrossRef
13. Halstead SB. Dengue haemorrhagic fever, a public health problem and a field for research. Bull World Health Organ 1980; 58: 1-21. Abstract | Full Text | PDF (1056 KB) | MEDLINE
14. Rico-Hesse R. Molecular evolution and distribution of dengue viruses types 1 and 2 in nature. Virology 1990; 174: 479-493. Abstract | Full Text | PDF (358 KB) | MEDLINE | CrossRef
15. Fergson N, Anderson R, Gupta S. The effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens. Proc Natl Acad Sci USA 1999; 96: 790-794. Abstract | Full Text | PDF (57 KB) | CrossRef
16. Ketterlinus R, Wiegers K, Dernick R. Revertants of poliovirus escape mutant: new insights into antigenic structures. Virology 1993; 192: 525-533. Abstract | Full Text | PDF (57 KB) | MEDLINE | CrossRef
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DOI:10.1016/S0140-6736(00)02303-5
Do escape mutants explain rapid increases in dengue casefatality rates within epidemics?
Prof Mar?a G Guzm?n
a , Prof Gustavo Kouri a and Prof Scott B Halstead bSummary
During the Cuban dengue epidemics of 1981 and 1997, significant monthly Increases were observed in the proportion of total cases that presented as dengue haemorrhagic fever or dengue shock syndrome (DHF/DSS), and in case-fatality rates for both dengue fever and DHF/DSS. We believe that theses increases can be explained by the hypothesis that some of the population of antibodies against dengue 1 virus raised after natural primary infections react with ?neutralisation? determinants found on dengue 2 viruses. These heterotypic antibodies do not prevent secondary dengue 2 infections, but serve to down-regulate the disease to mild illness or symptomless infections. A population of dengue 2 viruses that replicates in dengue-1-immune hosts escape heterotypic neutralisation. When inoculated into a new dengue-1-immune host, these viruses are free to interact with the more abundant infection-enhancing antibodies to produce severe disease.
Back to top
During the 1981 and 1997 dengue epidemics in Cuba, two or more of the following quantitative measures of disease severity increased month by month: mortality due to dengue, the proportion of dengue cases that presented as dengue haemorrhagic fever or dengue shock syndrome (DHF/DSS), and the proportion of DHF/DSS cases resulting in death (table).1,2 Each of these epidemics was caused by dengue 2 viruses of the southeast Asian genotype in people previously infected by dengue 1 virus during its only appearance in Cuba (during 1977-79).3,4 Extensive clinical and epidemiological studies have been done on both epidemics.3,4
Click to view table
Table. Monthly increases in disease severity during dengue epidemics in Cuba
Because these increases in severity were seen during two epidemics, they are not likely to be merely artefacts of the reporting process. So what might be the underlying mechanism? A common explanation for rapid phenotypic change in a microorganism is the selection of neutralisation-escape mutants.5?8 Neutralisation-escape mutants of the type 3 poliomyelitis virus have been seen after the administration of trivalent oral polio vaccine to partly immune individuals; these mutants presumably arose after just one replicative cycle.5 Neutralisation-escape mutants in polio-vaccine viruses are biologically important because limited aminoacid changes also result in reversion to strains expressing wild-type virulence.5,6 Analogous mechanisms have been postulated for the selection of variant polio viruses in naturally immune populations.7 Antigenic drift in influenza might also be explained by this mechanism, since neutralisation-escape mutants can be readily selected with polyclonal mouse immune serum.8
In dengue, a likely mechanism for selection of neutralisation-escape mutants is the occurrence of heterotypic dengue neutralising antibodies after a primary dengue infection. Experiments in human volunteers suggest that such antibodies even provide some protection against challenge with a heterotypic dengue virus. Symptomatic dengue-2-virus infections did not occur until 3 or more months after dengue-1-virus infections in cross-challenged adult volunteers.' The epidemiological setting for the 1981 and 1997 epidemics recapitulates this experiment. Dengue 2 viruses were transmitted in populations in which a high proportion of individuals were monotypically immune to dengue 1 infection.1 Each dengue-2-virus infection in an individual immune to dengue 1 virus could be expected to select viruses that ?escape? from heterotypic neutralising antibodies. The progeny of this selectant, when inoculated into a dengue-1-immune host, would be expected to interact readily with antibodies that enhance infection and disease. Kliks and colleagues10 have shown that heterotypic neutralising antibodies play a central role in downregulating secondary dengue infections. When serum samples were tested at low dilutions, heterotypic neutralising antibodies were powerful predictors of protection against severe secondary dengue-2-virus infection. A similar phenomen has been described for the passively acquired dengue antibodies that modify primary dengue-2-virus infections in infants.11 With the passage of time after infection, antibodies are known to show progressively maturation affinity.12 This phenomenon and its corollary?decreased heterotypic reactivity?could explain why DHF/DSS case-fatality rates were higher in 1997 than in 1981 (unpublished observations).
Assessment of the appearance of neutralisationescape mutants during the 1997 outbreak should be possible, as should the reproduction of the phenomenon in vitro. We isolated several dengue 2 viruses early in the 1997 outbreak, as well as more strains later in the epidemic. Full-length sequencing of these isolates from viraemic serum samples and frozen autopsy tissues might reveal consistent genomic changes over the course of the epidemic. We are currently using these serum samples in in-vitro studies to select and sequence dengue 2 escape mutants.
Obviously, dengue escape mutants would not be expected to occur solely between dengue 1 antibodies and dengue 2 viruses. In southeast Asia, all four dengue viruses co-circulate.13 The hyperendemic circulation of multiple dengue serotypes has coincided with the emergence of DHF/DSS. Of interest, each of the four dengue serotypes circulate as more than one genotype.14 The twin survival advantages of neutralisation escape plus the push of antibody-dependent infection-enhancement may underlie the genetic and epidemiological complexity that contributes to periodic DHF/DSS mega-epidemics.15 As is well established for other viruses, the study of escape mutants could lead to a comprehensive understanding of structure and function in the dengue group.16
We thank Jose Bravo for statistical analysis.
<!--start simple-tail=-->References
1. Kouri GP, Guzm?n MG, Bravo JR. Why dengue haemorrhagic fever in Cuba? II: an integral analysis. Trans R Soc Trop Med Hyg 1987; 81: 821-823. Abstract | Full Text | PDF (163 KB) | MEDLINE | CrossRef
2. Valdes L, Guzm?n MG, Kour? G, et al. La epidemiologia del dengue en Cuba en 1997. Rev Panam Salud Publica 1999; 6: 16-25. Abstract | Full Text | PDF (57 KB)
3. Guzm?n MG, Kour? G, Bravo J, Soler M, Vazquez S, Morier L. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiological study. Am J Trop Med Hyg 1990; 42: 179-184(in press).. Abstract | Full Text | PDF (72 KB) | MEDLINE
4. Guzm?n MG, Kour? G, Valdes L, et al. Epidemiological studies on dengue in Santiago de Cuba. Am J Epidemiol 1997;.
5. Ogra PL, Faden HS, Abraham R, Duffy LC, Sun M, Minor RD. Effect of prior immunity on the shedding of virulent revertant virus in the feces after oral immunization with live-attenuated poliovirus vaccines. J Infect Dis 1991; 164: 191-194. Abstract | Full Text | PDF (57 KB) | MEDLINE
6. Minor PD. Attenuation and reversion of the Sabin vaccine strains of poliovirus. Dev Biol Stand 1993; 78: 17-26. Abstract | Full Text | PDF (57 KB) | MEDLINE
7. Huovilainen A, Hovi T, Kinnunen L, Takkinen K, Ferguson M, Minor P. Evolution of poliovirus during an outbreak: sequential type 3 poliovirus isolates from several persons show shifts of neutralization determinants. J Gen Virol 1987; 68: 1373-1379. Abstract | Full Text | PDF (57 KB)
8. Lambkin R, McLain L, Jones SE, Aldridge SL, Dimmock NJ. Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism of antigenic drift. J Gen Virol 1994; 75: 3493-3502. Abstract | Full Text | PDF (57 KB)
10. Kliks S, Nisalak A, Brandt WE, et al. Antibody dependent enhancement of dengue virus in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 1989; 40: 444-451. Abstract | Full Text | PDF (213 KB) | MEDLINE
11. Kliks S, Nisalak A, Brandt WE, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 1988; 38: 411-419. Abstract | Full Text | PDF (213 KB) | MEDLINE
12. Bottiger JB, Jensen IP. Maturation of rubella IgG avidity over time after acute rubella infection. Clin Diagn Virol 1997; 8: 105-111. Abstract | Full Text | PDF (532 KB) | CrossRef
13. Halstead SB. Dengue haemorrhagic fever, a public health problem and a field for research. Bull World Health Organ 1980; 58: 1-21. Abstract | Full Text | PDF (1056 KB) | MEDLINE
14. Rico-Hesse R. Molecular evolution and distribution of dengue viruses types 1 and 2 in nature. Virology 1990; 174: 479-493. Abstract | Full Text | PDF (358 KB) | MEDLINE | CrossRef
15. Fergson N, Anderson R, Gupta S. The effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens. Proc Natl Acad Sci USA 1999; 96: 790-794. Abstract | Full Text | PDF (57 KB) | CrossRef
16. Ketterlinus R, Wiegers K, Dernick R. Revertants of poliovirus escape mutant: new insights into antigenic structures. Virology 1993; 192: 525-533. Abstract | Full Text | PDF (57 KB) | MEDLINE | CrossRef
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