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DENGUE HEMORRHAGIC FEVER CAUSED BY SEQUENTIAL DENGUE 1–3 VIRUS INFECTIONS OVER A LONG TIME INTERVAL: HAVANA EPIDEMIC, 2001–2002

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  • 1 Department of Virology, Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and its Vector, Department of Virology, Pedro Kourí Tropical Medicine Institute, Havana, Cuba; Pediatric Dengue Vaccine Initiative, Bethesda, Maryland

A dengue epidemic caused by dengue virus 3 (DENV-3) occurred in Cuba in 2001–2002. It included cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). We report neutralizing antibody studies on sera from 54 of 78 DHF/DSS patients that provide evidence of infections occurring in the sequence DENV-1 followed by DENV-3. No sera showed infection in the sequence DENV-2 followed by DENV-3. Some sera showed a pattern of infection in the sequence DENV-1 followed by DENV-2 and then DENV-3. However definitive categorization of a tertiary infection was not possible because of broadly reactive antibodies, which could have been raised by infections in the sequence DENV-1 then DENV-3.

INTRODUCTION

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality with an increasing incidence over the last decades. Any of the four dengue virus (DENV-1–4) serotypes can produce an asymptomatic infection or cause two main clinical diseases (DF or DHF/DSS).1 Infection induces life-long protective immunity to the infecting serotype, accompanied by short-term cross-protective immunity against the other viruses.2 DHF/DSS was first recognized as a children’s disease in southeast Asia. However, with its emergence in the Americas, severe disease has been reported both in children and adults. Secondary infection is considered the main individual risk factor for DHF/DSS.311

After the introduction and transmission of DENV-1 in Cuba in 1977–1979, DENV-2 and DENV-3 have resulted large or small DHF/DSS epidemics on three occasions. The first was caused by an Asian genotype of DENV-2 that was introduced in 1981 and produced a major island-wide epidemic with more than 300,000 cases of DF, 10,000 cases of DHF/DSS, and 158 deaths.12 A comprehensive mosquito control program terminated this epidemic in 1981 and kept the nation free of dengue until 1997. In that year, a second Asian genotype (DENV-2) was introduced and caused a small epidemic that was limited to the city of Santiago de Cuba and its surroundings.13 Seroepidemiologic studies showed there were 13,116 primary DENV-2 infections, nearly all of which were asymptomatic. However, all secondary DENV-2 infections resulted in clinical disease: 4,608 cases of DF, 191 cases of DHF/DSS, and 11 deaths, all in DENV-1-immune individuals ≥ 18 years of age.13 In June 2001, a third epidemic occurred with DF and DHF/DSS cases in Havana and its surroundings; this epidemic continued until February 2002. During this outbreak, there were 12,889 cases of DF and 78 cases of DHF/ DSS, and 3 deaths. All but three of the DHF/DSS cases were admitted to the hospital at the Pedro Kouri Tropical Medicine Institute. Nearly all were identified as secondary dengue infections. Polymerase chain reaction or virus isolation identified DENV-3 as the cause in both DF and DHF cases.14

In 2000, a small number of DF cases were reported to health authorities in Havana. Of the 138 DF cases with sera submitted for serologic analysis and virus isolation, 6 DENV-3 and 18 DENV-4 were isolated.1,4 With mosquito control, this outbreak quickly died out. No cases of DHF/DSS were reported during this outbreak.

Population-based studies conducted in 1983 among residents of an area in central Havana detected antibodies to DENV-1 in 40–50% of all age groups of the population; approximately 24% with antibodies to DENV-2 in persons ≤ 45 years of age, and 40–50% in persons ≥ 45 years of age due to one or more dengue epidemics that occurred around the time of World War II.15 On the basis of these studies, it is evident that the 2001–2002 DENV-3 epidemics occurred in a population partially immune to DENV-1 and/or DENV-2. Therefore, it was possible that the DHF/DSS cases may have been caused by the infection sequences DENV-1 then DENV-3, DENV-2 then DENV-3, or DENV-1, DENV-2, DENV-3.

On the basis of neutralizing antibody tests, we report that most of the DHF/DSS cases were persons who were immune to DENV-1 and infected with DENV-3 at an interval of 24 years. No unequivocal evidence was obtained that any DHF/ DSS case occurred in the sequence DENV-2 then DENV-3 either at intervals of 20 years or greater than 60 years. Limits to interpretation of secondary type antibody responses make it difficult to determine if tertiary infections occurred.

MATERIALS AND METHODS

Serum specimens.

Sera included in this study were collected from DHF cases classified according to the Pan American Health Organization/World Health Organization Guidelines of Dengue and Dengue Hemorrhagic Fever for Control and Prevention in the Americas.16 Illness occurred during the Havana DENV-3 epidemic in 2001–2002. All clinical cases were serologically analyzed for IgM and IgG antibodies to DENV using a capture IgM enzyme-linked immunosorbent assay (ELISA) (MAC ELISA)17 and an ELISA inhibition method (EIM).18 A serum sample obtained ≥ 5 days after onset of fever with an IgG antibody titer ≤ 20 was considered a case of primary infection and a serum sample with an IgG antibody titer ≥ 1,280 was considered a case of secondary infection.

Three groups of sera were studied by the EIM and a plaque-reduction neutralization technique: 1) acute-phase serum samples obtained 4–6 consecutive days after onset of fever from six patients (three with DF and three with DHF; 2) acute-phase and late convalescent-phase serum samples from 17 adults with DHF/DSS; and 3) late convalescent-phase serum samples obtained ≥ 16 months after illness from 37 adults with DHF/DSS. Acute-phase serum samples were stored at −20°C. Informed consent was obtained from all subjects included in the study.

Neutralization assay.

Neutralizing antibodies were measured using 50% endpoint plaque-reduction neutralization titers to the four virus serotypes. The method of Morens and others19 with modifications20 was used. Briefly, baby hamster kidney 21 clone 15 cells were grown at 37°C in complete medium (minimum essential medium with Earle’s balance salts) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 10% l-glutamine, and sodium bicarbonate to adjust the medium pH to 7.4–7.8. For antibody titration, 100 μL of diluted (in minimal essential medium) serum was incubated for 1 hour at 37°C with 100 μL of virus dilution calculated to give 10–20 plaque-forming units/50 μL of the final volume of virus-serum mixture. After incubation for one hour at 37°C, 50 μL of virus-serum mixtures was added to the cell suspension in triplicate. The pH during virus adsorption was 7–7.5. After incubation for 4 hours at 37°C in an atmosphere of 4.5% CO2, 0.5 mL of 3% medium viscosity carboxymethyl cellulose in Earle’s minimum essential medium without phenol red plus 10% heat-inactivated fetal bovine serum, 1% (2 mM) l-glutamine, 100 units of penicillin, and 100 μg/mL of streptomycin was added to each sample. Infected cells were incubated for 5–9 days using the same conditions as above depending on the virus serotype (7–9 days for DENV-1 and DENV-3, 5 days for DENV-2, and 6 days for DENV-4). After incubation, samples were rinsed gently under tap water and fixed and stained with a solution of naphthol blue black and acetic acid.

Calculations of 50% endpoint plaque-reduction neutralization titers were made using log probit paper by the method of Russell and others.21 According to previously established criteria,22 acute-phase or late convalescent-phase sera with neutralizing antibody titers ≥ 30 were considered more than one dengue virus infection. Results obtained with late convalescent-phase sera allowed determination of the sequence of infection. Late convalescent-phase sera with neutralizing antibody titers ≥ 30 to two serotypes were considered secondary infections. When the acute-phase serum was monovalent DENV-1 or the late convalescent-phase serum was bivalent DENV-1 and DENV-3, the case was classified as secondary DENV-1–3. When sera had antibodies to DENV-1 + -2 + -3 and antibody titers to DENV-2 were < 30, these cases were classified as consistent with DENV-1–3 secondary dengue infections. Patients whose late convalescent-phase sera had antibodies to DENV-1, -2, and -3 and antibody titers to DENV-2 ≥ 30 were classified as possible tertiary DENV-3 infections. Table 1 shows the virus strains used in this study.

RESULTS

Table 2 shows serologic data from six patients with documented DENV-3 infections who were hospitalized during the 2001–2002 outbreak. These patients were admitted to the hospital early in the course of their disease and blood samples were obtained on several consecutive days. This permitted us to test for neutralizing antibodies found early in infection. Given their specificity to a virus known to have circulated in Cuba at an earlier period and that was different from the infecting virus, it is logical to assume that these antibodies identify the individual’s first dengue virus infection. Three individuals had monovalent antibodies to DENV-1 early in their infections. Two others in this group had heterotypic antibodies on day 3 that resembled antibody patterns observed on day 4 in individuals who earlier had monovalent neutralizing antibodies. The sixth case displayed bivalent antibodies to DENV-1 and DENV-3, a pattern that was found on day 5 in another case in this series (case 5). Assuming that immune response kinetics differ between individuals, the early appearance of antibodies to DENV-1 and DENV-2 is consistent with a normal heterovalent antibody response that follows a DENV-3 infection in persons immune to DENV-1.

Table 3 shows titers in acute-phase sera and late convalescent-phase sera from 17 cases with DHF/DSS. Late convalescent-phase sera showed either no detectable levels or low levels of antibodies to DENV-2. This resulted in 11 cases being classified as secondary DENV-1–3 infections or consistent with secondary DENV-1–3 infections. When DENV-2 neutralizing antibody titers were ≥ 1:30, these cases were classified as possible tertiary DENV-3 infections.

A larger series of 37 late convalescent-phase sera from DHF/DSS cases was also studied. Three showed a primary DENV-3 infection pattern, eight showed a bivalent DENV-1 and DENV-3 infection, 22 showed a pattern consistent with secondary DENV-1–3 infections, and 4 had fairly high antibody titers to DENV-1, -2, and -3. None of these sera suggested infections in the sequence DENV-2 followed by DENV-3. Table 4 shows examples of the results obtained.

DISCUSSION

Epidemiologic studies showed that residents of Havana could have been immune to three of the four dengue viruses prior to the epidemic of DENV-3 in 2001–2002. DENV-1 circulated in 1977–1979. DENV-2 infections occurred in 1981 and sometime during or before the 1940s. A small outbreak of DENV-4 occurred in 2000 that was limited to Havana. This resulted in a situation in which there were a number of immunologic options for acquiring DENV-3 infections during the Havana outbreak: 1) infections in flavivirus-naive persons, 2) infections in DENV-1-immune persons, 3) infections in DENV-2-immune persons from the 1981 epidemic, 4) infections in DENV-2-immune persons from the 1940s, 5) infections in DENV-4-immune persons, 6) infections in DENV-1-immune persons who had also been infected with DENV-2 in 1981, 7) infections in DENV-1 immune persons who had been infected with DENV-2 in the 1940s, or 8) infections in DENV-1-immune persons who were previously infected with DENV-2 and DENV-4. In the studied samples, there was only one person old enough to have been infected in the 1940s, a 65-year-old woman with evidence of DENV-1 and DENV-3 infection. Of the persons with DHF/DSS reported during the 2001–2002 epidemic, only five were more than 60 years of age.

Because all but 3 of the 78 patients with DHF/DSS in the 2001–2002 Havana outbreak were hospitalized at our facility, we had an unique opportunity to study dengue serologic responses in sera collected during hospitalization or in special serum sets to attempt to reconstruct infection histories of these patients. Since neutralizing antibody responses in humans become more type specific with time, we studied neutralizing antibody patterns in sera obtained 16–29 months after clinical dengue illnesses. Eleven of 17 persons studied had antibody patterns consistent with infection in the sequence DENV-1 then DENV-3. Six persons had high titers of antibodies to DENV-1 and DENV-3 and titers ≥ 30 to DENV-2. These results could indicate a past infection with DENV-1 and DENV-2 prior to infection with DENV-3. However, these cases could also represent the upper end of the spectrum of heterovalent antibodies produced after only two dengue virus infections. Similar results were obtained in 34 late convalescent-phase sera from DHF cases with secondary infections. Thus, although tertiary infection resulting in DHF was possible, it can not be unequivocally established, at least in these serum samples.

In 40 late convalescent-phase sera obtained from DF cases infected with DENV-3 during the 2001–2002 outbreak, antibody patterns were observed that clearly indicate prior infection either with DENV-1 alone (60%), DENV-2 alone (15%), or with DENV-1 and DENV-2 followed by DENV-3 (15%). This demonstrates that primary infection with DENV-2 followed by a secondary infection with DENV-3 resulted in overt disease but was limited only to cases with DF (Table 5). These data also suggest that some persons may have had DENV-3 infections after earlier infections with DENV-1 and DENV-2 (tertiary infections). The result of comparison of proportions of DENV-1/DENV-3 infections with persons with DF and DHF was statistically significant (P < 0.005, by chi-square test).

We can make two conclusions from this study. First, some persons with overt DHF were infected with DENV-3 after a DENV-1 infection. Second, it should be noted that these infections, which was spaced by an interval of 24 years, resulted in classic cases of DHF/DSS. This study included 73% of all DHF/DSS cases in Havana. In no instance was there unequivocal evidence of infection of DHF/DSS cases in the sequence DENV-2 then DENV-3, although immunologic evidence of this infection sequence was observed in a few cases of DF. It is possible that some cases of DHF/DSS occurred in persons infected with DENV-3 in a background of immunity to DENV-1 and DENV-2 from either the 1981 epidemic or dengue epidemics during or before the 1940s. Further studies are required to confirm if tertiary infection is associated with DHF/DSS, as well as to understand why DENV-2 primary infections do not sensitize persons to severe DENV-3 infections. Severe disease caused by DENV-3 several decades after primary DENV 1 infection adds to concerns about hazards potentially associated with dengue vaccine–derived immunity.

Table 1

Details of the dengue virus (DENV) strains used in the neutralization assays

SerotypeStrainPlace and year of isolationPassage history*
* P = passage; C636 = Aedesalbopictus cell line; Vero = green monkey kidney cell line; MB = mouse brain; HT = high temperature.
† Kindly supplied by Dr. R. Shope, University of Texas Medical Branch (Galveston, TX).
DENV 1AngolaAngola 1998†4P C636 2P Vero 1P C636
DENV 2A15/1981Cuba 19814 P MB 4P C636
DENV 3116/00Cuba 20003P C636
DENV 4DominicaDominica 1981†1P C636 6P C636 HT
Table 2

Virologic and serologic results in six patients with overt dengue virus (DENV) infections*

PRNT50 reciprocal titer
Case no.Day of onsetVirus isolationPCRIgM†IgG‡Day 1Day 2Day 3Day 4Pattern in early acute-phase sera
* PRNT = plaque reduction neutralization test; PCR = polymerase chain reaction; DHF = dengue hemorrhagic fever; DF = dengue fever.
† IgM optical density ratio.
‡ Reciprocal antibody titer.
1 (DHF)3DENV 3DENV 30.954042< 10< 10< 10Monovalent DENV 1 antibodies
410.95,1201001904233
52.95,1201304412017
613.620,4801,7005,6003,30060
7440,960760960740120
212.820,480390780760210
2 (DHF)3DENV 3DENV 31.811603935< 10< 10Bivalent DENV 1 and 2 antibodies
4DENV 3DENV 32.12,56039074< 10< 10
516.320,4802,50042033022
615.82,560120340Es< 10
716.840,9606204901,200< 10
816.440,96027074029037
2155,1203,60041030060
3 (DHF)3DENV 3DENV 31.084058150< 10< 10Bivalent DENV 1 and 2 antibodies
4DENV 3DENV 31.44320180110< 10< 10
513.820,4801,100410470< 10
66.95,120680760820< 10
77.720,4803,3005402,700< 10
87.910,2402,0005401,700< 10
215.210,24050033060< 10
4 (DF)3DENV 3DENV 31.444011< 10< 10< 10Monovalent DENV 1 antibodies
4DENV 3DENV 35.711,280264218< 10
511.15,1209226033019
614.110,24042056033086
713.540,960450580510180
813.220,4804,8001,0001,800140
5 (DF)3DENV 3DENV 31.672022< 10< 10< 10Monovalent DENV 1 antibodies
4DENV 3DENV 31.78054< 10< 10< 10
53.1764010012200< 10
672,56010023062012
79.810,2401,3002402,600< 10
810.420,4801504201,000< 10
215.5220,4802107401,800< 10
6 (DF)3DENV 3DENV 33.433206010130< 10PRNT antibodies predominantly against DENV 1 and 3
4DENV 3DENV 38.91,28049064540< 10
513.810,240540700640< 10
615.120,4801,200390370< 10
715.140,9601201,2001,000< 10
814.920,4803505401,600< 10
Table 3

Dengue virus (DENV) 1–4 PRNT50 antibodies in acute-phase and late-convalescent sera obtained from 17 adult patients hospitalized with DHF/DSS in Havana, Cuba, 2001–2002*

PRNT50 reciprocal titer
Case no.Date of onsetTime after onsetDENV 1DENV 2DENV 3DENV 4Interpretation
* PRNT = plaque reduction neutralization test; DHF = dengue hemorrhagic fever; DSS = dengue shock syndrome.
325229/23/013 days
 16 months> 1,000
 24013
 22> 1,000
 6850
 < 10Consistent with DENV 1–3 secondary infection
3195111/17/014 days
 17 months> 1,000
 > 1,000200
 1270
 25310
 < 10Consistent with DENV 1–3 secondary infection
3406412/01/014 days
 17 months170
 160< 10
 < 1010
 110< 10
 < 10DENV 1–3 secondary infection
3406311/30/015 days
 17 months800
 41039
 17100
 6017
 < 10Consistent with DENV 1–3 secondary infection
3469812/05/015 days
 16 months100
 400490
 70> 1,000
 700180
 < 10DENV 1 + 2–3 possible tertiary infection??
3534512/06/015 days
 17 months> 1,000
 360210
 42820
 9098
 < 10DENV 1 + 2–3 possible tertiary infection??
3575312/07/015 days
 17 months> 1,000
 400170
 21> 1,000
 300< 10
 < 10Consistent with DENV 1–3 secondary infection
3575612/08/015 days
 17 months> 1,000
 92< 10
 10> 1,000
 170< 10
 < 10DENV 1–3 secondary infection
3057412/31/015 days
 17 months540
 16047
 16430
 11044
 < 10Consistent with DENV 1–3 secondary infection
3061911/17/015 days
 17 months> 1,000
 > 1,000> 1,000
 80> 1,000
 250240
 12DENV 1 + 2–3 possible tertiary infection???
10671/03/025 days
 17 months> 1,000
 14019
 < 10420
 16090
 < 10DENV 1–3 secondary infection
3365911/30/016 days
 17 months> 1,000
 25068
 12150
 4252
 < 10Consistent with DENV 1–3 secondary infection
3599912/09/016 days
 17 months10
 780< 10
 15< 10
 88< 10
 < 10DENV 1–3 secondary infection
3773312/06/016 days
 17 months> 1,000
 560450
 35> 1,000
 150220
 < 10DENV 1 + 2–3 possible tertiary infection??
3057612/07/016 days
 19 months> 1,000
 130140
 44> 1,000
 15< 10
 < 10DENV 1 + 2–3 possible tertiary infection??
2996011/04/017 days
 18 months> 1,000
 25080
 30490
 26098
 < 10DENV 1 + 2–3 possible tertiary infection??
3668712/13/017 days
 19 months> 1,000
 170< 10
 < 1064
 100< 10
 < 10DENV 1–3 secondary infection
Table 4

Dengue virus (DENV) 1–4 PRNT50 antibodies in selected late-convalescent sera from 10 adult patients hospitalized with DHF/DSS in Havana, Cuba, 2001–2002*

PRNT50 reciprocal titer
Case no.Date of onsetTime after onsetDENV 1DENV 2DENV 3DENV 4Interpretation
* For definitions of abbreviations, see Table 3.
325219/23/0115 months2402268< 10Consistent with DENV 1–3 secondary infection
3090411/10/0117 months33010340< 10Consistent with DENV 1–3 secondary infection
1553309/05/0120 months110< 1076< 10DENV 1–3 secondary infection
184969/19/0121 months2,0002780< 10Consistent with DENV 1–3 secondary infection
14710/16/0120 months3601166< 10Consistent with DENV 1–3 secondary infection
3332511/24/0121 months800< 10230< 10DENV 1–3 secondary infection
4439/21/0129 months1704090< 10DENV 1 + 2–3 tertiary infection???
3385111/28/0118 months7405226018DENV 1 + 2–3 tertiary infection???
3771012/21/0118 months1,0001005814DENV 1 + 2–3 tertiary infection???
3269011/21/0127 months64040170< 10DENV 1 + 2–3 tertiary infection???
Table 5

Dengue virus (DENV) 1–4 PRNT50 antibodies in selected late-convalescent sera from 10 adult patients hospitalized with DF in Havana, Cuba, 2001–2002*

PRNT50 reciprocal titer
Case no.Time after onsetDENV 1DENV 2DENV 3DENV 4Interpretation
* PRNT = plaque reduction neutralization test; DF = dengue fever.
1> 20 months1202356< 10Consistent with DENV 1–3 secondary infection
2> 20 months25016350< 10Consistent with DENV 1–3 secondary infection
3> 20 months2402480< 10Consistent with DENV 1–3 secondary infection
4> 20 months184560< 10Consistent with DENV 2–3 secondary infection
5> 20 months< 105480< 10Consistent with DENV 2–3 secondary infection
6> 20 months1878360< 10Consistent with DENV 2–3 secondary infection
7> 20 months273984< 10Consistent with DENV 2–3 secondary infection
8> 20 months13016060< 10DENV 1 + 2–3 possible tertiary infection???
9> 20 months27078150< 10DENV 1 + 2–3 possible tertiary infection???
10> 20 months250140390< 10DENV 1 + 2–3 possible tertiary infection???

*

Address correspondence to Maria G. Guzman, Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and its Vector, Department of Virology, Pedro Kourí Tropical Medicine Institute, Autopista Novia del Mediodía y Carretera Central km 61/2, La Lisa, PO Box 601, Marianao 13, Havana, Cuba. E-mail: lupe@ipk.sld.cu

Authors’ addresses: Mayling Alvarez, Rosemari Rodriguez-Roche, Lídice Bernardo, Susana Vázquez, Luis Morier, Daniel Gonzalez, Osvaldo Castro, Gustavo Kouri, and Maria G. Guzman, Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and its Vector, Department of Virology, Pedro Kourí Tropical Medicine Institute, Autopista Novia del Mediodía y Carretera Central km 61/2, La Lisa, PO Box 601, Mariano 13, Havana, Cuba. Telephone: 53-7-202-0450, Fax: 53-7-204-6051, E-mail: lupe@ipk.sld.cu. Scott B. Halstead, Pediatric Dengue Vaccine Initiative, Bethesda, MD 20852.

REFERENCES

  • 1

    Guzman MG, Kouri G, 2002. Dengue: an update. Lancet Infect Dis 2 :33–42.

  • 2

    Sabin AB, 1952. Research on dengue during World War II. Am J Trop Med Hyg 1 :30–50.

  • 3

    Nimmannitya S, Thisyakorn U, Hemsrichart V, 1987. Dengue haemorrhagic fever with unusual manifestations. Southeast Asian J Trop Med Public Health 18 :398–406.

    • Search Google Scholar
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