Natural History of Dengue ��� JID 2007:195 (1 April) ��� 1007 M A J O R A R T I C L E Natural History of Dengue Virus (DENV)���1 and DENV-4 Infections: Reanalysis of Classic Studies Hiroshi Nishiura1,2 and Scott B. Halstead3,4 1Department of Medical Biometry, University of Tu ��bingen, Tu ��bingen, Germany 2Research Center for Tropical Infectious Diseases, Nagasaki University Institute of Tropical Medicine, Nagasaki, Japan 3Department of Preventive Medicine and Biometrics, Uniformed Services University of Health Sciences, Bethesda, Maryland 4Pediatric Dengue Vaccine Initiative, International Vaccine Institute, Seoul, Korea Background. The natural history of wild-type dengue virus (DENV) infections of humans, including incubation and infectious periods, requires further study. Methods. Two experimental studies in the Philippines of DENV-4 (1924���1925) and DENV-1 (1929���1930) were reexamined. The intrinsic incubation periods were fitted to log-normal distribution using the maximum likelihood method, and the infectious and extrinsic incubation periods were assessed by proportions of successful transmissions causing clinically apparent dengue. Correlations between the intrinsic incubation period and other variables and univariate associations between clinical severity and serotype were also examined. Results. incubation periods were and days for DENV-4 and DENV-1, re- Mean SD 6.0 1.4 5.7 1.5 spectively. Significant negative correlations were observed between the incubation period and duration of fever ( and 0.33). Even 1 and 2 days before the onset of fever, 80.0% (95% confidence interval [CI], 44.9%��� r p 0.43 100%) and 25.0% (CI, 0%���67.4%) of biting experiments caused clinically apparent dengue. DENV-1 infections resulted in a significantly longer duration of fever than DENV-4 infections ( P ! .01). Conclusions. Incubation period was negatively correlated with disease severity, potentially reflecting a dose- response mechanism. The historical data provided useful details concerning serotype differences in the natural history of primary DENV infections. Dengue fever (DF) is a vectorborne disease caused by 4 closely related dengue viruses (DENV 1���4) [1, 2]. DF is distributed in most tropical and subtropical areas, where Aedes aegypti and/or A. albopictus are abundant [3]. Infection with DENV can also cause dengue hem- orrhagic fever (DHF), a syndrome characterized by in- creased vascular permeability, plasma leakage, hypo- volemia, and shock [4, 5]. Although the pathogenesis of DHF is not fully understood, several risks have been reported: secondary infection with heterologous strains [6, 7], primary infection in infants born to dengue- immune mothers [8], differing virulence of the strain [9], and differing human susceptibility according to Received 1 September 2006 accepted 22 October 2006 electronically published 15 February 2007. Potential conflicts of interest: none reported. Financial support: Banyu Life Science Foundation International (to H.N.). Reprints or correspondence: Dr. Hiroshi Nishiura, Dept. of Medical Biometry, University of Tu ��bingen, Westbahnhofstr. 55, Tu ��bingen, D-72070, Germany (nishiura .hiroshi@uni-tuebingen.de). The Journal of Infectious Diseases 2007 195:1007���13 2007 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2007/19507-0014$15.00 DOI: 10.1086/511825 race or genetic factors [10, 11]. Because DENV infec- tions are often clinically inapparent, it is difficult to clarify the epidemiology of DF and dengue patho- physiology without virological support [12, 13]. The natural history of DF involves the time from infection to onset and recovery from disease. However, although the clinical symptoms of DF and DHF have been relatively well described [14, 15], the incubation and infectious periods for different DENVs have yet to be examined in detail. This is partly attributable to the difficulty in identifying the time of infection after a mosquito bite. Detailed information on the above may assist in quantification of the transmission potentialand contribute to an understanding of the epidemiologic processes of this disease [16]. Before World War II, a number of studies were con- ducted to prove the mode of transmission and further basic knowledge on dengue. These studies were per- formed by US Army commissions [17] and by Austra- lian [18] and Japanese [19, 20] researchers. Successful studies include those of Ashburn and Craig [21] and Cleland, Bradley, and McDonald [18], the former of which was recently reviewed in the Journal of Infectious

1008 ��� JID 2007:195 (1 April) ��� Nishiura and Halstead Figure 1. Frequency distributions of the intrinsic incubation periods of dengue fever cases. Distributions of the incubation periods of dengue fever with dengue virus (DENV)���4 (A and DENV-1 (C infections based on maximum likelihood estimations assuming log-normal distributions. n p 47) n p 80) Observed (bars) and predicted (solid lines) frequencies are shown. The whiskers extend from the ends of each box to the outermost data points that fall within the distances computed. The bracket along the edge of each box identifies the shortest half, which represents the highest density (50%) of the observations. Log-normal quantile plots of the incubation periods of DENV-4 (B) and DENV-1 (D) infections are also given. The diagonal reference lines indicate the line of fit, and the 2 dashed lines denote the 95% confidence interval (0.001���0.99). Diseases [22]. In the present study, we reanalyzed data from 2 human volunteer experiments in which the DENV etiology was known. METHODS Brief historical background. Both dengue studies were per- formed in the Philippines and involved large numbers of hu- man volunteers recruited from US Army personnel. The first study, conducted by Joseph Franklin Siler, Milton Weston Hall, and Arthur Parker Hitchens took place in 1924���1925, whereas the latter was organized by James Stevens Simmons, Joseph Harold St. John, and Francois Hiie Kari Reynolds in 1929���1930 (the latter study is available at: http://plaza.umin.ac.ip/infepi/ simmons1.pdf). Both studies were originally published in the Philippine Journal of Science [23, 24] and were reprinted with appendices by the Bureau of Printing, Manila [25, 26]. In 1971��� 1972, Halstead obtained blood samples from 4 and 5 vol- unteers involved in each study and demonstrated specific neu- tralizing antibodies to DENV-4 and DENV-1, respectively [27]. Following these experimental studies of dengue, J. F. Siler (1875���1960) reported the efficacy of antityphoid vac- cines [28], whereas J. S. Simmons (1890���1954) contributed to the advancement of epidemiology during and after World War II, as chief of the Preventive Medicine Service, Office of the Surgeon General and, thereafter, as dean and professor of the Harvard School of Public Health [29]. The transmission of DENV by arthropods was first dem- onstrated by Graham, who claimed that Culex quinquefasciatus was the vector [30]. This finding was supported by Ashburn and Craig [21, 22], but not by the Australian researchers Ban- croft [31] and Cleland et al. [18], who suggested instead that A. aegypti was the major vector, with very different implications for mosquito control of dengue transmission. In the studies of Ashburn and Craig, mosquitoes were refed on susceptible vol- unteers within 24 h after viremic blood meals, too short a period for transmission of the virus [21, 22]. Given this knowl- edge, Siler et al. reexamined DENV transmission by A. aegypti at different extrinsic incubation and human infectious periods