DENGUE DENV-1, DENV-2, and DENV-3 circulated in Puerto Rico at the same time (8). In 2000, during an interepidemic peri- od, 1 isolate of DENV-3 was reported in Saint Martin (http://www.carec.org/annrep00/index.html), but to our knowledge its sequence was not determined. During the last 3 years, no further DENV isolates have been reported on the island. However, in December 2003, an outbreak of febrile illness was reported after heavy rains. An estimated 108 persons were infected, and DENV was suspected to be the causative agent by Saint Martin Lepers Laboratory (9). During this period, blood samples collected at early and late stages of infections were examined at the Tropical Medicine Institute of the French Armed Forces Medical Service (Institut de Médecine Tropicale du Service de Santé des Armées [IMTSSA]) tropical virology laboratory in Marseille to identify the etiologic agent. DENV-3 was isolated on C6/36 cells (Aedes albopictus). Partial genom- ic sequences were determined for each isolated virus to evaluate the origin and diversity of its spread. This approach should help clarify the geographic distribution of isolates in the Caribbean islands as well as the virus’ circu- lation in and transmission to humans. Materials and Methods The Outbreak In December 2003, an outbreak of febrile illness was reported (9) after heavy rainfalls in Saint Martin (29,000 inhabitants in the French part and 36,000 in the Dutch part). The outbreak seemed to start first in the northeastern part of the island (“Baie orientale” and “Cul de sac” neigh- borhoods), then spread through the island (Figure 1). The temporal distribution of the outbreak is shown in Figure 2 (10). We used a commercial enzyme-linked immunosor- bent assay (ELISA) (Eurobio, Courtaboeuf, France) in Saint Martin Lepers Laboratory to determine that, from October 2003 to April 2004, a total of 108 persons were infected; a DENV was suspected as the etiologic agent (10). Twelve persons manifesting thrombocytopenia with- out hemorrhagic signs were hospitalized (9). Collection and Transport of Samples Sera from 26 patients with dengue-like syndromes were collected at a local laboratory and transported (by air, at 4°C) to the IMTSSA laboratory in Marseille for serologic diagnosis and etiologic agent identification. All these sam- ples were collected from December 2003 to January 2004, the peak of the outbreak. Serologic Diagnosis In-house immunoglobulin (Ig) M-capture enzyme immunoassays (MAC-ELISA) and IgG sandwich ELISA were used to detect serum IgM and IgG antibodies to RESEARCH Toscana virus, dengue viruses, West Nile virus, and St. Louis encephalitis virus. Briefly, IgM antibodies were captured with rabbit anti-human IgM antibodies (Interchim, Montluçon, France). Toscana, dengue, West Nile, and Saint Louis antigens, prepared on Vero cells and inactivated by betapropiolactone (Sigma-Aldrich, St Quentin Fallavier, France), were added. Specific binding was demonstrated by using a Toscana virus, dengue viruses, West Nile virus, and St. Louis encephalitis virus mouse hyperimmune ascitic fluid virus (11) and a goat anti-mouse peroxidase-labeled conjugate (Interchim). For IgG detection, antibodies to Toscana virus, dengue viruses, West Nile virus, and St. Louis encephalitis virus were captured by goat anti-mouse IgG antibodies; viral antigens were followed by test sera; and specific binding was demonstrated by using a peroxi- dase-labeled goat anti-human IgG conjugate. Serum samples were considered positive if the optical density at 450 nm was >3-fold the mean of negative sera with an OPTImax spectrophotometer (Molecular Devices, Saint Gregoire, France). Virus Isolation and Propagation Work with the infectious virus was carried out in a biosafety level 3 laboratory. Virus isolation was attempted when the first sample could be collected <5 days after onset of illness and when enough serum was collected for serologic analysis (12). DENV-3 strains (see Online Appendix, available from http://www.cdc.gov/ncidod/ EID/vol11no05/04-0959_app.htm) were all isolated from leukocytes of patients. Leukocytes of the samples were directly coincubated with C6/36 cells (Ae. albopictus) grown at 28°C in Leibowitz’s L15 medium (BioWhittaker Europe, Verniers, Belgium) supplemented with 1% L-glu- tamine and 2% tryptose phosphate broth. Fetal bovine serum (5% final) was added 1 h later. Supernatants were collected on day 5 after infection. Figure 2. Temporal distribution of the outbreak (adapted from [10]). Bars indicate the suspected dengue patients observed by sentinel physicians; dark squares (curve) indicate laboratory-confirmed cases. 758 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 5, May 2005