Genetic Relatedness of Dengue Viruses in Key West, Florida, USA, 2009–2010

Jorge L. Munoz Jordan, Gilberto A. Santiago, Harold Margolis, and Lillian Stark

Author affiliations: Centers for Disease Control and Prevention, San Juan, Puerto Rico (J.L. Munoz Jordan, G.A. Santiago, H. Margolis); Florida Department of Health, Tampa, Florida, USA (L. Stark)

Abstract

Sequencing of dengue virus type 1 (DENV-1) strains isolated in Key West/Monroe County, Florida, indicate endemic transmission for >2 years of a distinct and predominant sublineage of the American–African genotype. DENV-1 strains isolated elsewhere in Florida grouped within a separate Central American lineage. Findings indicate endemic transmission of DENV into the continental United States.

Dengue is the most common mosquito-borne viral disease; cases have been reported from ≈100 countries, and there are indications of increased incidence and severity worldwide (1). The United States has reported year-round transmission of dengue virus (DENV) in Puerto Rico, the US Virgin Islands, and American Samoa and occasional transmission along the Texas–Mexico border. In the continental United States, DENV is the most frequent cause of febrile illness among travelers returning from the Caribbean, South America, and Asia (2,3). These frequent introductions of dengue infections and the increased presence of vectors (i.e., Aedes aegypti and Ae. albopictus mosquitoes) in many US regions may portend the reintroduction and extended transmission of DENV into the continental United States.
In September 2009, the Florida Department of Health (FDOH) and the Centers for Disease Control and Prevention (San Juan, Puerto Rico) investigated a case of DENV type 1 (DENV-1) infection in a person (index patient) who, as confirmed by reverse transcription PCR (RT-PCR), acquired the virus while traveling to Key West in Monroe County, Florida, USA. DENV-1 infections were subsequently confirmed in 2 Monroe County residents without histories of recent travel. In addition, among 13 other cases in the county that were identified by serologic methods, 2 were confirmed as DENV-1 infections (4). Thus, a total of 5 DENV-1 cases were confirmed in Key West during 2009, and ≈5% of the serosurveyed population in Key West had evidence of recent DENV infection (4,5). In 2010, additional dengue cases from Monroe County were reported, and DENV-1 was isolated from a mosquito pool (6) and a blood donor from Key West (7); isolates from the mosquito pool and blood donor appeared to be phylogenetically related (7). This study determined the genetic relatedness of the DENV-1 isolates from dengue patients in Key West and 4 other Florida counties during 2009–2010, including the blood donor and mosquito isolates.

The Study

During 2009–2010, serum samples from patients with suspected dengue were received by the FDOH for dengue diagnostic testing; the samples came from 16 of Florida’s 67counties. All samples were tested by using DENV serotype–specific, real-time RT-PCR (8) and IgM anti-DENV ELISA (9). Samples with highly positive RT-PCR results were spread onto cultured Ae. albopictus C6/36 cells, and the presence of virus and genome were confirmed by immunofluorescence (10) and RT-PCR, respectively (11). Isolates were further propagated and viral RNA was extracted from culture supernatants by using the Universal BioRobot System (QIAGEN, Valencia, CA, USA). The envelope glycoprotein (E) gene was amplified (Technical Appendix

[PDF - 27 KB - 2 pages]), and the E gene open-reading frame (1,485 bp) was sequenced. All sequences were submitted to GenBank; accession numbers are shown in Technical Appendix

[PDF - 27 KB - 2 pages]. Multiple sequence alignments were performed by using the MUSCLE module available in MEGA 5 (www.megasoftware.net). Evolutionary history was inferred by using maximum likelihood and phylogenetic trees constructed by using neighbor-joining methods. Evolutionary distances were computed, and several E gene sequences from GenBank were included in the phylogenetic tree to support tree topology by genotype (Technical Appendix

[PDF - 27 KB - 2 pages]).