Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene (C-prM) protocol (R. S. Lanciotti, C. H. Calisher, D. J. Gubler, G.-J. Chang, A. V. Vorndam, J. Clin. Microbiol. 30:545-551, 1992). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3′ noncoding region (3′NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3′NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and 3′NC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the 3′NC (91%) protocol.

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Airport fever screening in Taiwan, July 2003–June 2004, identified 40 confirmed dengue cases. Results obtained by capture immunoglobulin (Ig) M and IgG enzyme-linked immunoassay, real time 1-step polymerase chain reaction, and virus isolation showed that 33 (82.5%) of 40 patients were viremic. Airport fever screening can thus quickly identify imported dengue cases.

We present our surveillance results on imported dengue cases in Taiwan during 2008–2010. A total of 734 imported dengue patients were identified. The travelers were arriving from 18 countries, including Southeast Asia, the Indian subcontinent, South Pacific islands, and Latin America. Gene sequences from 358 dengue virus (DENV) isolates were used to perform phylogenetic analyses, thus, providing an updated view of the geographic distribution and dynamic transmission of DENV strains circulating in these countries. Our results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant DENV strains circulating in Southeast Asian countries. The DENV-3 Genotype III strain was found to be newly emerging in several Southeast Asian countries, however, the Asian genotype 2 and the Asian/American genotype of DENV-2 strains appeared to be less prevalent in Southeast Asia. Furthermore, imported dengue viruses are representative of the overall patterns of serotype/genotype frequencies of dengue outbreaks that occurred in Taiwan.

We presented our surveillance results on imported dengue cases in Taiwan during 2003–2007. A total of 542 imported dengue patients were identified. The travelers were infected in 17 countries in Southeast Asia, the Indian subcontinent, East African islands, South Pacific islands, and Central America. Most of these imported cases were infected in Southeast Asian countries. Phylogenetic analyses were conducted to examine 288 imported dengue virus (DENV) strains introduced from 13 countries. The results provide an updated view on the geographic distribution and dynamic transmission of epidemic DENV stains circulated in Southeast Asian countries. Although the geographic distributions of genotypes of DENV-3 isolated from Southeast Asian countries remain unchanged, the introductions and local expansions of epidemic DENV-1, DENV-2, and DENV-4 strains into new areas in Asia were observed. These findings highlight the importance to strengthen laboratory-based dengue surveillance for better understanding of transmission dynamics and molecular evolution of DENVs.

We present the results of laboratory-based dengue surveillance in Taiwan for 2005. A phylogenetic study showed that multiple dengue epidemics were caused by three different imported dengue virus (DENV) strains. A strain of DENV-3 (genotype I) imported from the Philippines first appeared in the southern part of Kaohsiung City and later spread to Kaohsiung County from August to December, which resulted in 77 cases of dengue. Another strain of DENV-3 (genotype II) imported from Vietnam first appeared in the central part of Kaohsiung City and later spread to Kaohsiung County from September to December, which resulted in 35 cases of dengue. A strain of DENV-2 (American/ Asian genotype) imported from Vietnam first appeared in Tainan City and later spread to Kaohsiung City/County from October to December, which resulted in 60 cases of dengue. This study provides molecular epidemiologic evidence that most dengue in Taiwan is caused by imported strains of the virus.