Rapid subtyping of dengue viruses by restriction site-specific (RSS)-PCR

Dengue is a major public health problem worldwide. It is caused by four dengue virus serotypes, each further divided into distinct genetic subtypes. Strain typing is important for understanding the epidemiology and viral factors associated with disease transmission. However, most of the existing subtyping methods are expensive and technically unwieldy for timely, practical applications in developing countries. Here we describe a simple, rapid, PCR-based subtyping method, restriction site-specific (RSS)-PCR, which we used to analyze dengue virus serotypes 2 and 3. For each serotype, four primers targeted to sequences spanning polymorphic endonuclease restriction sites in the envelope gene were used to reverse transcribe and amplify viral RNA. These RT-PCR products generated distinct electrophoretic band patterns for different strains. Analysis of 73 dengue-2 strains and 54 dengue-3 strains representing a broad geographic distribution over several decades revealed that the RSS-PCR fingerprints reproducibly fell into 7 and 3 groups, respectively. These groups correlated well with previous phylogenetic classifications. This one-step assay should be widely accessible and allow more detailed epidemiologic investigations in dengue-endemic countries. This novel PCR approach to subtyping organisms based on restriction site polymorphisms should be applicable to other pathogens.