TEXT

Top Abstract Text References

Dengue viruses are single-stranded, enveloped RNA flaviviruses which are traditionally classified into four serotypes, designated dengue-1, -2, -3, and -4, based on antigenic characteristics (2, 19). Numerous methods, including RNase T₁ oligonucleotide fingerprinting, restriction enzyme analysis, and nucleotide sequencing of different genomic fragments, have demonstrated strain variation within each dengue serotype, dividing them into distinct genetic subtypes (4, 5, 10, 11, 12, 13, 16, 17, 18). Dengue virus classification into subtypes is useful for studying the global distribution and movement of dengue serotypes, which contributes to the identification of viral factors that influence disease severity and risk factors associated with the transmission of particular strains.

Accurate characterization of strain difference usually requires labor-intensive typing procedures, which are difficult to perform in a timely manner during epidemic periods. Recently, a new PCR-based approach to rapidly subtype dengue viruses was developed (8) and consists of a single reverse transcriptase PCR (RT-PCR) amplification using four primers that target regions spanning polymorphic endonuclease restriction sites within the envelope (E) gene. This method, called restriction site-specific PCR (RSS-PCR), is simple, rapid, requires minimal laboratory equipment, and uses widely available reagents. The successful results obtained with dengue-2 and dengue-3 led us to develop the RSS-PCR method for dengue-1 and dengue-4 strains.

Dengue-1 (Table 1) and dengue-4 (Table 2) strains representing a broad geographical distribution were obtained from existing collections; Brazilian and Nicaraguan viruses were isolated from sera by inoculation into the Aedes albopictus cell line C6/36 (9) and were identified by immunofluorescence using type-specific monoclonal antibodies (6). Viral seeds were propagated once in C6/36 cells grown in Leibovitz-15 or minimal essential medium (Gibco BRL, Grand Island, N.Y.) containing 10% fetal bovine serum. Primers were designed based on the sequence around polymorphic restriction sites in the E gene region of dengue-1 and dengue-4 as described previously (8). The sequences and genomic positions of primers RSS9 to RSS12 (dengue-1) and RSS21 to RSS24 (dengue-4) are listed in Table 3.

Viral RNAs were extracted from the supernatants of infected cells using a QIAamp Viral RNA Mini Kit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's protocol or by lysis with guanidine isothiocyanate, extraction with organic solvents, and ethanol precipitation (7). The reaction mixture and electrophoresis conditions were as described previously (8), except that 25-µl reaction volumes were used. Briefly, 2.5 µl of viral RNA was added to 22.5 µl of an RT-PCR mixture consisting of 50 mM potassium chloride, 10 mM Tris (pH 8.5), 0.01% gelatin, 200 µM concentrations of each of the four deoxynucleoside triphosphates, 1.5 mM magnesium chloride, 30 mM tetramethylammonium chloride, 0.5 M betaine, 5 mM dithiothreitol, 0.5 µM concentrations of each of four RSS-PCR primers (RSS9 to -12 for dengue-1 and RSS21 to -24 for dengue-4), 0.025 U of RT RAV-2 (Amersham Corp., Arlington Heights, Ill.) per µl and 0.025 U of Taq DNA polymerase (AmpliTaq; Perkin-Elmer Corp., Foster City, Calif.) per µl. Reverse transcription was conducted at 42°C for 60 min, followed directly by 30 amplification cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 5 min. Amplification was conducted with 0.5-ml tubes (USA Scientific, Ocala, Fla.) in a model PTC-200 thermocycler (MJ Research, Inc., Watertown, Mass.).

We selected sets of strains in our collection that represented each of three dengue-1 genotypes previously described by E-NS1 sequence pairwise comparison (13), with one set including strains from Southeast Asia and the South Pacific, another containing viruses from Thailand and Taiwan, and a third containing isolates from the Americas, Africa, and Southeast Asia. The bands generated by the RSS-PCR assay using primers RSS9 to -12 showed distinct patterns for American and Asian strains. Figure 1 shows representative examples of each RSS-PCR pattern for dengue-1 viruses, along with a schematic diagram summarizing the results. The first group (type A), which includes viruses from the Philippines (1983 to 1984), Indonesia (1976 to 1978), Thailand (1975, 1979, and 1980), and the western Pacific (1980), was divided into two subgroups (A1 and A2), depending on the presence of an ~200-bp fragment. The second group (type B) contains isolates from Thailand (1973, 1974, and 1980), and the third group (type C) is composed of strains from the Americas, Africa, and Sri Lanka. These RSS primers are specific for dengue-1, as dengue-2, -3, and -4 did not generate amplified products in this assay (Fig. 1, lanes 1 to 3).

View larger version (37K): [in this window] [in a new window]   FIG. 1.   RSS-PCR patterns of dengue-1 strains of different geographic origins. (A) Agarose gel electrophoresis of RSS-PCR products. Lane 1, dengue-2 Brazil 1998 (64022); lane 2, dengue-3 Indonesia 1989 (430); lane 3, dengue-4 Puerto Rico 1986 (081); lane M, 100-bp ladder (Gibco BRL); lanes 4 to 16, dengue-1 [lane 4, Indonesia 1977 (407-1); lane 5, Philippines 1984 (901); lane 6, Thailand 1979 (213); lane 7, Thailand 1973 (735); lane 8, Thailand 1974 (106); lane 9, Western Pacific 1980; lane 10, Nigeria 1985; lane 11, Sri Lanka 1985; lane 12, Mexico 1982 (086); lane 13, Brazil 1986 (543); lane 14, Brazil RJ 1995 (49217); lane 15, Brazil ES 1998 (60548); lane 16, Brazil BA 1996 (57291)]. Lanes 4, 5, and 9, pattern A1; lane 6, pattern A2; lanes 7 to 8, pattern B; lanes 10 to 16, pattern C. (B) Schematic diagram representing the different RSS-PCR patterns.

As with dengue-1, several primer sets were tested with dengue-4 strains from our collection that matched strains previously classified by sequence analysis of the E gene (10) with respect to location and date of isolation. The best results were obtained with primers RSS21 to -24 (Table 3), which were used to analyze the rest of our dengue-4 strains. Two patterns were generated, as shown in Fig. 2 and Table 2, one (type A) representing strains from Thailand (1977 to 1988) and the other (type B) representing viruses isolated in Mexico and the Caribbean. Type A was divided into two subgroups due to variation in one of the amplified fragments from a Thai isolate from 1984. Again, no product was obtained with the other three serotypes (Fig. 2).

View larger version (35K): [in this window] [in a new window]   FIG. 2.   RSS-PCR patterns of dengue-4 strains of different temporal and geographic origins. (A) Agarose gel electrophoresis of RSS-PCR products. Lane 1, dengue-1 Brazil 1986 (543); lane 2, dengue-2 Brazil 1998 (63731); lane 3, dengue-3 Indonesia 1989 (430); lane M, 100-bp ladder (Gibco BRL); lanes 4 to 9, dengue-4 [lane 4, Thailand 1985 (789); lane 5, Thailand 1977 (377); lane 6, Thailand 1988 (280); lane 7, Puerto Rico 1986 (437); lane 8, Mexico 1984 (440); lane 9, Nicaragua 1999 (703)]. Lane 4, pattern A1; lanes 5 to 6, pattern A2; lanes 7 to 9, pattern C. (B) Schematic diagram representing the different RSS-PCR patterns. The dotted line designates bands that display sample-to-sample variation.

The RSS-PCR method for dengue-1 and -4 fulfills the requisites of a molecular typing assay: the primers were specific to the serotype to which they were developed, the patterns were stable over time, repeated amplification of the same specimens reproducibly yielded the same results, and geographic and temporal clustering was observed. In addition, in areas with extensive dengue transmission, such as Thailand, the cocirculation of two distinct subtypes and of genetic variants within the same subtype was observed, which is consistent with previous reports (13, 15).

The dengue-1 RSS-PCR results, categorized according to country and year of isolation, are essentially the same as the sequence analysis results showing geographic and temporal clustering. A phylogenetic analysis of 40 dengue-1 strains from different geographic areas based on a 240-nt region from the E-NS1 junction defined three main genotypes and two additional ones, each represented by a single virus isolate (13). Similarly, another study comparing the sequences of a 179-nt region of the E genes of 35 dengue-1 isolates yielded three genotypes (4). The largest genotypic group in both analyses consisted of dengue-1 strains from the Americas, Africa, and Southeast Asia, and it corresponds to our RSS-PCR type C. The second genotype, containing viruses from the Philippines, Indonesia, Thailand, and the South Pacific, coincides with RSS-PCR type A, and the third group, consisting of Thai and Taiwanese viruses, corresponds to RSS-PCR type B. RSS-PCR revealed two distinct subtypes (A and B) circulating simultaneously in Thailand, in agreement with previous observations (13).

The dengue-4 isolates we analyzed by RSS-PCR fell into two subtypes, consistent with the two genotypes that resulted from sequence analysis of the entire E gene (10). By both methods, American isolates were contained in a different group than that of Thai viruses. Another phylogenetic analysis derived from sequence comparison of a small 179-nt region of the E gene revealed two subgroups that differed in sequence by 4.9%, again grouping the isolates from the Americas, South Pacific, and Indonesia separately from those from the Philippines, Sri Lanka, and Thailand (4). That dengue-4 yielded fewer subtypes than dengue-1, -2, and -3 is not surprising, since dengue-4 is reported to have less sequence variation in the E gene than the other dengue serotypes (4). A minor difference was observed in pattern A (the absence of a 420 bp-fragment in type A1 compared to A2), indicating a certain degree of genetic variation in Thai isolates (1984).

The occurrence of new dengue epidemics every year emphasizes the need for a simple assay that can facilitate analysis of a large number of samples in order to obtain more detailed epidemiologic information during epidemic periods. The relation between dengue subtype and disease severity has not been extensively studied for dengue-1 or dengue-4 strains, but it warrants further investigation since an association between viruses of Southeast Asian origin and dengue hemorrhagic fever has been reported for dengue-2 and -3 (4, 11, 14). RSS-PCR generates a classification of dengue virus subtypes similar to that obtained using the more labor-intensive and costly sequence analysis approach. In this report, RSS-PCR proved useful in quickly identifying recent isolates of dengue-1 from Brazil (type C) and dengue-4 from Nicaragua (type B). This technique should be valuable as a simple alternative for the rapid characterization of viral isolates and for epidemiologic analysis.