| Dengue (DEN) viruses belong to the genus Flavivirus. There are four closely related, but antigenically distinct, virus serotypes (DEN-1, DEN-2, DEN-3 and DEN-4). Viral genome is a positive-sense single-stranded RNA approximately 11,000 nucleotides long, encoding 10 distinct proteins. The gene order is 5′-C-prM (M)-E-NS1-NS2A-NS2B-NS3- NS4A-NS4B-NS5-3′, expressed as a single polyprotein that is cleaved by both viral and cellular proteases to form the viral polypeptides, including three structural proteins: capsid (C), membrane (M) and envelope (E) protein, and the remaining seven are nonstructural (NS) proteins. DEN viruses cause classical dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DF is a nonspecific viral syndrome while DHF/DSS is severe and fatal hemorrhagic disease. Up to 100 million cases of DF occur annually, with some 2 billion people at risk of infection in tropical and subtropical regions of Africa, Asia, and the Americas. The pathogenesis of DEN virus infection is not clear. Although many factors are proposed to involve in DEN virus epidemiology and occurrence of DHF/DSS, evolution of DEN viral virulence and spread of DEN viruses between different geographic areas are two important features in epidemiology and pathogenesis of the disease. Studies have suggested that specific viral structures might contribute to replication capability in target cells and to transmission. Co-circulation of novel DEN genotype, either different genotype of the same serotype or different serotypes of DEN viruses in same area might lead to displacement of native genotype by new genotype and occurrence of DHF/DSS.Several studies have shown that the phylogenetic trees obtained from small gene This work was partially supported by grants no. 30170848 and no. 30300303 from the National Science Foundation of China (NSFC).fragment sequences are congruent with the trees obtained from an entire gene sequence although there might be minor rearrangements in the terminal branches. Using partial sequence from Envelope-Nonstructural protein 1 (E/NS1) junction region, Rico-Hesse defined five genotypes for DEN-1 viruses isolated worldwide: I) America, Africa, and Southeast Asia; II) Sri Lanka; III) Japan; IV) Southeast Asia, the South Pacific, Australia and Mexico; V) Taiwan and Thailand. Recently, Goncalvez set up phylogeny of 36 DEN-1 viruses according full E gene sequences. Statistical analyses of validity of branching patterns (bootstrap) also suggested five classification: 1) from Asia, 2) from Thailand, 3) from sylvatic/Malaysia, 4) from South Pacific and 5) from Americas/Africa, in which three genotypes indicated as 1), 4) and 5) were consistent to I, IV and V referred by Rico-Hesse. Aviles-G and collaborators sequenced Capsid-pre Membrane (C/prM) and E/NS1 regions of 24 recent isolates of DEN-1 virus from South America, suggesting that the recent epidemics in Argentina and Paraguay were due to re-mergence of a previously circulated strain. These all indicated that small gene fragment sequences are available for analysis of viral genetic characterization.In this study, we isolated and identified virus from the serum samples of patients with suspected DF in Guangzhou province in 2002. Firstly, 20 patients with suspected DEN virus infection admitted to the Eighth People's Hospital of Guangzhou were enrolled, including 12 male and 8 female range 23 to 57 years. The blood samples were collected at day 1 to 11 after fever onset. The sera were separated by centrifugation and stored at -70 °C until use. The serum was incubated with the Aedes albopictus cell line, C6/36, for isolation of virus and 5 of the 20 serum samples caused cytopathologic effects on C6/36 cells.Then, a 539-nucleotide (nt) sequence in the NS1 region (positions 2503 to 3041) of the isolated virus genome was amplified using universal primers of 4 serotypes of DEN viruses. The universal primer sequences are:5′-GTG CAC ACA TGG ACA GAA CA-3′ (forward) and 5′-CTT TCT ATC CAA TAA CCC AT-3′ (reverse). After sequencing t... |
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