Molecular evolution, distribution and genetic relationship among the dengue 2 viruses isolated from different clinical severity.

Twenty-two strains of dengue 2 virus, isolated in China, Latin America, New Guinea and Thailand were subjected to phylogenetic analysis. The UPGMA analysis was carried out on each gene region of dengue virus and demonstrated that outcome from most of the gene regions showed similar results except those from NS4B and YUTR with very short nucleotide length. Among ten regions examined, the results from E gene documented the geographical differences of the virus strains most clearly and all the American strains (Mara 4, IQT1797 and S1) were distantly related to the Asian isolates. As for the 16 Thai strains isolated in 1993, they were clustered into three groups and a strain from a DSS patient formed a distinct branch compared to the other two groups. This finding from phylogenetic analysis is consistent with earlier conclusion and support the severity related subtyping of dengue 2 virus based on amino acid changes.

Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations.

The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.

Genotype determination of three dengue type 2 virus strains from Myanmar by sequencing E/NSI gene junction.

Genotype of three dengue-2 virus strains from Myanmar was determined as genotype II by sequencing 240 nucleotide long fragment across the E/NS1 gene junction by the primer extension dideoxy chain termination method, applying direct sequencing of the PCR product. These strains were isolated from a dengue shock syndrome (DSS) patient and two patients with dengue hemorrhagic fever (DHF) grade 1, in Yangon (Rangoon), Myanmar (Burma), in 1987. Sequence homology of all three strains were highest (96%) to New Guinea C strain (genotype II), lesser homology (93%) to Jamaican 1409 strain (genotype III), and the least homology (91%) to PR 159/S1 strain (genotype I). Two DHF strains revealed only 2 nucleotide and 3 nucleotide differences compared with DSS strain, all at the 3rd position of the codons which resulted in silent mutations.

The use of polymerase chain reaction (PCR) as a diagnostic tool for dengue virus.

This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.

Comparative nucleotide and deduced amino acid sequence of the envelope glycoprotein gene among three dengue virus type 2 strains isolated from patients with different disease severities in Maha Sarakham, northeast Thailand.

The nucleotide (nt) sequence of the envelope glycoprotein (E) gene of dengue virus type 2 was determined by the primer-extension dideoxy chain-termination method for 3 dengue virus type 2 (D2) strains which had been isolated from patients with dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), in Maha Sarakham, Northeast Thailand, in 1986-1987. Their nt sequences were essentially the same except for a single silent nt replacement in each DHF and DSS strain compared with DF strain. Therefore, these 3 strains possessed identical deduced amino acid (AA) sequences in their E protein. The result indicated that the primary structure of the E protein of D2 virus is not related to the clinical severity of the infected patients. Eleven nt replacements which resulted in 4 amino acid replacements were found to be unique to these 3 Northeast Thai strains. Sequence similarity showed that the 3 Northeast Thai strains were closest to the DSS isolate (H) followed by the DHF isolate (D) identified in Bangkok in 1980.