IgM-capture ELISA of serum samples collected from Filipino dengue patients.

Viral antigens for 4 dengue serotypes were produced in C6/36 Aedes albopictus cells. These were used as assay antigens for IgM-capture ELISA to detect IgM antibodies in sera of dengue patients from 3 hospitals in Metro Manila, Philippines. A total of 378 serum samples came from National Children's Hospital (NCH), San Lazaro Hospital (SLH), and St Luke's Medical Center (SLMC), from January to November 1995. Three hundred and four (304) out of 378 serum samples, or 80.42% showed positive IgM ELISA titer against at least one of the 4 assay antigens. Dengue type 4 (D4) antigen detected antibodies in 61.90% (234/378) of these serum samples, whereas type 1 (D1), type 3 (D3), and type 2 (D2) had detection rates of 60.05% (227/378), 50.79% (192/378) and 49.47% (187/378) respectively. Although the results show that both D1 and D4 are the most effective antigens in identifying dengue infections for this batch of samples, the use of a cocktail of antigens is still recommended. The results of this study are the basis for the IgM-capture ELISA protocol presently applied for the laboratory confirmation of dengue cases in the Philippines.

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.

Infection of dengue 2 virus strains isolated from patients exhibiting different disease severities to human peripheral blood leukocytes and production of cytokines in the infected culture supernatant.

Three strains of type 2 dengue virus (DV-2), which had been isolated from patients exhibiting different disease severity, were inoculated to primary culture of human peripheral blood leukocytes from 3 healthy donors. The percentage of dengue antigen positive cells was highest for the strain isolated from a case of dengue shock syndrome, followed by the strain isolated from a case of dengue hemorrhagic fever, and lowest for the strain isolated from a mild case of dengue fever (DF). Generally, similar trend was observed for the amount of some cytokines released into infected culture supernatant, such as interleukin 6, and tumor necrosis factor-alpha. However, such a trend was not observed for interleukin 1 beta.

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.