The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.

Objective. In 1998, an enterovirus 71 (EV71) epidemic in Taiwan was associated with hand, foot, and mouth disease (HFMD)/herpangina and involved 78 fatal cases. We measured EV71 seroprevalence rates before and after the epidemic and investigated risk factors associated with EV71 infection and illness. Methods. Neutralizing antibodies to EV71 were assayed for 539 people before the epidemic and 4619 people of similar ages after the epidemic. Questionnaires, which were completed during household interviews after the epidemic, solicited demographic variables, exposure history, and clinical manifestations. Results. A total of 129 106 cases of HFMD were reported during the epidemic. Age-specific pre-epidemic EV71 seroprevalence rates were inversely related to age-specific periepidemic mortality rates (r = −0.82) or severe case rates (r = −0.93). Higher postepidemic EV71 seropositive rates among children who were younger than 3 years positively correlated with higher mortality rates in different areas (r = 0.88). After the epidemic, 51 (56%) of 91 younger siblings of elder siblings who were EV71-seropositive were EV71-seropositive; otherwise, 2.2% (4 of 186) of younger siblings were EV71-seropositive (matched odds ratio [OR]: 10; 95% confidence interval [CI]: 3.4–29). Stepwise multiple logistic regression revealed other factors associated with EV71 infection to be older age (adjusted OR: 2.5; 95% CI: 1.9–3.4), attendance at kindergartens/child care centers (adjusted OR: 1.8; 95% CI: 1.3–2.5), contact with HFMD/herpangina (adjusted OR: 1.6; 95% CI: 1.2–2.1), greater number of children in a family (adjusted OR: 1.4; 95% CI: 1.1–1.7), and rural residence (adjusted OR: 1.4; 95% CI: 1.2–1.6). Twenty-nine percent of preschool children who were infected with EV71 developed HFMD/herpangina. Younger age and contact with HFMD/herpangina were significant factors for the development of EV71-related HFMD/herpangina in these children. Conclusions. An increased incidence of EV71 infection in young children occurred more often in geographic areas with increased mortality rates. Intrafamilial and kindergarten transmissions among preschool children were major modes of disease transmission during the widespread EV71 epidemic in Taiwan in 1998.

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Background. Although previous studies have revealed the contribution of an initial high level of dengue virus replication to the severe and potential