Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

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.

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2.

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.

Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses.

In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.

Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to be transmitted primarily through dispersal of droplets, but little is known about the load of SARS-CoV in oral droplets. We examined oral specimens, including throat wash and saliva, and found large amounts of SARS-CoV RNA in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/mL) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/mL) from all specimens of 17 consecutive probable SARS case-patients, supporting the possibility of transmission through oral droplets. Immunofluorescence study showed replication of SARS-CoV in the cells derived from throat wash, demonstrating the possibility of developing a convenient antigen detection assay. This finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for SARS diagnosis.

Site-directed mutagenesis in a conserved motif of Epstein-Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases.

Sequence alignment of human herpesvirus DNases revealed that they share several conserved regions. One of these, the conserved motif D203...E225XK227 (D.EXK) in the sequence of Epstein-Barr virus (EBV) DNase, has a striking similarity to the catalytic sites of some other nucleases, including type II restriction endonucleases, lambda exonuclease and MutH. The predicted secondary structures of these three residues were shown to resemble the three catalytic residues of type II restriction endonucleases. Site-directed mutagenesis was carried out to replace each of the acidic residues near the motif by residues with different properties. All substitutions of D203, E225 and K227 were shown to cause significant reductions in nuclease activity. Six other acidic residues, within the conserved regions, were also replaced by Asn or Gln. Five of these six variants retained nuclease activity and mutant D195N alone lost nuclease activity. The four charged residues, D195, D203, E225 and K227, of EBV DNase were found to be important for nuclease activity. Biochemical analysis indicated that the preference for divalent cations was altered from Mg2+ to Mn2+ for mutant E225D. The DNA-binding abilities of D203E, E225D and E225Q were shown to be similar to that of wild-type. However, K227 mutants were found to have variable DNA-binding abilities: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability. Comparison of the biochemical properties of the corresponding substitutions among EBV DNase and type II restriction enzymes indicated that the D...EXK motif is most likely the putative catalytic centre of EBV DNase.