Yow-Cheong Chan

Full-length cDNA sequence of dengue type 1 virus (Singapore strain S275/90)☆

The complete nucleotide sequence and the deduced amino acid sequence of the genome of dengue virus type 1 (Singapore strain S275/90) were determined from cDNA clones. The single-stranded, positive-sense RNA is 10,718 nucleotides in length and contains a single long open reading frame of 10,188 nucleotides encoding a polyprotein of 3396 amino acids. The genomic size and organization were found to be similar to that of other dengue virus serotypes. Both the nucleotide and deduced amino acid sequences were compared with the partial sequence of DEN1 (Nauru Island) and complete sequences of DNE2 (Jamaica), DEN3 (H87), and DEN4 (Dominica) virus genomes.

EXPRESSION OF THE DENGUE VIRUS STRUCTURAL PROTEINS IN PICHIA PASTORIS LEADS TO THE GENERATION OF VIRUS-LIKE PARTICLES

We have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1.13 g/cm3 when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.

Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens

BACKGROUND More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. OBJECTIVE To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera. STUDY DESIGN A first round of single-step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID(50)-titrated virus-infected tissue culture fluid, and total RNA extracted from C6/36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera. RESULTS The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation. CONCLUSIONS This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.

A survey of Blastocystis in reptiles.

A total of 28 species of reptiles were investigated forBlastocystis using light microscopy and in vitro culture in biphasic egg slant medium.Blastocystis species were detected in 8 (28.6%) of these 28 species in 3 tortoises (Geochelone elephantopus, G. elegans andG. carbonaria), 3 snakes (Boiga dendrophilla, Python reticulatus andElaphe radiata), 1 crocodile (Crocodylus porosus) and 1 iguana lizard (Cyclura cornuta). The reptilianBlastocystis appeared to be morphologically similar toB. hominis.

A Blastocystis species from the sea-snake, Lapemis hardwickii (Serpentes: Hydrophiidae)

Observations were made on Blastocystis isolated from the sea-snake, Lapemis hardwickii. Exponential growth of the organism was observed between 2 and 4 days of culture. Vacuolated, amoeboid and granular forms were observed in cultures, similar to B. hominis. The optimal growth temperature for the sea-snake Blastocystis was 24 degrees C compared with 37 degrees C for B. hominis. The karyotypic patterns of B. hominis and the sea-snake Blastocystis were studied in the clamped homogeneous electric field (CHEF) technique and found to be different. Based on the above differences, the sea-snake Blastocystis was designated as Blastocystis lapemi sp. nov.

Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus.

Antibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the non-structural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies prepared against NS3 of dengue 1 virus (Hawaiian). Five monoclonal antibodies that were authenticated to be reactive to NS3 were used to immunize 13- to 14-day old mice intraperitoneally. Thereafter, the mice were challenged intracerebrally with 100 LD50 of neurotropic dengue 1 virus and the survival indices of the mice were calculated. Significant decreases in survival indices (P less than 0.05), indicating increases in survival times were observed with four of five monoclonal antibodies tested. Monoclonal antibodies to NS3 of dengue 1 virus are able to increase the survival time of mice challenged with a lethal dose of dengue 1 virus, although the mechanism remains to be defined.

Adult dengue deaths in Singapore

BACKGROUND Dengue haemorrhagic fever (DHF) is a leading cause of hospitalization of children in Southeast Asia and regarded mainly as a problem of childhood. In Singapore, however, both dengue fever (DF) and DHF now occur most frequently in those aged 16-25 years and case fatality rates are higher among adults than children. OBJECTIVE To describe adults who died from DHF in Singapore. STUDY DESIGN The clinical, laboratory and, where performed, autopsy records of adults reported to the Ministry of the Environment to have died from DHF were reviewed. RESULTS Four of 10 adults had clinical, serological and/or virological evidence of acute dengue virus infection. All 4 patients, who were between 27 and 58 years old, had dengue IgM antibodies. Two of them had elevated dengue IgG antibodies consistent with a recent infection. Dengue type 2 virus was isolated from one of these two patients who had bled into the lungs, skin, pericardial and pleural surfaces and succumbed to shock. The other patient had no evidence of overt bleeding. A third patient, who suffered massive intractable retroperitoneal haemorrhage and shock, seroconverted in the haemagglutination inhibition test for dengue antibodies. The fourth patient had bleeding into her skin, urinary and gastrointestinal tracts, acute pulmonary oedema, ascites and hypotension. Her stillborn baby also had ascites. CONCLUSION Deaths from DHF are not mainly a childhood occurrence. Adults do die from severe DHF, whether the infection be primary or secondary.

Prevalence of Human Papillomavirus Types 16 and 18 in Cervical Carcinomas: A Study by Dot and Southern Blot Hybridization and the Polymerase Chain Reaction

Histologically classified biopsies from 83 women with invasive cervical carcinoma were analyzed by dot blot hybridization for human papillomavirus (HPV) types 16 and 18 infection. Sixty of the 83 (72.3%) were found to contain HPV DNA, of which 43 (51.8%) contained HPV 16 DNA, 12 (14.5%) contained HPV 18 DNA and 5 (6.0%) contained both HPV 16 and 18 DNAs. Southern blot analysis on 65 specimens gave similar results. Of 23 specimens negative by dot blot, 21 were tested by the polymerase chain reaction. Seventeen of the 21 were positive for HPV DNA, of which 13 contained HPV 16 DNA and 4 contained both HPV 16 and 18 DNAs. In all, 95.1% (77/81) were positive for HPV 16 and/or 18 DNA sequences.

A shortened dengue IgM capture ELISA using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody.

A shortened IgM capture ELISA for the detection of dengue IgM antibodies using simultaneous incubation of antigen and peroxidase-labeled monoclonal antibody was described. The shortened two-step assay was compared with the four-step IgM capture ELISA on sera from dengue patients confirmed by the hemagglutination inhibition (HI) test. When paired acute and convalescent sera were tested, the shortened ELISA showed 100% agreement with HI results. It detected dengue IgM antibodies in the acute sera of 66% of patients with a primary dengue infection, 60% of patients with a secondary infection, and 98% of patients with a presumptive secondary infection. When the results of 151 dengue patients were combined, 75% of the acute sera were positive by the shortened IgM capture ELISA.

Virus-like particles in a Blastocystis species from the sea-snake, Lapemis hardwickii

Abstract A 5.0 kb band was consistently observed in purified nucleic acid samples from a sea-snake Blastocystis. This band was resistant to DNase I treatment, and eliminated by 0.2 n -NaOH treatment. That it was highly susceptible to pancreatic ribonuclease A, with sensitivity increasing with decreasing ionic strength, and was undigested by mung bean nuclease indicated that it was a dsRNA band. Transmission electron microscopy of cesium chloride fractions revealed icosahedral virus-like particles 30 nm in diameter. These icosahedral virus-like particles were not found in isolates of B. hominis .