A novel method for the preparation of large cDNA fragments from dengue-3 RNA genome by long RT-PCR amplification.

For many years, dengue viruses were among the most difficult flaviviruses to isolate and to identify, but technical advances in the past 20 years have facilitated this process. Dengue viruses are usually recovered from specimens by the infection of mosquito-cell cultures. The virus may be passaged several times in cell cultures until a sufficient infectivity titer is attained. The viral nucleocapsid consists of capsid protein and an RNA genome. The dengue genome is a single stranded messenger (positive) sense RNA of approximately 11 kb in length. The isolation of dengue genomic RNA from various sources requires precautions to avoid RNases. RNases are released during cell disruption, and their activity must be inhibited as quickly as possible by using guanidinium thiocyanate in the presence of 2-mercaptoethanol. There has recently been a revolution in molecular biology with the development of the powerful reverse transcriptase (RT) and polymerase chain reaction (PCR) technology. Advanced studies on RT technique lead to much further improvement of the reverse transcriptase enzyme by genetic engineering. The Superscript II RNase H- RT (GIBCO BRL, USA) is genetically engineered DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA. DNA or an RNA-DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene constructed by the introduction of point mutation in the RNase H active center. The selective mutations within the RNase H domain maintain full polymerase activity. This structural modification eliminates degradation of RNA molecules during the first strand cDNA synthesis. The combination of thermostable DNA polymerase with and without proofreading activity (3'-exonuclease activity), improved buffer conditions and thermal cycling profiles overcome the length limitation of PCR. On the basis of these findings, we have developed a long RT-PCR system for preparing large cDNA fragments of dengue 3 virus (H-87) by using the Superscript II RNase H- RT for reverse transcription and a mixture of Taq and Pwo DNA polymerases for PCR. Three large cDNA fragments covered the full genomic RNA from the 5'-end to the 3'-end of dengue-3 virus (H-87; 10,696 bps) could be successfully prepared as the lengths of 2.437 bps, 3,980 bps and 4,337 bps respectively. The ability of our developed long RT-PCR will bring speed and simplicity to genomic mapping and sequencing and facilitate studies in molecular genetics of dengue viruses.

Highly conserved nucleotide sequence and its deduced amino acids of the 5'-noncoding region and the capsid protein of a Bangkok isolate dengue-3 virus.

The dengue-3 virus genome encodes an uninterrupted open reading frame (ORF) flanked by 5' and 3' non-coding regions. The order of proteins encoded in dengue-3 virus ORF, as with other flaviviruses, is: Cap 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. The nucleotide sequence of the 5'-noncoding region and the capsid protein of dengue-3virus (a Bangkok isolate: CH53489 isolated by USAMC-AFRIMS in 1973) has been analyzed in both forward and reverse directions. The PCR-based cycle sequencing technique by the enzymatic method of Sanger et al (1977) using a sequencing primer 5'-end labeled with gamma32P-ATP is the method of our choice for sequencing analysis. One cDNA template was prepared by RT-PCR technique starting from the 5'-end nucleotide 1-465 of the dengue-3 genome. In our cycle sequencing experiments, the substitution of 7-deaza-dG was used for dG in DNA eliminated much of the secondary structures that produced gel artifacts. The final sequence result of this cDNA template was established from its sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequence as well as its deduced amino acid sequence of the Bangkok dengue-3 virus and the published sequence data of the dengue-3 prototype (H87) was manipulated by the PC-DOS-GIBIO-DNASIS TM 06-00 (Hitachi Software). According to the deduced amino acid sequence of the Bangkok dengue-3 virus, its C protein was found to be highly positively charged because of large numbers of lysine and arginine. The homology of the nucleotide sequence between the two dengue-3 virus revealed 97%. The deduced amino acid sequences from the nucleotides 95-465 of the two viruses showed the same indicating highly conserved capsid proteins. Multiple alignment of the nucleotide sequences as well as the deduced amino acid sequences among the Bangkok dengue-3 virus and other dengue 3 viruses also confirmed the highly conserved 5'-noncoding regions and the capsid proteins.

B cells are the principal circulating mononuclear cells infected by dengue virus.

Although dengue virus infects a variety of cells in vitro, little is known about cell types infected in vivo. Since blood is a readily accessible tissue, we chose to determine which circulating blood cells are infected by dengue viruses. We collected blood mononuclear cells from acutely ill dengue patients and separated the cells by flow cytometry into subsets for virus isolation. Cells were sorted into groups corresponding to the cluster designations CD3, CD14, CD16 and CD20. Virus was isolated from sorted groups by inoculation into Toxorhynchites splendens mosquitos. The majority of the virus was recovered from the CD20 or B cell positive subset. Little virus was isolated from monocytes, NK cells or T cells. Virus was isolated from B cells regardless of the age or sex of the patient, virus serotype isolated, or the patient's history of dengue virus infection. The location of cell associated virus was determined by proteolytic digestion of surface virus. There was an equal distribution of virus between the intracellular compartment and the surface of B cells. The intracellular localization of virus was confirmed by immunocytochemistry. Since this study focused on circulating cells, no inferences were made regarding infection of cells in solid tissues.

Causes of death and unintentional injury among schoolchildren in Thailand.

Few prospective studies of mortality among children in developing countries have been published. Here we quantify and describe mortality and injury morbidity among a cohort of schoolchildren in rural Southeast Asia. Deaths among a cohort of 40,119 schoolchildren in Thailand were prospectively monitored over a two year period from January 1991. Additionally, data were collected with a questionnaire from a subset of 6,378 children asking them to recall all injuries over a one-year period. There were 40 deaths for an annual incidence of 50/100,000. Fifty percent of all deaths were due to injury; 25% to infectious diseases. Sixty percent of the injury deaths were due to motor vehicles and 35% to drowning. Sixty-six percent of the children reported one or more accidents. The leading categories of non-fatal injuries, in decreasing order, were: animal bite, puncture wound, burn, near-drowning, fall from a height. Boys experienced more injuries than girls for almost every type of injury. Injuries are replacing infectious diseases as the most important cause of deaths in developing countries. Additional public health initiatives to reduce childhood accidents may be warranted.

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 3 virus, Bangkok genotype.

The nucleotide sequence of the nonstructural protein gene (1,610 bases) of dengue 3 virus (Bangkok genotype; CH53489 isolated in 1973) has been determined in both forward and reverse directions. The PCR based cycle sequencing technic by the enzymatic method of Sanger et al using a sequencing primer 5'-end labeled with gamma-32P-ATP was the method of our choice for sequence analysis. Two cDNA templates were prepared by RT-PCR technique starting from the nucleotides 6,306-6,969 and 6,925-7,915 of the dengue 3 genome with the lengths of 663 and 990 base pairs respectively. In our cycle sequencing experiments, it has been observed that the substitution of 7-deaza-dG for dG in DNA eliminated most of the secondary structures that produce gel artifacts. The final sequence results of these two cDNA templates were established from their sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequences as well as their deduced amino acid sequences of the Bangkok dengue 3 (CH53489) virus and the published sequence data of the dengue 3 prototype (H87) was manipulated by the PC-DOS-GIBIO DNASIS TM 06-00 software. The homology of the nucleotide sequences between the two dengue 3 viruses was 96.65%. The deduced amino acid sequence from nucleotides 6,306-7,915 of the two viruses showed conserved amino acids of the nonstructural protein NS4a and 6 amino acid changes in NS4b and NS5.

IgM capture ELISA for detection of IgM antibodies to dengue virus: comparison of 2 formats using hemagglutinins and cell culture derived antigens.

The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.

Human immune responses to dengue viruses.

Dengue fever (DF) and dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) are major public health problems in many areas of the world. We are analyzing the human immune responses to dengue viruses, in order to understand the mechanism of recovery from dengue virus infections and the pathogenesis of DHF/DSS. Human natural killer (NK) cells lyse dengue virus-infected cells to a greater degree than uninfected cells. Antibodies to dengue viruses augment the lysis of dengue virus-infected cells by NK cells. Dengue virus-infected monocytes produce high levels of interferon alpha (IFN alpha). DR+ lymphocytes also produce high levels of IFN alpha after contact with dengue virus-infected monocytes. The IFN alpha produced protects uninfected monocytes from dengue virus infection. These results suggest that NK cells and IFN alpha may play an important role in controlling primary dengue virus infection. Dengue virus-specific CD4+CD8(-)T lymphocytes and CD4(-)CD8+T lymphocytes are present in the peripheral blood mononuclear cell population from donors who were infected with dengue virus. Most of CD4+T lymphocytes are dengue serotype-crossreactive. They lyse dengue virus-infected autologous cells in an HLA class II-restricted fashion, and produce interferon gamma (IFN gamma). IFN gamma augments dengue virus infection of monocytic cells in the presence of antidengue virus antibodies by increasing the number of Fc gamma receptors. Dengue virus-specific CD8+T lymphocytes lyse dengue virus-infected autologous cells in an HLA class I-restricted fashion. These CD8+T lymphocytes are also dengue serotype-crossreactive.(ABSTRACT TRUNCATED AT 250 WORDS)