Molecular serotyping of dengue viruses in field-caught Aedes mosquitos by in-house RNA extraction/RT-PCR reagent kits.

We developed in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. Mosquitos that showed positive results by ELISA or IFA were selected for the identification of dengue viruses in order to predict the distribution of the four dengue serotypes. Total RNA was extracted from one whole mosquito as well as from one dissected mosquito by our in-house RNA extraction reagents using the modified method of guanidinium thiocyanate denaturation and isopropanol precipitation. The extracted RNA was amplified by our in-house RT-PCR reagents specific for each dengue serotype under optimized conditions. Dengue viral RNA extracted from a single mosquito as well as from the head and thorax of one dissected mosquito could be detected successfully; it could not be found in the abdomen, however. These results indicated that most of the dengue viruses were located in the head and thorax rather than in the abdomen. The results of dengue serotyping showed a pure specific PCR product for each dengue serotype at 490, 230, 320 and 398 bp for DEN-1, DEN-2, DEN-3, and DEN-4 respectively. In addition, the detection sensitivity was very high: an amount of RNA template equivalent to approximately 1/80 of a single mosquito could be detected by agarose gel electrophoresis and ethidium bromide staining. The coupling of RT-PCR-based surveillance of dengue viral infection with disease mapping data (Geograpical Information System, GIS) could serve as an alternative epidemiological means of providing an early warning of dengue fever/dengue hemorrhagic fever epidemics.

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.

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.

Prenatal sex determination in Thai pregnant subjects by nested-PCR amplification of fetal Y chromosome-specific sequence.

Detection of a Y-specific sequence in either amniotic cells or the maternal circulation has clinical importance for prenatal sex diagnosis, especially in mothers with severe X-linked disorders. Deoxyribonucleic acid sequences of human ZFY (zinc-finger-Y) gene, a Y-chromosome-specific gene and candidate for the testis-determining factor, were amplified by in vitro nested PCR technique, in amniotic cells or peripheral blood of Thai pregnant subjects carrying male fetuses. This technique permits detection of ZFY gene DNA sequences in as few as a single male cell among 100,000 female cells. The subjects in this study were a group of Thai pregnant women who had been given prenatal fetal sex diagnosis by the cytogenetic method. The results herein demonstrated the usefulness of PCR detection of Y-specific sequences in amniotic cells. The ZFY gene detection method for prenatal sex prediction by nested PCR has been developed experimentally. However, further investigation is necessary to increase reliability of clinical application. With properly designed guidelines, the established nested-PCR technique may be an alternative for the determination of the fetal sex for those pregnancies at risk of X-linked genetic disorders.

Treatment of hypertension with chinese celery.

Twenty mild essential hypertension patients 2 males and 18 females (B.P. 150/95-179/110 mmHg.) aged 34-69 years were tried on crude Chinese celery and its extract. When the crude Chinese celery was extracted, two fractions were found to reduce blood pressure. The fraction which gave the best reduction of blood pressure was used for this trial. The results of the trial showed that the crude Chinese celery had a statistically significant on lowering the blood pressure, but the extract did not. This lack of effect might be due to the fact that only one fraction of the extract was used in the trial. However, there were no changes in the biochemistry of the kidney and liver functions, including electrolytes and lipids, and no side-effect was observed.