Evolutionary relationship of 5'-untranslated regions among Thai dengue-3 viruses, Bangkok isolates, during 24 year-evolution.

OBJECTIVE: To study evolutionary relationship of the 5'untranslated regions (5'UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution (1977-2000) comparing to the DEN3 prototype (H87). METHODS: The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced. Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing to the H87 prototype were done in human (HepG2) and the mosquito (C6/36) cell lines. RESULTS: Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90T, and two minor SNPs including A109G and A112G were found. The C90T of Thai DEN3, Bangkok isolates was shown predominantly before 1977. Five Thai DEN3 candidates with the predominant C90T were shown to replicate in human (HepG2) and the mosquito (C6/36) cell lines better than the H87 prototype. However, their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human. CONCLUSIONS: Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high-order secondary structure and the predominant C90T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.

Predominant genotypes and alleles of two functional polymorphisms in the manganese superoxide dismutase gene are not associated with Thai cervical or breast cancer.

BACKGROUND: Defects of manganese superoxide dismutase (MnSOD) have long been implicated in generation of oxidative stress and risk susceptibility to various cancers. Two functional polymorphisms within the MnSOD gene, including the Val-9Ala of the mitochondrial targeting sequence (MTS) and the Ile58Thr of the exon-3, have been proposed to reduce its enzyme activity and antioxidant potential. MATERIALS AND METHODS: A high- throughput multiplex SNaPshot® system was developed herein for simultaneous analyses of Val-9Ala and Ile58Thr in a single reaction. Genomic DNA extracted from each whole blood sample of 248 patients including 107 with cervical cancer and 141 with breast cancer and from 136 healthy women as controls was analyzed by the multiplex SNaPshot® system. RESULTS: The Val/Val, Val/Ala genotypes and the Val allele of the MTS were predominant in patients with cervical or breast cancer as well as healthy women in Thailand. The Ile/Ile genotype and the Ile allele of the exon-3 were found in all of them whereas none of the Ile/Thr, the Thr/Thr genotypes and the Thr allele was detected. Genotypic association of both Val-9Ala and Ile58Thr polymorphisms with cervical cancer and breast cancer of these patients comparing to healthy women was not statistically significant (p<0.05). CONCLUSIONS: The Val/Val, Val/Ala genotypes and the Val allele of the MTS were found predominantly but the Ile/Ile genotype and the Ile allele of the exon-3 were detected in patients with cervical cancer, breast cancer and healthy women in Thailand. These two functional polymorphisms (Val-9Ala and Ile58Thr) in MnSOD gene did not associate with susceptibility risk of these cancer patients in Thailand.

Molecular genetic relationship of the 3' untranslated region among Thai dengue-3 virus, Bangkok isolates, during 1973-2000.

Dengue virus serotype 3 (DENV-3) was associated with severe dengue epidemics in Thailand during 1973-1999. We studied Thai DENV-3 viruses isolated from hospitalized children in Bangkok with differing disease severity during that period. Viruses were sequenced at their 5' and 3' untranslated regions (UTRs), which are regions that play a pivotal role in viral replication. Our results indicated that the primary sequences as well as the secondary structures at both ends of Thai DENV-3 viruses were highly conserved over almost three decades. We found nucleotide insertions and deletions at the variable region (VR) that is located just downstream of the nonstructural protein 5 (NS5) stop codon among these viruses. The phylogenetic tree derived from the size heterogeneity of VR in the 3' UTR divided DENV-3 into four genotypes, and Thai DENV-3 viruses in this study belonged to genotype II. The replication efficiency of the candidate viruses with different lengths at the VR were assessed in the mosquito (C6/36) and human (HepG2) cell lines. Our results show that the viruses with nucleotide insertions at VR replicated better than the virus that contained deletions. Our findings indicate that Thai DENV-3 demonstrated a remarkable conservation of nucleotides over 28 years. Correlation with disease severity suggests that both primary sequences and secondary structures of the 3' UTR do not appear correlated with disease severity in humans.

ELISA as an alternative tool for epidemiological surveillance for dengue in mosquitoes: a report from Thailand.

BACKGROUND & OBJECTIVES: Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the re-emerging infectious diseases caused by the four serotypes of dengue (DEN) virus, type 1 to 4, belonging to the family Flaviviridae and genus Flavivirus. In the absence of a safe and effective mass immunisation, the prevention and control of dengue outbreaks depend upon the surveillance of cases and mosquito vector. The aim of this work is to test enzyme-linked immunosorbent assay (ELISA) tool for the virological surveillance of dengue. METHODS: Virus-infected Aedes mosquitoes were collected from the field in order to serve as an early warning monitoring tool for dengue outbreaks. In a prospective field study conducted from April to September 2000, female adult Aedes mosquitoes were caught from selected dengue-sensitive area in Chombung district, Ratchaburi province and assayed by ELISA. RESULT: Approximately 18.3% were found positive for dengue virus. CONCLUSION: This can imply that ELISA can be an alternative tool for epidemiological surveillance for dengue in mosquitoes.

Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway.

The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

Epigenetic regulation of manganese superoxide dismutase expression in human breast cancer cells.

Malignant breast cancer cells often exhibit lower expression and activity of manganese superoxide dismutase (MnSOD) than their normal cell counterparts; however, the mechanism(s) responsible for this change remains unclear. We examined whether SOD2, the gene encoding MnSOD, was epigenetically repressed in breast cancer cell lines by DNA methylation and histone acetylation. RT-PCR analysis of SOD2 mRNA showed the nontumorigenic breast epithelial cell line MCF-10A to have two to three fold higher expression levels than either UACC-893 or MDA-MB-435 breast carcinoma cells. Analysis of a region in the SOD2 promoter by sodium bisulfite genomic sequencing demonstrated significantly higher levels of CpG methylation in both human breast carcinoma cell lines assessed than in MCF-10A cells. CREB binding in vitro to a cognate site derived from this region was repressed by DNA methylation, and CREB binding to the 5' regulatory region of the SOD2 gene in vivo as determined by ChIP was significantly lower in breast carcinoma cells than in MCF-10A. Increased cytosine methylation was also accompanied by a significant decrease in the level of acetylated histones in the same region of the SOD2 promoter. Finally, a causal link between cytosine methylation and transcriptional repression was established by increasing MnSOD mRNA, protein and activity in breast carcinoma cells using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. These findings indicate that epigenetic silencing of SOD2 constitutes one mechanism leading to the decreased expression of MnSOD observed in many breast cancers.

Expression and evolutionary analysis of West Nile virus (Merion strain).

The authors report a new strain of West Nile virus (WNV) with the expression analysis of its individual open reading frames. Since its sudden appearance in the summer of 1999 in New York City, the virus has spread rapidly across the continental United States into Canada and Mexico. Besides, its rapid transmission by various vectors, the spread of this virus through organ transplantation, blood transfusion, and mother-child transmission through breast milk is of concern. In order to understand molecular variations of WNV in North America and to generate new tools for understanding WNV biology, a complete clone of WNV has been constructed. Investigations so far have focused only on half of its genes products and a detailed molecular and cell biological aspects on all of WNV gene have yet to be clearly established. The open reading frames of WNV were recovered through an reverse transcriptase-polymerase chain reaction (RT-PCR)-PCR using brain tissue from a dead crow collected in Merion, PA, and cloned into a mammalian expression vector. The deduced amino acid sequences of individual open reading frames were analyzed to determine various structural motifs and functional domains. Expression analysis shows that in neuronal cells, C, NS1, and NS5 proteins are nuclear localized whereas the rest of the antigens are confined to the cytoplasm when they are expressed in the absence of other viral antigens. This is the first report that provides an expression analysis as well as intracellular distribution pattern for all of WNV gene products, cloned from an infected bird. Evolutionary analysis of Merion strain sequences indicates that this strain is distinct phylogenetically from the previously reported WNV strains.

The geographic information system as an epidemiological tool in the surveillance of dengue virus-infected Aedes mosquitos.

A Geographic Information System (GIS) was used as analysis tool to study the spatial distribution of dengue virus-infected Aedes mosquitos in Thailand. Global Positioning System (GPS) instruments were used to map villages involved in dengue epidemiological studies in Ratchaburi Province, Thailand. Differentially processed GPS data, with a spatial resolution of approximately 1 meter, were incorporated into a GIS for analysis and mapping. Databases associated with a village GIS included village number, Aedes aegypti populations, and test results. Epidemiological surveillance for dengue infection through the detection of the dengue virus type(s) infecting Aedes mosquitos during epidemic periods constitutes a reliable sentinel system for dengue outbreaks. Various techniques were applied including: enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA), and reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the virologic surveillance of the type-specific detection of dengue viruses in artificially infected and in field-caught adult Aedes mosquitos. In laboratory experiments, all assays showed sufficient sensitively to detect one virus infected mosquito and the rapid RT-PCR clearly showed serotype-specificity with very high detection sensitivity. In the field study conducted from April to September 2000, female adult Aedes mosquitos were collected from selected dengue-sensitive areas in Chom Bung district, Ratchaburi Province and assayed by ELISA, IFA and RT-PCR with 18.3% (44/240), 28.98% (20/69) and 15% (3/20) positive for dengue virus, respectively. Geographic distribution of the virus-infected Aedes mosquitos and household locations were demonstrated by the GPS and the GIS. The development of disease mapping data coupled with RT-PCR laboratory-based surveillance of dengue virus infection can successfully serve as epidemiologic tools in an early warning system for dengue hemorrhagic fever (DHF) epidemics.

A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.

A novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.