Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.
 ...
PMID:Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. 137 17

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.
 ...
PMID:Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. 171 65

A complementary DNA copy of DEN-1 RNA was synthesized using reverse transcriptase and a random primer. The double-stranded DNA copy was cloned at the Sma I site of the pUC13 vector and was used to transform Escherichia coli JM83. Among the transformants selected for characterization by nucleotide sequence analysis, we report here one that codes for a region of nonstructural hydrophilic protein, NS-3. Computer analysis of this sequence (967 bp) showed about 87% conservation at the amino acid level and 79% at the nucleotide level when compared with dengue serotypes 2, 3 and 4 and Japanese encephalitis virus. This suggests an important function which is common to all four serotypes. Comparison of the cloned region with sequences of the above strains also suggested conservation of hydrophobic regions.
 ...
PMID:Partial sequence analysis of type 1 dengue virus coding for the nonstructural hydrophilic protein NS-3. 182 70

Dengue virus (DEN) is a member of flaviviruses and contains a single, (+)-strand RNA of approx. 11 kb. Complementary DNA copy of the RNA was synthesized using reverse transcriptase and oligo(dT) as primer. The double-stranded DNA copy was cloned at the PstI site of pUC13'-1 vector and was used to transform Escherichia coli JM83. Eleven transfomants were found to contain DEN insert as screened by colony hybridization. Three clones were chosen for further characterization by nucleotide (nt), sequence analysis. Two of these clones overlapped by 470 bp. Sequences of these three clones totalling about 4.6 kb were obtained. Translation of this DNA in all possible reading frames revealed the presence of long open reading frames spanning the entire length of the cDNA clones. The putative polypeptides derived from the nt sequence are 885 and 643 amino acids in length and show homology to the region of polyprotein coded by the yellow fever virus genome corresponding to the non-structural proteins [Rice et al., Science 229 (1985) 726-733]. The significant homology between these two viruses in the regions coding for the non-structural proteins NS3 and NS5 suggests an important role for these two proteins in the life cycle of these viruses.
 ...
PMID:Partial sequence analysis of cloned dengue virus type 2 genome. 380 28

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.
 ...
PMID:An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses. 751 96

A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.
 ...
PMID:Ultra-rapid, simple, sensitive, and economical silica method for extraction of dengue viral RNA from clinical specimens and mosquitoes by reverse transcriptase-polymerase chain reaction. 768 35

In order to better understand human T-cell responses to dengue viruses (DV), we analyzed T-cell receptor (TCR) V beta gene usage in DV-specific T lymphocytes. Peripheral blood T lymphocytes from a DV type 4 immune donor were stimulated in vitro with DV type 4 antigen, and TCR usage was examined by reverse transcriptase PCR. TCR V beta 17 was preferentially used (P = 0.020) among T cells stimulated by DV type 4 in bulk culture. Furthermore, 8 of 19 DV type 4-specific CD4+ T-cell clones established from the peripheral blood mononuclear cells expressed V beta 17 (P = 0.008). Preferential usage of TCR V alpha was not found among T cells expressing V beta 17. These results indicate that there is preferential usage of TCR V beta 17 among DV-specific T cells in this donor and suggest that T cells with certain TCRs may be important in immune responses to DV.
 ...
PMID:Preferential usage of T-cell receptor V beta 17 by dengue virus-specific human T lymphocytes in a donor with immunity to dengue virus type 4. 793 52

The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
 ...
PMID:Molecular evolution and epidemiology of dengue-3 viruses. 811 41

The double-stranded replicative form of dengue 2 virus (DEN-2) RNA (Tonga strain) was used as a substrate to produce DNA clones of the NS1-NS2A genes via reverse transcriptase synthesis of full length cDNA followed by polymerase chain reaction amplification of the NS1-NS2A region. Products were cloned into pTZ18R for sequencing and into baculovirus for expression studies. The deduced amino acid sequence of the NS1-NS2A was almost identical to that of the S1 attenuated strain of DEN-2 Puerto Rico 159, differing in only four amino acids. The NS1 protein expressed in insect cells [Spodoptera frugiperda (Sf-9)] from the baculovirus recombinant was indistinguishable from authentic NS1 of DEN-infected Aedes albopictus cells in glycosylation, dimerization, cellular presentation and antigenicity. Mice injected with the expressed protein developed NS1-specific complement-fixing antibodies and were partially protected against neurological residua after intracranial challenge. The protective effect was mouse strain- and gender-specific.
 ...
PMID:Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system. 842 52

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera. 847 56


1 2 3 4 5 6 7 8 9 10 Next >>